Patent application title: Compositions Comprising A Polypeptide Having Cellulolytic Enhancing Activity And A Liquor And Uses Thereof
Inventors:
Jason Quinlan (Woodland, CA, US)
Jason Quinlan (Woodland, CA, US)
Feng Xu (Davis, CA, US)
Feng Xu (Davis, CA, US)
Matthew Sweeney (Sacramento, CA, US)
Matthew Sweeney (Sacramento, CA, US)
Don Higgins (Franklinton, NC, US)
Hui Xu (Wake Forest, NC, US)
Hui Xu (Wake Forest, NC, US)
Assignees:
Novozymes North America, Inc.
NOVOZYMES, INC.
IPC8 Class: AC12P1914FI
USPC Class:
1 1
Class name:
Publication date: 2021-10-14
Patent application number: 20210317494
Abstract:
The present invention relates to compositions comprising: a polypeptide
having cellulolytic enhancing activity and a liquor. The present
invention also relates to methods of using the compositions.Claims:
1. A method for degrading or converting a cellulosic material,
comprising: treating the cellulosic material with an enzyme composition
in the presence of a polypeptide having cellulolytic enhancing activity
and a liquor, wherein the combination of the polypeptide having
cellulolytic enhancing activity and the liquor enhances hydrolysis of the
cellulosic material by the enzyme composition.
2. The method of claim 1, further comprising recovering the degraded cellulosic material.
3. A method for producing a fermentation product, comprising: (a) saccharifying a cellulosic material with an enzyme composition in the presence of a polypeptide having cellulolytic enhancing activity and a liquor, wherein the combination of the polypeptide having cellulolytic enhancing activity and the liquor enhances hydrolysis of the cellulosic material by the enzyme composition; (b) fermenting the saccharified cellulosic material with one or more fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation.
4. A method of fermenting a cellulosic material, comprising: fermenting the cellulosic material with one or more fermenting microorganisms, wherein the cellulosic material is saccharified with an enzyme composition in the presence of a polypeptide having cellulolytic enhancing activity and a liquor, wherein the combination of the polypeptide having cellulolytic enhancing activity and the liquor enhances hydrolysis of the cellulosic material by the enzyme composition.
5. (canceled)
6. (canceled)
7. The method of claim 1, wherein the liquor is obtained from a material that is the same as or different from the cellulosic material subjected to saccharification by the enzyme composition.
8. (canceled)
9. The method of claim 1, wherein the liquor is obtained from a material that is the same as the cellulosic material subjected to saccharification by the enzyme composition, and the treatment conditions used to produce the liquor are the same as or different from the pretreatment conditions of the cellulosic material, and optionally the liquor is further processed to remove inhibitors of a cellulase, a hemicellulase, or a combination thereof.
10. (canceled)
11. (canceled)
12. The method of claim 1, wherein the liquor is obtained from a material that is different than the cellulosic material subjected to saccharification by the enzyme composition, and the treatment conditions used to produce the liquor are the same as or different from the pretreatment conditions of the cellulosic material, and optionally the liquor is further processed to remove inhibitors of a cellulase, a hemicellulase, or a combination thereof.
13. (canceled)
14. (canceled)
15. The method of claim 1, wherein the liquor is obtained from a lignocellulose material, a hemicellulose material, a lignacious material, monosaccharides of the lignocellulose material, monosaccharides of the hemicellulose material, or a combination thereof, and optionally the liquor is further processed to remove inhibitors of a cellulase, a hemicellulase, or a combination thereof.
16. (canceled)
17. (canceled)
18. An isolated liquor, which in combination with a polypeptide having cellulolytic enhancing activity enhances hydrolysis of a cellulosic material by a cellulolytic enzyme.
19. A composition comprising a polypeptide having cellulolytic enhancing activity and a liquor, wherein the combination of the polypeptide having cellulolytic enhancing activity and the liquor enhances hydrolysis of a cellulosic material by a cellulolytic enzyme.
20. The composition of claim 19, which further comprises one or more enzymes selected from the group consisting of a cellulase, a hemicellulase, an esterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin.
21. The composition of claim 19, wherein the liquor is obtained from a lignocellulose material, a hemicellulose material, a lignacious material, monosaccharides of the lignocellulose material, monosaccharides of the hemicellulose material, or a combination thereof.
22. The method of claim 3, wherein the liquor is obtained from a material that is the same as or different from the cellulosic material subjected to saccharification by the enzyme composition.
23. The method of claim 3, wherein the liquor is obtained from a material that is the same as the cellulosic material subjected to saccharification by the enzyme composition, and the treatment conditions used to produce the liquor are the same as or different from the pretreatment conditions of the cellulosic material, and optionally the liquor is further processed to remove inhibitors of a cellulase, a hemicellulase, or a combination thereof.
24. The method of claim 3, wherein the liquor is obtained from a material that is different than the cellulosic material subjected to saccharification by the enzyme composition, and the treatment conditions used to produce the liquor are the same as or different from the pretreatment conditions of the cellulosic material, and optionally the liquor is further processed to remove inhibitors of a cellulase, a hemicellulase, or a combination thereof.
25. The method of claim 3, wherein the liquor is obtained from a lignocellulose material, a hemicellulose material, a lignacious material, monosaccharides of the lignocellulose material, monosaccharides of the hemicellulose material, or a combination thereof, and optionally the liquor is further processed to remove inhibitors of a cellulase, a hemicellulase, or a combination thereof.
26. The method of claim 4, wherein the liquor is obtained from a material that is the same as or different from the cellulosic material subjected to saccharification by the enzyme composition.
27. The method of claim 4, wherein the liquor is obtained from a material that is the same as the cellulosic material subjected to saccharification by the enzyme composition, and the treatment conditions used to produce the liquor are the same as or different from the pretreatment conditions of the cellulosic material, and optionally the liquor is further processed to remove inhibitors of a cellulase, a hemicellulase, or a combination thereof.
28. The method of claim 4, wherein the liquor is obtained from a material that is different than the cellulosic material subjected to saccharification by the enzyme composition, and the treatment conditions used to produce the liquor are the same as or different from the pretreatment conditions of the cellulosic material, and optionally the liquor is further processed to remove inhibitors of a cellulase, a hemicellulase, or a combination thereof.
29. The method of claim 4, wherein the liquor is obtained from a lignocellulose material, a hemicellulose material, a lignacious material, monosaccharides of the lignocellulose material, monosaccharides of the hemicellulose material, or a combination thereof, and optionally the liquor is further processed to remove inhibitors of a cellulase, a hemicellulase, or a combination thereof.
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application Ser. No. 61/373,124, filed Aug. 12, 2010, U.S. Provisional Application Ser. No. 61/373,128, filed Aug. 12, 2010, U.S. Provisional Application Ser. No. 61/373,145, filed Aug. 12, 2010, U.S. Provisional Application Ser. No. 61/373,150, filed Aug. 12, 2010, U.S. Provisional Application Ser. No. 61/373,157, filed Aug. 12, 2010, U.S. Provisional Application Ser. No. 61/373,166, filed Aug. 12, 2010, U.S. Provisional Application Ser. No. 61/373,170, filed Aug. 12, 2010, and U.S. Provisional Application Ser. No. 61/373,210, filed Aug. 12, 2010, which applications are incorporated herein by reference.
REFERENCE TO A SEQUENCE LISTING
[0003] This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.
BACKGROUND OF THE INVENTION
Field of the Invention
[0004] The present invention relates to compositions comprising a polypeptide having cellulolytic enhancing activity and a liquor, and to methods of using the compositions.
Description of the Related Art
[0005] Cellulose is a polymer of the simple sugar glucose covalently linked by beta-1,4-bonds. Many microorganisms produce enzymes that hydrolyze beta-linked glucans. These enzymes include endoglucanases, cellobiohydrolases, and beta-glucosidases. Endoglucanases digest the cellulose polymer at random locations, opening it to attack by cellobiohydrolases. Cellobiohydrolases sequentially release molecules of cellobiose from the ends of the cellulose polymer. Cellobiose is a water-soluble beta-1,4-linked dimer of glucose. Beta-glucosidases hydrolyze cellobiose to glucose.
[0006] The conversion of lignocellulosic feedstocks into ethanol has the advantages of the ready availability of large amounts of feedstock, the desirability of avoiding burning or land filling the materials, and the cleanliness of the ethanol fuel. Wood, agricultural residues, herbaceous crops, and municipal solid wastes have been considered as feedstocks for ethanol production. These materials primarily consist of cellulose, hemicellulose, and lignin. Once the lignocellulose is converted to fermentable sugars, e.g., glucose, the fermentable sugars are easily fermented by yeast into ethanol.
[0007] WO 2005/074647, WO 2008/148131, WO 2011/035027 disclose isolated GH61 polypeptides having cellulolytic enhancing activity and the polynucleotides thereof from Thielavia terrestris. WO 2005/074656 and WO 2010/065830 disclose isolated GH61 polypeptides having cellulolytic enhancing activity and the polynucleotides thereof from Thermoascus aurantiacus. WO 2007/089290 discloses an isolated GH61 polypeptide having cellulolytic enhancing activity and the polynucleotide thereof from Trichoderma reesei. WO 2009/085935, WO 2009/085859, WO 2009/085864, and WO 2009/085868 disclose isolated GH61 polypeptides having cellulolytic enhancing activity and the polynucleotides thereof from Myceliophthora thermophila. WO 2010/138754 discloses isolated GH61 polypeptides having cellulolytic enhancing activity and the polynucleotides thereof from Aspergillus fumigatus. WO 2011/005867 discloses isolated GH61 polypeptides having cellulolytic enhancing activity and the polynucleotides thereof from Penicillium pinophilum. WO 2011/039319 discloses isolated GH61 polypeptides having cellulolytic enhancing activity and the polynucleotides thereof from Thermoascus sp. WO 2011/041397 discloses isolated GH61 polypeptides having cellulolytic enhancing activity and the polynucleotides thereof from Penicillium sp. WO 2011/041504 discloses isolated GH61 polypeptides having cellulolytic enhancing activity and the polynucleotides thereof from Thermoascus crustaceous. WO 2008/151043 discloses methods of increasing the activity of a GH61 polypeptide having cellulolytic enhancing activity by adding a soluble activating divalent metal cation to a composition comprising the polypeptide.
[0008] It would be advantageous in the art to improve the ability of polypeptides having cellulolytic enhancing activity to enhance enzymatic hydrolysis of lignocellulosic feedstocks.
[0009] The present invention relates to compositions comprising a polypeptide having cellulolytic enhancing activity and a liquor, and to methods of using the compositions.
SUMMARY OF THE INVENTION
[0010] The present invention relates to compositions comprising: (a) a polypeptide having cellulolytic enhancing activity; and (b) a liquor, wherein the combination of the polypeptide having cellulolytic enhancing activity and the liquor enhances hydrolysis of a cellulosic material by a cellulolytic enzyme.
[0011] The present invention also relates to methods for degrading or converting a cellulosic material, comprising: treating the cellulosic material with an enzyme composition in the presence of a polypeptide having cellulolytic enhancing activity and a liquor, wherein the combination of the polypeptide having cellulolytic enhancing activity and the liquor enhances hydrolysis of the cellulosic material by the enzyme composition.
[0012] The present invention also relates to methods for producing a fermentation product, comprising:
[0013] (a) saccharifying a cellulosic material with an enzyme composition in the presence of a polypeptide having cellulolytic enhancing activity and a liquor, wherein the combination of the polypeptide having cellulolytic enhancing activity and the liquor enhances hydrolysis of the cellulosic material by the enzyme composition;
[0014] (b) fermenting the saccharified cellulosic material with one or more (e.g., several) fermenting microorganisms to produce the fermentation product; and
[0015] (c) recovering the fermentation product from the fermentation.
[0016] The present invention also relates to methods of fermenting a cellulosic material, comprising: fermenting the cellulosic material with one or more (e.g., several) fermenting microorganisms, wherein the cellulosic material is saccharified with an enzyme composition in the presence of a polypeptide having cellulolytic enhancing activity and a liquor, wherein the combination of the polypeptide having cellulolytic enhancing activity and the liquor enhances hydrolysis of the cellulosic material by the enzyme composition.
BRIEF DESCRIPTION OF THE FIGURES
[0017] FIG. 1 shows the fractional hydrolysis of washed and unwashed pretreated corn stover by a Trichoderma reesei cellulase composition with various concentrations of Thermoascus aurantiacus GH61A polypeptide having cellulolytic enhancement activity. Open symbols: 1 day of hydrolysis; closed symbols: 3 days of hydrolysis; squares: milled, water-washed pretreated corn stover; circles: milled, unwashed pretreated corn stover; triangles: hot-water washed, milled pretreated corn stover.
[0018] FIGS. 2A and 2B show the effect of acid-pretreated corn stover liquor on GH61 polypeptide-enhancement of cellulolysis of milled, water-washed pretreated corn stover by a T. reesei cellulase composition. Panel A: fractional hydrolysis. White bars: 1 day hydrolysis; gray bars: 3 day hydrolysis. Panel B: open symbols, 1 day hydrolysis; solid symbols: 3 day hydrolysis. Circles: no added liquor; squares: 2% (v/v) liquor; diamonds: 5% (v/v) liquor; triangles: 10% liquor; inverted triangles: 15% liquor. Data were fit linearly or by a modified saturation-binding model as described.
[0019] FIGS. 3A and 3B show acid-pretreated corn stover liquor dependence of T. aurantiacus GH61A polypeptide-enhancement of hydrolysis of pretreated corn stover. Panel A: AVICEL.RTM.+various concentrations of the acid-pretreated corn stover liquor as a function of GH61 polypeptide concentrations. Panel B: AVICEL.RTM.+synthetic liquor as a function of GH61 polypeptide concentration. Open symbols: 1 day hydrolysis; closed symbols: 3 days hydrolysis. Circles: no liquor; diamonds: 5% (v/v) liquor; triangles: 10% liquor; inverted triangles: 15% liquor; squares: 5% synthetic liquor containing no phenol; right triangles: 15% synthetic liquor containing no phenol. Data were fit linearly or using Equation 2, as described.
[0020] FIGS. 4A, 4B, and 4C show the effect of enzymatically treated or not enzymatically-treated pretreated corn stover liquors on enhancement of cellulolysis of pretreated corn stover by the Thelavia terrestris GH61E polypeptide. Panel A: un-treated liquor; Panel B: T. reesei cellulase-treated liquor; and Panel C: T. reesei cellulase and Thelavia terrestris GH61E polypeptide-treated liquor. Circles: no added liquor; squares: 5% (v/v) liquor; diamonds: 10% (v/v) liquor; triangles: 15% (v/v) liquor. Data were fit linearly or using Equation 2, as described.
[0021] FIGS. 5A and 5B show the fractional hydrolysis of microcrystalline cellulose by the T. reesei cellulase composition with various T. aurantiacus GH61A polypeptide concentrations, comparing impact of addition of dilute-acid and steam pretreatment liquors at 5 days of hydrolysis. Panel A: NREL acid-pretreated corn stover liquor; Panel B: steam explosion-pretreated corn stover liquor. Open symbols: 5% (v/v) liquor; closed symbols: 15% (v/v) liquor. Circles: whole liquor; squares: low molecular weight fraction, triangles: high molecular weight fraction. Data were fit using Equation 2, as described.
[0022] FIG. 6 shows the effect of retentates and flow-through samples of molecular weight-filtered acid-pretreated corn stover liquor on GH61 polypeptide cellulolytic enhancing activity. White bars: 1 day of saccharification; gray bars: 6 days of saccharification. Concentrations listed refer to the T. reesei cellulase composition and the GH61 polypeptide concentration, respectively, in mg per gram cellulose.
[0023] FIG. 7 shows (A) the fractional hydrolysis of microcrystalline cellulose by individual T. reesei cellulase monocomponents and mixtures thereof, and the effects of the T. aurantiacus GH61A polypeptide and NREL acid-pretreated corn stover liquor thereon and (B) the GH61 effect on the individual cellulases and mixtures of cellulases. White bars: 3 days of hydrolysis; gray bars: 5 days of hydrolysis; black bars: 7 days of hydrolysis.
[0024] FIG. 8A shows HPLC chromatography of NREL acid-pretreated corn stover liquor. Fractional hydrolysis and absorbance are plotted for the various HPLC fractions. Solid line: fractional hydrolysis; light dashed line: absorbance at 210 nm; heavy dashed line: absorbance at 280 nm. The average hydrolysis for all samples is indicated by the solid, horizontal line. FIG. 8B shows the fractional hydrolysis for the T. reesei cellulase composition with increasing GH61A polypeptide concentrations in the presence of 3 kDa MWCO flow-through fractions of NREL acid-pretreated corn stover liquor incubated with microcrystalline cellulose and eluted with successive water and organic solvent washes. Gray bars: 3 days of hydrolysis; black bars: 7 days of hydrolysis.
[0025] FIG. 9 shows a standard curve of AVICEL.RTM. height vs. added mass.
[0026] FIG. 10 shows the effect of various pooled, HPLC-separated NREL acid-pretreated corn stover liquor fractions and the T. aurantiacus GH61A polypeptide on hydrolysis of microcrystalline cellulose by the T. reesei cellulase composition. Solid lines: height of AVICEL.RTM. incubated with the GH61 polypeptide; dashed lines: A.sub.(600 nm) of washed, BCA reagent-reacted AVICEL.RTM..
[0027] FIG. 11 shows (A) a standard curve of reducing sugar equivalents and (B) the reducing sugar equivalents measured in the solid microcrystalline cellulose incubated with the T. aurantiacus GH61A polypeptide and the indicated HPLC fractions.
[0028] FIG. 12 shows the results of a microcrystalline cellulose hydrolysis assay with the T. reesei cellulase composition in the presence of the T. aurantiacus GH61A polypeptide and chromatographed fractions of acid-pretreated xylan. Solid line: fractional hydrolysis with GH61; dashed line: mean fractional hydrolysis.
[0029] FIG. 13 shows a LC-MS chromatogram of a representative HPLC fraction of acid-pretreated xylan.
[0030] FIG. 14 shows liquid chromatography-mass spectrometry chromatograms of GH61 polypeptide-affinity enriched acid-pretreated corn stover. Panel A: diode array detection; Panel B: TOF MS/MS total ion current 17.5; Panel C: TOF MS/MS ES-total ion current 273; Panel D: TOF MS ES-total ion survey.
[0031] FIG. 15 shows ion chromatograms of microcrystalline cellulose or phosphoric acid-swollen cellulose incubated with the T. aurantiacus GH61A polypeptide and NREL acid-pretreated corn stover liquor. Panel A: reaction samples; Panel B: comparison to analytical standards. Panel A: solid lines: AVICEL.RTM. incubations; dashed lines: PASC incubations; light gray: AVICEL.RTM.; medium gray: AVICEL.RTM.+NREL acid-pretreated corn stover liquor; dark gray: AVICEL.RTM.+GH61 polypeptide; black: AVICEL.RTM.+GH61 polypeptide+liquor. Panel B: black: AVICEL.RTM.+GH61 polypeptide+liquor; dark gray solid lines: liquor; dark gray dashed lines: cellopentaose, cellotetraose and cellotriose; light gray: cellopentaonic acid, cellotetraonic acid, gluconic acid, galactonic acid and xylotetraose, peaks as indicated.
[0032] FIG. 16A shows the fractional hydrolysis of AVICEL.RTM. by a T. reesei cellulase composition with the indicated T. aurantiacus GH61A polypeptide concentration in the presence of alkaline-pretreated corn stover generated using the indicated pretreatment concentration of sodium hydroxide. White bars: 1 day of hydrolysis; gray bars: 3 days of hydrolysis; black bars: 7 days of hydrolysis. FIG. 16B shows the fractional hydrolysis of AVICEL.RTM. by the T. reesei cellulase composition with the indicated GH61 polypeptide concentration in the presence of water-extracted, acid-pretreated corn stover liquors generated using the indicated extraction temperature. Gray bars: 1 day of hydrolysis; white bars: 3 days of hydrolysis.
[0033] FIGS. 17A and 17B show the fractional hydrolysis of AVICEL.RTM. by a T. reesei cellulase composition with various concentrations of the T. aurantiacus GH61A polypeptide in the presence of acid-pretreated components of biomass or mixtures thereof. White bars: 1 day of hydrolysis; dark gray bars: 5 days of hydrolysis.
[0034] FIG. 18 shows the fractional hydrolysis of AVICEL.RTM. by a T. reesei cellulase composition with various concentrations of the T. aurantiacus GH61A polypeptide in the presence of post-fermentation liquors. White bars: 1 day of hydrolysis; gray bars: 3 days of hydrolysis; black bars: 7 days of hydrolysis.
[0035] FIGS. 19A and 19B show the fractional hydrolysis of AVICEL.RTM. by a T. reesei cellulase composition with various concentrations of the T. aurantiacus GH61A polypeptide in the presence of various severity acid-pretreatments of corn stover. Gray bars: 3 days of hydrolysis; black bars: 7 days of hydrolysis. FIG. 19C shows the fractional hydrolysis of AVICEL.RTM. by the T. reesei cellulase composition with T. aurantiacus GH61A polypeptide and liquors generated by acid pretreatment of cellulose at various severities between 110.degree. C. and 190.degree. C. at GH61A polypeptide concentrations of 50% ( ), 24% ( ); 8% ( ); 4% ( ); 2% ( ) and 0 ( )(w/w).
[0036] FIGS. 20A, 20B, and 20C shows the fractional hydrolysis of AVICEL.RTM. by T. reesei cellulase compositions with increasing GH61 polypeptide concentrations plus added acid-pretreated xylan of various severities. Panel A shows 7 days of hydrolysis data for a broad range of severities. Panel B shows more severe pretreatments, gray bars: 3 days of hydrolysis; black bars: 7 days of hydrolysis. Panel C shows the fractional hydrolysis of AVICEL.RTM. by the T. reesei cellulase composition with T. aurantiacus GH61A polypeptide and liquors generated by acid pretreatment of xylan at various severities between 110.degree. C. and 190.degree. C. at GH61A polypeptide concentrations of 50% ( ), 24% ( ); 8% ( ); 4% ( ); 2% ( ) and 0 ( )(w/w).
[0037] FIG. 21 shows the fractional hydrolysis of AVICEL.RTM. by T. reesei cellulase compositions with and without 50% (w/w) T. aurantiacus GH61A polypeptide concentrations plus added solid-phase extracted NREL acid-pretreated corn stover or acid-pretreated xylan as indicated. Gray bars: 3 days of hydrolysis; black bars: 7 days of hydrolysis.
[0038] FIG. 22 shows the fractional hydrolysis of AVICEL.RTM. by a T. reesei cellulase composition with various concentrations of T. aurantiacus GH61A in the presence of 10% (v/v) of the indicated pretreatment liquor or electrodialyzed pretreatment liquor. Gray bars: 3 days of hydrolysis; black bars: 7 days of hydrolysis.
[0039] FIG. 23 shows the fractional hydrolysis of various biomass substrates by the T. reesei cellulase composition with increasing concentrations of the T. aurantiacus GH61A polypeptide with and without NREL acid-pretreated corn stover liquor. Panel A: low and medium severity organosolv ethanol pretreated corn stover; Panel B: medium severity glycerol and water pretreated corn stover, and 5% total solids water pretreated corn stover; Panel C: 5% total solids sugarcane bagasse; Panel D: alkaline pretreated corn stover plus no liquor and NREL milled washed pretreated corn stover controls. White bars: 1 day of hydrolysis; gray bars: 3 days of hydrolysis; black bars: 7 days of hydrolysis.
[0040] FIG. 24 shows the fractional hydrolysis of AVICEL.RTM. by the T. reesei cellulase composition with either zero or 24% T. aurantiacus GH61A polypeptide in the presence of the indicated acid-pretreated monosaccharides. Gray bars: 3 days of hydrolysis; black bars: 7 days of hydrolysis.
[0041] FIG. 25 shows the fractional hydrolysis of AVICEL.RTM. by the T. reesei cellulase composition with various concentrations of the indicated GH61 polypeptides with 10% (v/v) NREL PCS liquor. White symbols: 3 days of hydrolysis; black symbols: 7 days of hydrolysis; Thermoascus aurantiacus GH61A: circles; Aspergillus fumigatus GH61B polypeptide: diamonds; Penicillium pinophilum GH61 polypeptide: squares.
[0042] FIG. 26 shows the glucose produced by hydrolysis of AVICEL.RTM. by a T. reesei cellulase composition with T. aurantiacus GH61A polypeptide in the presence or absence of Kraft lignin. Solid symbols: T. reesei cellulase composition+T. aurantiacus GH61A polypeptide. Open symbols: T. reesei cellulase composition+T. aurantiacus GH61A polypeptide supplemented with additional 15% (w/w) T. aurantiacus GH61A polypeptide. Circles: no lignin; squares: 0.1% (w/w) Kraft lignin; diamonds: 0.1% (w/w) oxidized Kraft lignin.
[0043] FIG. 27 shows the concentrations of glucose and xylose from 120 hours of saccharification of washed, milled alkaline pretreated corn stover by the T. reesei cellulase composition supplemented with T. aurantiacus GH61A polypeptide, replaced with increasing concentrations of T. aurantiacus GH61A. Solid squares: xylose, open diamonds: glucose.
[0044] FIG. 28 shows the conversion of high total solids (15% TS) dilute acid pretreated corn stover of various pretreatment severities as indicated. The pretreated corn stovers were hydrolyzed by either a composition containing a blend of an Aspergillus aculeatus GH10 xylanase and a Trichoderma reesei cellulase preparation containing Aspergillus fumigatus beta-glucosidase and Thermoascus aurantiacus GH61A polypeptide or this mixture replaced with 20% additional T. aurantiacus GH61A. For each severity pretreatment other than the least severe, replacement of the cellulase-GH61A polypeptide mixture with additional GH61A polypeptide yielded a greater conversion. Grey bars: 120 hours of saccharification; black bars: 216 hours of saccharification.
[0045] FIG. 29 shows the conversion of high total solids (15% TS) dilute acid pretreated Arundo donax of various pretreatment severities as indicated. The variously pretreated A. donax were hydrolyzed by either a composition containing a blend of an Aspergillus aculeatus GH10 xylanase (WO 94/021785) and a Trichoderma reesei cellulase preparation containing Aspergillus fumigatus beta-glucosidase (WO 2005/047499) and Thermoascus aurantiacus GH61A polypeptide (WO 2005/074656) or this mixture replaced by 20% additional T. aurantiacus GH61A polypeptide. For each severity pretreatment, replacement of the cellulase-GH61A polypeptide mixture with additional GH61A polypeptide yielded a greater conversion. Grey bars: 120 hours of saccharification; black bars: 216 hours of saccharification.
DEFINITIONS
[0046] Acetylxylan esterase: The term "acetylxylan esterase" means a carboxylesterase (EC 3.1.1.72) that catalyzes the hydrolysis of acetyl groups from polymeric xylan, acetylated xylose, acetylated glucose, alpha-napthyl acetate, and p-nitrophenyl acetate. For purposes of the present invention, acetylxylan esterase activity is determined using 0.5 mM p-nitrophenylacetate as substrate in 50 mM sodium acetate pH 5.0 containing 0.01% TWEEN.TM. 20 (polyoxyethylene sorbitan monolaurate). One unit of acetylxylan esterase is defined as the amount of enzyme capable of releasing 1 .mu.mole of p-nitrophenolate anion per minute at pH 5, 25.degree. C.
[0047] Allelic variant: The term "allelic variant" means any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.
[0048] Alpha-L-arabinofuranosidase: The term "alpha-L-arabinofuranosidase" means an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55) that catalyzes the hydrolysis of terminal non-reducing alpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzyme acts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)- and/or (1,5)-linkages, arabinoxylans, and arabinogalactans. Alpha-L-arabinofuranosidase is also known as arabinosidase, alpha-arabinosidase, alpha-L-arabinosidase, alpha-arabinofuranosidase, polysaccharide alpha-L-arabinofuranosidase, alpha-L-arabinofuranoside hydrolase, L-arabinosidase, or alpha-L-arabinanase. For purposes of the present invention, alpha-L-arabinofuranosidase activity is determined using 5 mg of medium viscosity wheat arabinoxylan (Megazyme International Ireland, Ltd., Bray, Co. Wicklow, Ireland) per ml of 100 mM sodium acetate pH 5 in a total volume of 200 .mu.l for 30 minutes at 40.degree. C. followed by arabinose analysis by AMINEX.RTM. HPX-87H column chromatography (Bio-Rad Laboratories, Inc., Hercules, Calif., USA).
[0049] Alpha-glucuronidase: The term "alpha-glucuronidase" means an alpha-D-glucosiduronate glucuronohydrolase (EC 3.2.1.139) that catalyzes the hydrolysis of an alpha-D-glucuronoside to D-glucuronate and an alcohol. For purposes of the present invention, alpha-glucuronidase activity is determined according to de Vries, 1998, J. Bacteriol. 180: 243-249. One unit of alpha-glucuronidase equals the amount of enzyme capable of releasing 1 .mu.mole of glucuronic or 4-O-methylglucuronic acid per minute at pH 5, 40.degree. C.
[0050] Beta-glucosidase: The term "beta-glucosidase" means a beta-D-glucoside glucohydrolase (E.C. 3.2.1.21) that catalyzes the hydrolysis of terminal non-reducing beta-D-glucose residues with the release of beta-D-glucose. For purposes of the present invention, beta-glucosidase activity is determined using p-nitrophenyl-beta-D-glucopyranoside as substrate according to the procedure of Venturi et al., 2002, Extracellular beta-D-glucosidase from Chaetomium thermophilum var. coprophilum: production, purification and some biochemical properties, J. Basic Microbiol. 42: 55-66. One unit of beta-glucosidase is defined as 1.0 .mu.mole of p-nitrophenolate anion produced per minute at 25.degree. C., pH 4.8 from 1 mM p-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mM sodium citrate containing 0.01% TWEEN.RTM. 20.
[0051] Beta-xylosidase: The term "beta-xylosidase" means a beta-D-xyloside xylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of short beta (1-4)-xylooligosaccharides, to remove successive D-xylose residues from non-reducing termini. For purposes of the present invention, one unit of beta-xylosidase is defined as 1.0 .mu.mole of p-nitrophenolate anion produced per minute at 40.degree. C., pH 5 from 1 mM p-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodium citrate containing 0.01% TWEEN.RTM. 20.
[0052] cDNA: The term "cDNA" means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.
[0053] Cellobiohydrolase: The term "cellobiohydrolase" means a 1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91) that catalyzes the hydrolysis of 1,4-beta-D-glucosidic linkages in cellulose, cellooligosaccharides, or any beta-1,4-linked glucose containing polymer, releasing cellobiose from the reducing or non-reducing ends of the chain (Teeri, 1997, Crystalline cellulose degradation: New insight into the function of cellobiohydrolases, Trends in Biotechnology 15: 160-167; Teeri et al., 1998, Trichoderma reesei cellobiohydrolases: why so efficient on crystalline cellulose?, Biochem. Soc. Trans. 26: 173-178). For purposes of the present invention, cellobiohydrolase activity is determined according to the procedures described by Lever et al., 1972, Anal. Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBS Letters, 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters, 187: 283-288; and Tomme et al., 1988, Eur. J. Biochem. 170: 575-581. In the present invention, the Lever et al. method can be employed to assess hydrolysis of cellulose in corn stover, while the methods of van Tilbeurgh et al. and Tomme et al. can be used to determine the cellobiohydrolase activity on a fluorescent disaccharide derivative, 4-methylumbelliferyl-.beta.-D-lactoside.
[0054] Cellulolytic enhancing activity: The term "cellulolytic enhancing activity" means a biological activity catalyzed by a GH61 polypeptide that enhances the hydrolysis of a cellulosic material by enzyme having cellulolytic activity. For purposes of the present invention, cellulolytic enhancing activity is determined by measuring the increase in reducing sugars or the increase of the total of cellobiose and glucose from the hydrolysis of a cellulosic material by cellulolytic enzyme under the following conditions: 1-50 mg of total protein/g of cellulose in PCS, wherein total protein is comprised of 50-99.5% w/w cellulolytic enzyme protein and 0.5-50% w/w protein of a GH61 polypeptide having cellulolytic enhancing activity for 1-7 days at 50.degree. C. compared to a control hydrolysis with equal total protein loading without cellulolytic enhancing activity (1-50 mg of cellulolytic protein/g of cellulose in PCS). In a preferred aspect, a mixture of CELLUCLAST.RTM. 1.5L (Novozymes A/S, Bagsv.ae butted.rd, Denmark) in the presence of 2-3% of total protein weight Aspergillus oryzae beta-glucosidase (recombinantly produced in Aspergillus oryzae according to WO 02/095014) or 2-3% of total protein weight Aspergillus fumigatus beta-glucosidase (recombinantly produced in Aspergillus oryzae as described in WO 2002/095014) of cellulase protein loading is used as the source of the cellulolytic activity.
[0055] The GH61 polypeptides having cellulolytic enhancing activity enhance the hydrolysis of a cellulosic material catalyzed by enzyme having cellulolytic activity by reducing the amount of cellulolytic enzyme required to reach the same degree of hydrolysis preferably at least 1.01-fold, more preferably at least 1.05-fold, more preferably at least 1.10-fold, more preferably at least 1.25-fold, more preferably at least 1.5-fold, more preferably at least 2-fold, more preferably at least 3-fold, more preferably at least 4-fold, more preferably at least 5-fold, even more preferably at least 10-fold, and most preferably at least 20-fold.
[0056] Cellulolytic enzyme or cellulase: The term "cellulolytic enzyme" or "cellulase" means one or more (e.g., several) enzymes that hydrolyze a cellulosic material. Such enzymes include endoglucanase(s), cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. The two basic approaches for measuring cellulolytic activity include: (1) measuring the total cellulolytic activity, and (2) measuring the individual cellulolytic activities (endoglucanases, cellobiohydrolases, and beta-glucosidases) as reviewed in Zhang et al., Outlook for cellulase improvement: Screening and selection strategies, 2006, Biotechnology Advances 24: 452-481. Total cellulolytic activity is usually measured using insoluble substrates, including Whatman No. 1 filter paper, microcrystalline cellulose, bacterial cellulose, algal cellulose, cotton, pretreated lignocellulose, etc. The most common total cellulolytic activity assay is the filter paper assay using Whatman No. 1 filter paper as the substrate. The assay was established by the International Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987, Measurement of cellulase activities, Pure Appl. Chem. 59: 257-68).
[0057] For purposes of the present invention, cellulolytic enzyme activity is determined by measuring the increase in hydrolysis of a cellulosic material by cellulolytic enzyme(s) under the following conditions: 1-20 mg of cellulolytic enzyme protein/g of cellulose in PCS for 3-7 days at 50.degree. C. compared to a control hydrolysis without addition of cellulolytic enzyme protein. Typical conditions are 1 ml reactions, washed or unwashed PCS, 5% insoluble solids, 50 mM sodium acetate pH 5, 1 mM MnSO.sub.4, 50.degree. C., 72 hours, sugar analysis by AMINEX.RTM. HPX-87H column (Bio-Rad Laboratories, Inc., Hercules, Calif., USA).
[0058] Cellulosic material: The term "cellulosic material" means any material containing cellulose. The predominant polysaccharide in the primary cell wall of biomass is cellulose, the second most abundant is hemicellulose, and the third is pectin. The secondary cell wall, produced after the cell has stopped growing, also contains polysaccharides and is strengthened by polymeric lignin covalently cross-linked to hemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thus a linear beta-(1-4)-D-glucan, while hemicelluloses include a variety of compounds, such as xylans, xyloglucans, arabinoxylans, and mannans in complex branched structures with a spectrum of substituents. Although generally polymorphous, cellulose is found in plant tissue primarily as an insoluble crystalline matrix of parallel glucan chains. Hemicelluloses usually hydrogen bond to cellulose, as well as to other hemicelluloses, which help stabilize the cell wall matrix.
[0059] Cellulose is generally found, for example, in the stems, leaves, hulls, husks, and cobs of plants or leaves, branches, and wood of trees. The cellulosic material can be, but is not limited to, agricultural residue, herbaceous material (including energy crops), municipal solid waste, pulp and paper mill residue, waste paper, and wood (including forestry residue) (see, for example, Wiselogel et al., 1995, in Handbook on Bioethanol (Charles E. Wyman, editor), pp. 105-118, Taylor & Francis, Washington D.C.; Wyman, 1994, Bioresource Technology 50: 3-16; Lynd, 1990, Applied Biochemistry and Biotechnology 24/25: 695-719; Mosier et al., 1999, Recent Progress in Bioconversion of Lignocellulosics, in Advances in Biochemical Engineering/Biotechnology, T. Scheper, managing editor, Volume 65, pp. 23-40, Springer-Verlag, New York). It is understood herein that the cellulose may be in the form of lignocellulose, a plant cell wall material containing lignin, cellulose, and hemicellulose in a mixed matrix. In a preferred aspect, the cellulosic material is any biomass material. In another preferred aspect, the cellulosic material is lignocellulose, which comprises cellulose, hemicelluloses, and lignin.
[0060] In one aspect, the cellulosic material is agricultural residue. In another aspect, the cellulosic material is herbaceous material (including energy crops). In another aspect, the cellulosic material is municipal solid waste. In another aspect, the cellulosic material is pulp and paper mill residue. In another aspect, the cellulosic material is waste paper. In another aspect, the cellulosic material is wood (including forestry residue).
[0061] In another aspect, the cellulosic material is arundo. In another aspect, the cellulosic material is bagasse. In another aspect, the cellulosic material is bamboo. In another aspect, the cellulosic material is corn cob. In another aspect, the cellulosic material is corn fiber. In another aspect, the cellulosic material is corn stover. In another aspect, the cellulosic material is miscanthus. In another aspect, the cellulosic material is orange peel. In another aspect, the cellulosic material is rice straw. In another aspect, the cellulosic material is switchgrass. In another aspect, the cellulosic material is wheat straw.
[0062] In another aspect, the cellulosic material is aspen. In another aspect, the cellulosic material is eucalyptus. In another aspect, the cellulosic material is fir. In another aspect, the cellulosic material is pine. In another aspect, the cellulosic material is poplar. In another aspect, the cellulosic material is spruce. In another aspect, the cellulosic material is willow.
[0063] In another aspect, the cellulosic material is algal cellulose. In another aspect, the cellulosic material is bacterial cellulose. In another aspect, the cellulosic material is cotton linter. In another aspect, the cellulosic material is filter paper. In another aspect, the cellulosic material is microcrystalline cellulose. In another aspect, the cellulosic material is phosphoric-acid treated cellulose.
[0064] In another aspect, the cellulosic material is an aquatic biomass. As used herein the term "aquatic biomass" means biomass produced in an aquatic environment by a photosynthesis process. The aquatic biomass can be algae, emergent plants, floating-leaf plants, or submerged plants.
[0065] The cellulosic material may be used as is or may be subjected to pretreatment, using conventional methods known in the art, as described herein. In a preferred aspect, the cellulosic material is pretreated.
[0066] Coding sequence: The term "coding sequence" means a polynucleotide, which directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon or alternative start codons such as GTG and TTG and ends with a stop codon such as TAA, TAG, and TGA. The coding sequence may be a DNA, cDNA, synthetic, or recombinant polynucleotide.
[0067] Control sequences: The term "control sequences" means all components necessary for the expression of a polynucleotide encoding a polypeptide. Each control sequence may be native or foreign to the polynucleotide encoding the polypeptide or native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a polypeptide.
[0068] Endoglucanase: The term "endoglucanase" means an endo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4) that catalyzes endohydrolysis of 1,4-beta-D-glycosidic linkages in cellulose, cellulose derivatives (such as carboxymethyl cellulose and hydroxyethyl cellulose), lichenin, beta-1,4 bonds in mixed beta-1,3 glucans such as cereal beta-D-glucans or xyloglucans, and other plant material containing cellulosic components. Endoglucanase activity can be determined by measuring reduction in substrate viscosity or increase in reducing ends determined by a reducing sugar assay (Zhang et al., 2006, Biotechnology Advances 24: 452-481). For purposes of the present invention, endoglucanase activity is determined using carboxymethyl cellulose (CMC) as substrate according to the procedure of Ghose, 1987, Pure and Appl. Chem. 59: 257-268, at pH 5, 40.degree. C.
[0069] Expression: The term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
[0070] Expression vector: The term "expression vector" means a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide and is operably linked to additional nucleotides that provide for its expression.
[0071] Family 61 glycoside hydrolase: The term "Family 61 glycoside hydrolase" or "Family GH61" or "GH61" means a polypeptide falling into the glycoside hydrolase Family 61 according to Henrissat B., 1991, A classification of glycosyl hydrolases based on amino-acid sequence similarities, Biochem. J. 280: 309-316, and Henrissat B., and Bairoch A., 1996, Updating the sequence-based classification of glycosyl hydrolases, Biochem. J. 316: 695-696. The enzymes in this family were originally classified as a glycoside hydrolase family based on measurement of very weak endo-1,4-beta-D-glucanase activity in one family member. The structure and mode of action of these enzymes are non-canonical and they cannot be considered as bona fide glycosidases. However, they are kept in the CAZy classification on the basis of their capacity to enhance the breakdown of lignocellulose when used in conjunction with a cellulase or a mixture of cellulases.
[0072] Feruloyl esterase: The term "feruloyl esterase" means a 4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) that catalyzes the hydrolysis of 4-hydroxy-3-methoxycinnamoyl (feruloyl) groups from esterified sugar, which is usually arabinose in "natural" substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate). Feruloyl esterase is also known as ferulic acid esterase, hydroxycinnamoyl esterase, FAE-III, cinnamoyl ester hydrolase, FAEA, cinnAE, FAE-I, or FAE-II. For purposes of the present invention, feruloyl esterase activity is determined using 0.5 mM p-nitrophenylferulate as substrate in 50 mM sodium acetate pH 5.0. One unit of feruloyl esterase equals the amount of enzyme capable of releasing 1 .mu.mole of p-nitrophenolate anion per minute at pH 5, 25.degree. C.
[0073] Hemicellulolytic enzyme or hemicellulase: The term "hemicellulolytic enzyme" or "hemicellulase" means one or more (e.g., several) enzymes that hydrolyze a hemicellulosic material. See, for example, Shallom, D. and Shoham, Y. Microbial hemicellulases. Current Opinion In Microbiology, 2003, 6(3): 219-228). Hemicellulases are key components in the degradation of plant biomass. Examples of hemicellulases include, but are not limited to, an acetylmannan esterase, an acetyxylan esterase, an arabinanase, an arabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, a galactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and a xylosidase. The substrates of these enzymes, the hemicelluloses, are a heterogeneous group of branched and linear polysaccharides that are bound via hydrogen bonds to the cellulose microfibrils in the plant cell wall, crosslinking them into a robust network. Hemicelluloses are also covalently attached to lignin, forming together with cellulose a highly complex structure. The variable structure and organization of hemicelluloses require the concerted action of many enzymes for its complete degradation. The catalytic modules of hemicellulases are either glycoside hydrolases (GHs) that hydrolyze glycosidic bonds, or carbohydrate esterases (CEs), which hydrolyze ester linkages of acetate or ferulic acid side groups. These catalytic modules, based on homology of their primary sequence, can be assigned into GH and CE families marked by numbers. Some families, with an overall similar fold, can be further grouped into clans, marked alphabetically (e.g., GH-A). A most informative and updated classification of these and other carbohydrate active enzymes is available in the Carbohydrate-Active Enzymes (CAZy) database. Hemicellulolytic enzyme activities can be measured according to Ghose and Bisaria, 1987, Pure & Appl. Chem. 59: 1739-1752.
[0074] Host cell: The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.
[0075] Isolated or Purified: The term "isolated" or "purified" means a polypeptide or polynucleotide that is removed from at least one component with which it is naturally associated. For example, a polypeptide may be at least 1% pure, e.g., at least 5% pure, at least 10% pure, at least 20% pure, at least 40% pure, at least 60% pure, at least 80% pure, at least 90% pure, or at least 95% pure, as determined by SDS-PAGE, and a polynucleotide may be at least 1% pure, e.g., at least 5% pure, at least 10% pure, at least 20% pure, at least 40% pure, at least 60% pure, at least 80% pure, at least 90% pure, or at least 95% pure, as determined by agarose electrophoresis.
[0076] Liquor: The term "liquor" means the solution phase, either aqueous, organic, or a combination thereof, arising from treatment of a lignocellulose and/or hemicellulose and/or lignacious material or feedstock, or monosaccharides thereof, e.g., xylose, arabinose, mannose, etc., under conditions as described herein, and the soluble contents thereof.
[0077] Mature polypeptide: The term "mature polypeptide" means a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. It is known in the art that a host cell may produce a mixture of two of more different mature polypeptides (i.e., with a different C-terminal and/or N-terminal amino acid) expressed by the same polynucleotide. The mature polypeptide can be predicted using the SignalP program (Nielsen et al., 1997, Protein Engineering 10: 1-6).
[0078] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" is defined herein as a nucleotide sequence that encodes a mature polypeptide having biological activity. The mature polypeptide coding sequence can be predicted using the SignalP program (Nielsen et al., 1997, supra).
[0079] Nucleic acid construct: The term "nucleic acid construct" means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic. The term nucleic acid construct is synonymous with the term "expression cassette" when the nucleic acid construct contains the control sequences required for expression of a coding sequence of the present invention.
[0080] Operably linked: The term "operably linked" means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs the expression of the coding sequence.
[0081] Polypeptide fragment: The term "fragment" means a polypeptide having one or more (e.g., several) amino acids deleted from the amino and/or carboxyl terminus of a mature polypeptide; wherein the fragment has biological activity.
[0082] Pretreated corn stover: The term "PCS" or "Pretreated Corn Stover" means a cellulosic material derived from corn stover by treatment with heat and dilute sulfuric acid, alkaline pretreatment, or neutral pretreatment.
[0083] Sequence Identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".
[0084] For purposes of the present invention, the degree of sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0, 5.0.0, or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
(Identical Residues.times.100)/(Length of Alignment-Total Number of Gaps in Alignment)
[0085] For purposes of the present invention, the degree of sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
(Identical Deoxyribonucleotides.times.100)/(Length of Alignment-Total Number of Gaps in Alignment)
[0086] Subsequence: The term "subsequence" means a polynucleotide having one or more (e.g., several) nucleotides deleted from the 5' and/or 3' end of a mature polypeptide coding sequence; wherein the subsequence encodes a fragment having biological activity.
[0087] Variant: The term "variant" means a polypeptide having cellulolytic enhancing activity comprising an alteration, i.e., a substitution, insertion, and/or deletion of one or more (e.g., several) amino acid residues at one or more (e.g., several) positions. A substitution means a replacement of an amino acid occupying a position with a different amino acid; a deletion means removal of an amino acid occupying a position; and an insertion means adding one or more (e.g., several) amino acids, e.g., 1-5 amino acids, adjacent to an amino acid occupying a position.
[0088] Xylan-containing material: The term "xylan-containing material" means any material comprising a plant cell wall polysaccharide containing a backbone of beta-(1-4)-linked xylose residues. Xylans of terrestrial plants are heteropolymers possessing a beta-(1-4)-D-xylopyranose backbone, which is branched by short carbohydrate chains. They comprise D-glucuronic acid or its 4-O-methyl ether, L-arabinose, and/or various oligosaccharides, composed of D-xylose, L-arabinose, D- or L-galactose, and D-glucose. Xylan-type polysaccharides can be divided into homoxylans and heteroxylans, which include glucuronoxylans, (arabino)glucuronoxylans, (glucurono)arabinoxylans, arabinoxylans, and complex heteroxylans. See, for example, Ebringerova et al., 2005, Adv. Polym. Sci. 186: 1-67.
[0089] In the methods of the present invention, any material containing xylan may be used. In a preferred aspect, the xylan-containing material is lignocellulose.
[0090] Xylan degrading activity or xylanolytic activity: The term "xylan degrading activity" or "xylanolytic activity" means a biological activity that hydrolyzes xylan-containing material. The two basic approaches for measuring xylanolytic activity include: (1) measuring the total xylanolytic activity, and (2) measuring the individual xylanolytic activities (e.g., endoxylanases, beta-xylosidases, arabinofuranosidases, alpha-glucuronidases, acetylxylan esterases, feruloyl esterases, and alpha-glucuronyl esterases). Recent progress in assays of xylanolytic enzymes was summarized in several publications including Biely and Puchard, Recent progress in the assays of xylanolytic enzymes, 2006, Journal of the Science of Food and Agriculture 86(11): 1636-1647; Spanikova and Biely, 2006, Glucuronoyl esterase --Novel carbohydrate esterase produced by Schizophyllum commune, FEBS Letters 580(19): 4597-4601; Herrmann, Vrsanska, Jurickova, Hirsch, Biely, and Kubicek, 1997, The beta-D-xylosidase of Trichoderma reesei is a multifunctional beta-D-xylan xylohydrolase, Biochemical Journal 321: 375-381.
[0091] Total xylan degrading activity can be measured by determining the reducing sugars formed from various types of xylan, including, for example, oat spelt, beechwood, and larchwood xylans, or by photometric determination of dyed xylan fragments released from various covalently dyed xylans. The most common total xylanolytic activity assay is based on production of reducing sugars from polymeric 4-O-methyl glucuronoxylan as described in Bailey, Biely, Poutanen, 1992, Interlaboratory testing of methods for assay of xylanase activity, Journal of Biotechnology 23(3): 257-270. Xylanase activity can also be determined with 0.2% AZCL-arabinoxylan as substrate in 0.01% TRITON.RTM. X-100 (4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol) and 200 mM sodium phosphate buffer pH 6 at 37.degree. C. One unit of xylanase activity is defined as 1.0 .mu.mole of azurine produced per minute at 37.degree. C., pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6 buffer.
[0092] For purposes of the present invention, xylan degrading activity is determined by measuring the increase in hydrolysis of birchwood xylan (Sigma Chemical Co., Inc., St. Louis, Mo., USA) by xylan-degrading enzyme(s) under the following typical conditions: 1 ml reactions, 5 mg/ml substrate (total solids), 5 mg of xylanolytic protein/g of substrate, 50 mM sodium acetate pH 5, 50.degree. C., 24 hours, sugar analysis using p-hydroxybenzoic acid hydrazide (PHBAH) assay as described by Lever, 1972, A new reaction for colorimetric determination of carbohydrates, Anal. Biochem 47: 273-279.
[0093] Xylanase: The term "xylanase" means a 1,4-beta-D-xylan-xylohydrolase (E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1,4-beta-D-xylosidic linkages in xylans. For purposes of the present invention, xylanase activity is determined with 0.2% AZCL-arabinoxylan as substrate in 0.01% TRITON.RTM. X-100 and 200 mM sodium phosphate buffer pH 6 at 37.degree. C. One unit of xylanase activity is defined as 1.0 .mu.mole of azurine produced per minute at 37.degree. C., pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6 buffer.
DETAILED DESCRIPTION OF THE INVENTION
[0094] The present invention relates to compositions comprising: (a) a polypeptide having cellulolytic enhancing activity; and (b) a liquor, wherein the combination of the polypeptide having cellulolytic enhancing activity and the liquor enhances hydrolysis of a cellulosic material by a cellulolytic enzyme. In one aspect, the compositions further comprise (c) one or more (e.g., several) enzymes selected from the group consisting of a cellulase, a hemicellulase, an esterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin.
[0095] The present invention also relates to methods for degrading or converting a cellulosic material, comprising: treating the cellulosic material with an enzyme composition in the presence of a polypeptide having cellulolytic enhancing activity and a liquor, wherein the combination of the polypeptide having cellulolytic enhancing activity and the liquor enhances hydrolysis of the cellulosic material by the enzyme composition. In one aspect, the method above further comprises recovering the degraded or converted cellulosic material. Soluble products of degradation or conversion of the cellulosic material can be separated from the insoluble cellulosic material using technology well known in the art such as, for example, centrifugation, filtration, and gravity settling.
[0096] The present invention also relates to methods for producing a fermentation product, comprising:
[0097] (a) saccharifying a cellulosic material with an enzyme composition in the presence of a polypeptide having cellulolytic enhancing activity and a liquor, wherein the combination of the polypeptide having cellulolytic enhancing activity and the liquor enhances hydrolysis of the cellulosic material by the enzyme composition;
[0098] (b) fermenting the saccharified cellulosic material with one or more (e.g., several) fermenting microorganisms to produce the fermentation product; and
[0099] (c) recovering the fermentation product from the fermentation.
[0100] The present invention also relates to methods of fermenting a cellulosic material, comprising: fermenting the cellulosic material with one or more (e.g., several) fermenting microorganisms, wherein the cellulosic material is saccharified with an enzyme composition in the presence of a polypeptide having cellulolytic enhancing activity and a liquor, wherein the combination of the polypeptide having cellulolytic enhancing activity and the liquor enhances hydrolysis of the cellulosic material by the enzyme composition. In one aspect, the fermenting of the cellulosic material produces a fermentation product. In another aspect, the method further comprises recovering the fermentation product from the fermentation.
Liquors
[0101] The term "liquor" means the solution phase, either aqueous, organic, ionic liquid, or combinations thereof, arising from treatment of a lignocellulose and/or hemicellulose and/or lignacious material or feedstock in a slurry, or monosaccharides thereof, e.g., xylose, arabinose, mannose, etc., under conditions as described herein, and the soluble contents thereof. A liquor for cellulolytic enhancement of a GH61 polypeptide can be produced by treating a lignocellulosic, hemicellulosic, or lignacious material (or feedstock) by applying heat and/or pressure, optionally in the presence of a catalyst, e.g., acid, optionally in the presence of an organic solvent, and optionally in combination with physical disruption of the material, and then separating the solution from the residual solids. Alternatively, the lignocellulosic, hemicellulosic, or lignacious material can be slurried and incubated in aqueous, organic, or ionic liquids or combinations thereof as either solutions or suspensions without addition of heat or pressure. Such conditions determine the degree of cellulolytic enhancement obtainable through the combination of liquor and a GH61 polypeptide during hydrolysis of a cellulosic substrate by a cellulase preparation. The liquor can be separated from the treated material using methods standard in the art, such as filtration, sedimentation, or centifugation.
[0102] In one aspect, the material for producing the liquor is herbaceous material. In another aspect, the material is agricultural residue. In another aspect, the material is forestry residue. In another aspect, the material is municipal solid waste. In another aspect, the material is waste paper. In another aspect, the material is pulp and paper mill residue. In another aspect, the material is pulping liquor. In another aspect, the material is mixed wood waste.
[0103] In another aspect, the material for producing the liquor is corn stover. In another aspect, the material is corn fiber. In another aspect, the material is corn cob. In another aspect, the material is orange peel. In another aspect, the material is rice straw. In another aspect, the material is wheat straw. In another aspect, the material is switch grass. In another aspect, the material is miscanthus. In another aspect, the material is sugar cane bagasse. In another aspect, the material is energy cane. In another aspect, the material is sorghum. In another aspect, the material is algae biomass.
[0104] In another aspect, the material for producing the liquor is softwood. In another aspect, the material is hardwood. In another aspect, the material is poplar. In another aspect, the material is pine. In another aspect, the material is spruce. In another aspect, the material is fir. In another aspect, the material is willow. In another aspect, the material is eucalyptus.
[0105] In another aspect, the material for producing the liquor is a hemicellulose. In another aspect, the material is a hemicellulose-rich lignocellulose. In another aspect, the material is a xylan. In another aspect, the material is beechwood xylan. In another aspect, the material is birch xylan. In another aspect, the material is spruce xylan. In another aspect, the material is arabinoxylan. In another aspect, the material is mannan. In another aspect, the material is glucomannan. In another aspect, the material contains beta-(1,4)-linked xylan. In another aspect, the material contains beta-(1,4)-linked mannan. In another aspect, the material contains branched beta-(1,4)-linked xylan, such as arabinoxylan and arabino-(glucoryono-) xylan. In another aspect, the material contains branched beta-(1,4)-linked mannan, such as galactoglucomannan.
[0106] In another aspect, the material for producing the liquor is a C5 monosaccharide (pentose). In another aspect, the material is arabinose. In another aspect, the material is xylose. In another aspect, the material is xylulose. In another aspect, the material is ribose. In another aspect, the material is ribulose. In another aspect, the material is acetyl-xylose. In another aspect, the material is ferulyl-xylose. In another aspect, the material is glucurono-xylose or methyl-glucurono-xylose.
[0107] In another aspect, the material for producing the liquor is a C6 monosaccharide (hexose). In another aspect, the material is glucose. In another aspect, the material is mannose. In another aspect, the material is fructose. In another aspect, the material is gulose. In another aspect, the material is allose. In another aspect, the material is altrose. In another aspect, the material is idose. In another aspect, the material is talose. In another aspect, the material is galactose. In another aspect, the material is gluconic acid. In another aspect, the material is glucuronic acid. In another aspect, the material is galactonic acid. In another aspect, the material is galacturonic acid. In another aspect, the material is psicose. In another aspect, the material is fructose. In another aspect, the material is sorbose. In another aspect, the material is tagatose.
[0108] In another aspect, the material for producing the liquor is a C4 monosaccharide. In another aspect, the material is erythrose. In another aspect, the material is threose. In another aspect, the material is erythrulose.
[0109] In another aspect, the material for producing the liquor is a C3 monosaccharide. In another aspect, the material is glyceraldehyde. In another aspect, the material is dihydroxyacetone.
[0110] In another aspect, the material for producing the liquor is lignin. In another aspect, the material is Kraft (Indulin) lignin. In another aspect, the material is p-hydroxyphenyl (H) rich lignin. In another aspect, the material is guaiacyl (G) rich lignin. In another aspect, the material is syringal (S) rich lignin. In another aspect, the material is lignosulfonate. In another aspect the material is black liquor or components thereof. In another aspect, the material is tall oil or components thereof.
[0111] In another aspect, the material for producing the liquor is a post-pretreatment residue of biomass (the solid waste after pretreatment. In another aspect, the material for producing the liquor is a post-saccharification residue of biomass (the solid waste after saccharification). In another aspect, the material for producing the liquor is a post-fermentation residue of biomass (the solid waste after fermentation). In another aspect, the material for producing the liquor is a post-distillation residue of biomass (the solid waste after distillation).
[0112] In a non-limiting aspect, the material is treated using acid in the range of about 0.5 to about 5% (w/v), e.g., about 0.5 to about 4.5% (w/v), about 0.75 to about 4% (w/v), about 1.0 to about 3.5% (w/v), about 1.25 to about 3.0% (w/v), or about 1.5 to about 2.5% (w/v); a pH of about 0 to about 3, e.g., about 0.5 to about 3, about 0.5 to about 2.5, about 1 to about 2, or about 1 to about 1.5; a time period of about 1 to about 15 minutes, e.g., about 1 to about 12 minutes, about 2 to about 10 minutes, about 3 to about 9 minutes, about 4 to about 9 minutes, about 5 to about 9 minutes, or about 6 to about 8 minutes; at a temperature at about 130.degree. C. to about 250.degree. C., e.g., about 140.degree. C. to about 220.degree. C., about 150.degree. C. to about 200.degree. C., about 160.degree. C. to about 190.degree. C., about 160.degree. C. to about 185.degree. C., about 165.degree. C. to about 180.degree. C., about 165.degree. C. to about 175.degree. C., or about 165.degree. C. to about 170.degree. C.; and a pressure of about 100 to about 600 psi, e.g., about 50 to about 1700 psi, about 100 to about 1500 psi, about 100 to about 1200 psi, about 100 to about 1000 psi, about 100 to about 500 psi, about 100 to about 400 psi, about 100 to about 300 psi, about 100 to about 200 psi, about 100 to about 150 psi, or about 100 to about 120 psi. In another aspect, the acid is sulfuric acid. In another aspect, the acid is hydrochloric acid. In another aspect, the acid is nitric acid. In another aspect, the acid is phosphoric acid. In another aspect, the acid is acetic acid. In another aspect, the acid is citric acid. In another aspect, the acid is succinic acid. In another aspect, the acid is tartaric acid. In another aspect, the acid is mixtures of any of the above acids. It is understood herein that the conditions described above may need to be optimized depending on the material being treated and the reactor used to produce the liquor. Such optimization is well within the skill in the art. Conditions used to pretreat a cellulosic material as described herein may also be used to generate liquor from a particular feedstock.
[0113] In a preferred aspect, the material is treated using 1.4% (w/v) sulfuric acid for 8 minutes at 165.degree. C. and 107 psi.
[0114] Treatment of a material to produce such liquor may also generate other compounds that are inhibitory to cellulases and/or hemicellulases, e.g., organic acids and lignin-derived compounds. Conditions can be selected that balance cellulolytic enhancing activity of a GH61 polypeptide with production of inhibitor compounds of cellulases and/or hemicellulases. Such conditions can vary depending on the material used for producing the liquor. However, the liquor can be subjected to a molecular weight filter(s) or dialysis, e.g. electrodialysis, using a membrane with nominal molecular weight cut-off of in the range of about 0.1 kDa to about 10 kDa, e.g., about 0.5 kDa to about 7 kDa, about 0.5 kDa to about 5 kDa, and about 1 kDa to about 3 kDa, to reduce the amount of the inhibitory compounds. Any method known in the art can be used to reduce the amount of the inhibitory compounds. In one aspect, the liquor is further processed to remove inhibitors of a cellulase, a hemicellulase, or a combination thereof.
[0115] In other aspects of the present invention, the liquor can be generated in situ by pretreating a cellulosic material that will be saccharified by a cellulase preparation. However, in such instances the amount of effective liquor generated in situ may be insufficient with regard to the GH61 polypeptide having cellulolytic enhancing activity, cellulolytic enzyme(s), and cellulose. For example, pretreatment of a cellulosic material under mild conditions, e.g., auto-catalyzed steam explosion, alkaline pretreatment, auto-hydrolysis, jet cooking, hot water-pretreatment, organosolv using ethanol, glycerol, etc., dilute acid pretreatment, and the like ("low severity conditions") or a pretreatment that includes a wash or rinse step or unpretreated cellulosic material compared to harsh conditions ("high severity conditions") to produce in situ a liquor may be inadequate for optimizing the cellulolytic enhancing activity of a GH61 polypeptide. Conditions employed to produce NREL preatreated corn stover, i.e., 1.4 wt % sulfuric acid for 8 minutes at 165.degree. C. and 107 psi (Example 1) would be considered high severity conditions. In such circumstances a liquor obtained using treatment conditions different from the pretreatment conditions of the cellulosic material can be added to the saccharification reaction. In other aspects of the invention, a low-severity extraction treatment that extracts liquor can be used instead of conventional treatment techniques listed herein, and this liquor can then be added to the saccharification reaction.
[0116] In one aspect, the liquor is obtained from a material that is the same as the cellulosic material to be subjected to saccharification by a cellulase composition. In another aspect, the liquor is obtained from a material that is different than the cellulosic material to be subjected to saccharification by a cellulase composition.
[0117] In another aspect, the liquor is obtained from a material that is the same as the cellulosic material to be subjected to saccharification by a cellulase composition, but the treatment conditions used to produce the liquor are different from the pretreatment conditions of the cellulosic material. In another aspect, the liquor is obtained from a material that is the same as the cellulosic material to be subjected to saccharification by a cellulase composition, and the treatment conditions used to produce the liquor are the same as the pretreatment conditions of the cellulosic material. In another aspect, the liquor is obtained from a material that is the same as the cellulosic material to be subjected to saccharification by a cellulase composition, and the treatment conditions used to produce the liquor are the same as the pretreatment conditions of the cellulosic material, and the liquor is further processed, e.g., concentrated, filtered to remove cellulase inhibitors, filtered and concentrated, etc.
[0118] In another aspect, the liquor is obtained from a material that is different than the cellulosic material to be subjected to saccharification by a cellulase composition, and the treatment conditions used to produce the liquor are different from the pretreatment conditions of the cellulosic material. In another aspect, the liquor is obtained from a material that is different than the cellulosic material to be subjected to saccharification by a cellulase composition, and the treatment conditions used to produce the liquor are the same as the pretreatment conditions of the cellulosic material. In another aspect, the liquor is obtained from a material that is different than the cellulosic material to be subjected to saccharification by a cellulase composition, and the treatment conditions used to produce the liquor are the same as the pretreatment conditions of the cellulosic material, and the liquor is further processed, e.g., concentrated, filtered to remove cellulase inhibitors, filtered and concentrated, etc. Further processing to remove cellulose inhibitors can be accomplished using any method known in the art.
[0119] In another aspect, liquors generated in situ may be washed or diluted and replaced with liquors generated ex situ to a greater or lesser extent, so the liquor composition/content is optimized for the cellulolytic enhancing effect of a GH61 polypeptide. In another aspect, the solids content of subsequent saccharifications is altered to optimize the liquor content for the cellulolytic enhancing effect of a GH61 polypeptide.
[0120] The effective amount of the liquor can depend on one or more (e.g., several) factors including, but not limited to, the mixture of component cellulolytic enzymes, the cellulosic substrate, the concentration of cellulosic substrate, the pretreatment(s) of the cellulosic substrate, non-cellulosic components (e.g., native or degraded lignin or hemicellulose), non-cellulase components, temperature, reaction time, and the liquor (e.g., filtered to remove cellulase and/or hemicellulase inhibitors).
[0121] The liquor is preferably present in an amount that is not limiting with regard to the GH61 polypeptide having cellulolytic enhancing activity, cellulolytic enzyme(s), and cellulose. In one aspect, the liquor is present in an amount that is not limiting with regard to the GH61 polypeptide having cellulolytic enhancing activity. In another aspect, the liquor is present in an amount that is not limiting with regard to the cellulolytic enzyme(s). In another aspect, the liquor is present in an amount that is not limiting with regard to the cellulose. In another aspect, the liquor is present in an amount that is not limiting with regard to the GH61 polypeptide having cellulolytic enhancing activity and the cellulolytic enzyme(s). In another aspect, the liquor is present in an amount that is not limiting with regard to the GH61 polypeptide having cellulolytic enhancing activity and the cellulose. In another aspect, the liquor is present in an amount that is not limiting with regard to the cellulolytic enzyme(s) and the cellulose. In another aspect, the liquor is present in an amount that is not limiting with regard to the GH61 polypeptide having cellulolytic enhancing activity, the cellulolytic enzyme(s), and the cellulose.
[0122] The liquor is preferably present in an amount that optimizes the cellulolytic enhancing activity of a GH61 polypeptide during saccharification with a cellulase composition. In one aspect, the liquor optimizes the cellulolytic enhancing activity of a GH61 polypeptide with a GH61 effect as defined by Equation 3 (the ratio of fractional hydrolysis in the presence to the absence of the GH61 polypeptide) of preferably at least 1.05, more preferably at least 1.10, more preferably at least 1.15, more preferably at least 1.2, more preferably at least 1.25, more preferably at least 1.3, more preferably at least 1.35, more preferably at least 1.4, more preferably at least 1.45, more preferably at least 1.5, more preferably at least 1.55, more preferably at least 1.6, more preferably at least 1.65, more preferably at least 1.7, more preferably at least 1.75, more preferably at least 1.8, more preferably at least 1.85, more preferably at least 1.9, most preferably at least 1.95, and even most preferably at least 2. An increase in the GH61 effect is obtained when liquor is added, relative to when liquor is not added, during hydrolysis. In another aspect, the amount of GH61 polypeptide is optimized for a given concentration of liquor. Such optimization is accomplished by varying the concentration of each component to determine the optimal ratio of the components during saccharification.
[0123] In another aspect, an effective amount of the liquor to cellulose is about 10.sup.-6 to about 10 g per g of cellulose, e.g., about 10.sup.-6 to about 7.5 g, about 10.sup.-6 to about 5, about 10.sup.-6 to about 2.5 g, about 10.sup.-6 to about 1 g, about 10.sup.-5 to about 1 g, about 10.sup.-5 to about 10.sup.-1 g, about 10.sup.-4 to about 10.sup.-1 g, about 10.sup.-3 to about 10.sup.-1 g, and about 10.sup.-3 to about 10.sup.-2 g per g of cellulose.
[0124] In another aspect, the amount of liquor present that minimizes inhibition of a cellulase composition and in combination with a GH61 polypeptide enhances hydrolysis by an enzyme composition is about 1 to about 20% (v/v), e.g., about 1 to about 15%, about 1 to about 10%, about 2 to about 7%, about 2 to about 5%, and about 3 to about 5%.
[0125] In the methods of the present invention, the term "liquor" encompasses one or more (e.g., several) liquors from different materials based on the same or different conditions of treatment to produce the liquors.
[0126] In the methods of the present invention, the liquor is preferably present when a GH61 polypeptide is present, for example, is added with the GH61 polypeptide. The liquor can also be added at different stages of a saccharification. The liquor can also be redosed at different stages of saccharification, e.g., daily, to maintain the presence of an effective concentration of the liquor. The liquor can also be removed or washed to various degrees, or may be diluted at different stages of saccharification. Liquors generated in situ may be washed and replaced with liquors generated ex situ to a greater or lesser extent, at various times during saccharification
[0127] In another aspect of the present invention, the liquor may be recycled from a completed saccharification or completed saccharification and fermentation to a new saccharification. The liquor can be recovered using standard methods in the art, e.g., filtration/centrifugation, sedimentation, and/or flocculation of solids materials pre- or post-distillation, to remove residual solids, cellular debris, etc. and then recirculated to the new saccharification.
Polypeptides Having Cellulolytic Enhancing Activity and Polynucleotides Thereof
[0128] In the methods of the present invention, any GH61 polypeptide having cellulolytic enhancing activity can be used.
[0129] In a first aspect, the polypeptide having cellulolytic enhancing activity comprises the following motifs:
[0130] [ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(4)-[HNQ] (SEQ ID NO: 127 or SEQ ID NO: 128) and [FW]-[TF]-K-[AIV],
wherein X is any amino acid, X(4,5) is any amino acid at 4 or 5 contiguous positions, and X(4) is any amino acid at 4 contiguous positions.
[0131] The isolated polypeptide comprising the above-noted motifs may further comprise:
[0132] H-X(1,2)-G-P-X(3)-[YW]-[AILMV] (SEQ ID NO: 129 or SEQ ID NO: 130),
[0133] [EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV] (SEQ ID NO: 131), or
[0134] H-X(1,2)-G-P-X(3)-[YW]-[AILMV] (SEQ ID NO: 132 or SEQ ID NO: 133) and [EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV] (SEQ ID NO: 134),
wherein X is any amino acid, X(1,2) is any amino acid at 1 position or 2 contiguous positions, X(3) is any amino acid at 3 contiguous positions, and X(2) is any amino acid at 2 contiguous positions. In the above motifs, the accepted IUPAC single letter amino acid abbreviation is employed.
[0135] In a preferred embodiment, the isolated GH61 polypeptide having cellulolytic enhancing activity further comprises H-X(1,2)-G-P-X(3)-[YW]-[AILMV] (SEQ ID NO: 135 or SEQ ID NO: 136). In another preferred embodiment, the isolated GH61 polypeptide having cellulolytic enhancing activity further comprises [EQ]X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV] (SEQ ID NO: 137). In another preferred embodiment, the isolated GH61 polypeptide having cellulolytic enhancing activity further comprises H-X(1,2)-G-P-X(3)-[YW]-[AILMV] (SEQ ID NO: 138 or SEQ ID NO: 139) and [EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV] (SEQ ID NO: 140).
[0136] In a second aspect, isolated polypeptides having cellulolytic enhancing activity, comprise the following motif:
[0137] [ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]X(3)-A-[HNQ] (SEQ ID NO: 141 or SEQ ID NO: 142),
wherein X is any amino acid, X(4,5) is any amino acid at 4 or 5 contiguous positions, and X(3) is any amino acid at 3 contiguous positions. In the above motif, the accepted IUPAC single letter amino acid abbreviation is employed.
[0138] In a third aspect, the polypeptide having cellulolytic enhancing activity comprises an amino acid sequence that has a degree of identity to the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, or SEQ ID NO: 166 of preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%, or at least 100% and even most preferably at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%.
[0139] In a preferred aspect, the mature polypeptide is amino acids 20 to 326 of SEQ ID NO: 2, amino acids 18 to 239 of SEQ ID NO: 4, amino acids 20 to 258 of SEQ ID NO: 6, amino acids 19 to 226 of SEQ ID NO: 8, amino acids 20 to 304 of SEQ ID NO: 10, amino acids 23 to 250 of SEQ ID NO: 12, amino acids 22 to 249 of SEQ ID NO: 14, amino acids 20 to 249 of SEQ ID NO: 16, amino acids 18 to 232 of SEQ ID NO: 18, amino acids 16 to 235 of SEQ ID NO: 20, amino acids 19 to 323 of SEQ ID NO: 22, amino acids 16 to 310 of SEQ ID NO: 24, amino acids 20 to 246 of SEQ ID NO: 26, amino acids 22 to 354 of SEQ ID NO: 28, amino acids 22 to 250 of SEQ ID NO: 30, or amino acids 22 to 322 of SEQ ID NO: 32, amino acids 24 to 444 of SEQ ID NO: 34, amino acids 26 to 253 of SEQ ID NO: 36, amino acids 20 to 223 of SEQ ID NO: 38, amino acids 18 to 246 of SEQ ID NO: 40, amino acids 20 to 334 of SEQ ID NO: 42, amino acids 18 to 227 of SEQ ID NO: 44, amino acids 22 to 368 of SEQ ID NO: 46, amino acids 25 to 330 of SEQ ID NO: 48, amino acids 17 to 236 of SEQ ID NO: 50, amino acids 17 to 250 of SEQ ID NO: 52, amino acids 23 to 478 of SEQ ID NO: 54, amino acids 17 to 230 of SEQ ID NO: 56, amino acids 20 to 257 of SEQ ID NO: 58, amino acids 23 to 251 of SEQ ID NO: 60, amino acids 19 to 349 of SEQ ID NO: 62, amino acids 24 to 436 of SEQ ID NO: 64, amino acids 21 to 344 of SEQ ID NO: 144, amino acids 21 to 389 of SEQ ID NO: 146, amino acids 22 to 406 of SEQ ID NO: 148, amino acids 20 to 427 of SEQ ID NO: 150, amino acids 18 to 267 of SEQ ID NO: 152, amino acids 21 to 273 of SEQ ID NO: 154, amino acids 21 to 322 of SEQ ID NO: 156, amino acids 18 to 234 of SEQ ID NO: 158, amino acids 24 to 233 of SEQ ID NO: 160, amino acids 17 to 237 of SEQ ID NO: 162, amino acids 20 to 484 of SEQ ID NO: 164, or amino acids 22 to 320 of SEQ ID NO: 166.
[0140] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 2 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 2. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 2. In another preferred aspect, the polypeptide comprises or consists of amino acids 20 to 326 of SEQ ID NO: 2, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 20 to 326 of SEQ ID NO: 2.
[0141] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 4 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 4. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 4. In another preferred aspect, the polypeptide comprises or consists of amino acids 18 to 239 of SEQ ID NO: 4, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 18 to 239 of SEQ ID NO: 4.
[0142] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 6 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 6. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 6. In another preferred aspect, the polypeptide comprises or consists of amino acids 20 to 258 of SEQ ID NO: 6, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 20 to 258 of SEQ ID NO: 6.
[0143] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 8 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 8. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 8. In another preferred aspect, the polypeptide comprises or consists of amino acids 19 to 226 of SEQ ID NO: 8, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 19 to 226 of SEQ ID NO: 8.
[0144] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 10 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 10. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 10. In another preferred aspect, the polypeptide comprises or consists of amino acids 20 to 304 of SEQ ID NO: 10, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 20 to 304 of SEQ ID NO: 10.
[0145] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 12 or an allelic variant thereof; or a fragment thereof having cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 12. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 12. In another preferred aspect, the polypeptide comprises or consists of amino acids 16 to 317 of SEQ ID NO: 12, or an allelic variant thereof; or a fragment thereof having cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 16 to 317 of SEQ ID NO: 12.
[0146] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 14 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 14. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 14. In another preferred aspect, the polypeptide comprises or consists of amino acids 23 to 250 of SEQ ID NO: 14, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 23 to 250 of SEQ ID NO: 14.
[0147] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 16 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 16. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 16. In another preferred aspect, the polypeptide comprises or consists of amino acids 20 to 249 of SEQ ID NO: 16, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 20 to 249 of SEQ ID NO: 16.
[0148] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 18 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 18. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 18. In another preferred aspect, the polypeptide comprises or consists of amino acids 18 to 232 of SEQ ID NO: 18, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 18 to 232 of SEQ ID NO: 18.
[0149] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 20 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 20. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 20. In another preferred aspect, the polypeptide comprises or consists of amino acids 16 to 235 of SEQ ID NO: 20, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 16 to 235 of SEQ ID NO: 20.
[0150] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 22 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 22. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 22. In another preferred aspect, the polypeptide comprises or consists of amino acids 19 to 323 of SEQ ID NO: 22, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 19 to 323 of SEQ ID NO: 22.
[0151] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 24 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 24. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 24. In another preferred aspect, the polypeptide comprises or consists of amino acids 16 to 310 of SEQ ID NO: 24, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 16 to 310 of SEQ ID NO: 24.
[0152] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 26 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 26. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 26. In another preferred aspect, the polypeptide comprises or consists of amino acids 20 to 246 of SEQ ID NO: 26, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 20 to 246 of SEQ ID NO: 26.
[0153] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 28 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 28. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 28. In another preferred aspect, the polypeptide comprises or consists of amino acids 22 to 354 of SEQ ID NO: 28, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 22 to 354 of SEQ ID NO: 28.
[0154] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 30 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 30. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 30. In another preferred aspect, the polypeptide comprises or consists of amino acids 22 to 250 of SEQ ID NO: 30, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 22 to 250 of SEQ ID NO: 30.
[0155] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 32 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 32. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 32. In another preferred aspect, the polypeptide comprises or consists of amino acids 22 to 322 of SEQ ID NO: 32, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 22 to 322 of SEQ ID NO: 32.
[0156] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 34 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 34. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 34. In another preferred aspect, the polypeptide comprises or consists of amino acids 24 to 444 of SEQ ID NO: 34, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 24 to 444 of SEQ ID NO: 34.
[0157] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 36 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 36. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 36. In another preferred aspect, the polypeptide comprises or consists of amino acids 26 to 253 of SEQ ID NO: 36, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 26 to 253 of SEQ ID NO: 36.
[0158] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 38 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 38. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 38. In another preferred aspect, the polypeptide comprises or consists of amino acids 20 to 223 of SEQ ID NO: 38, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 20 to 223 of SEQ ID NO: 38.
[0159] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 40 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 40. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 40. In another preferred aspect, the polypeptide comprises or consists of amino acids 18 to 246 of SEQ ID NO: 40, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 18 to 246 of SEQ ID NO: 40.
[0160] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 42 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 42. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 42. In another preferred aspect, the polypeptide comprises or consists of amino acids 20 to 334 of SEQ ID NO: 42, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 20 to 334 of SEQ ID NO: 42.
[0161] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 44 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 44. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 44. In another preferred aspect, the polypeptide comprises or consists of amino acids 18 to 227 of SEQ ID NO: 44, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 18 to 227 of SEQ ID NO: 44.
[0162] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 46 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 46. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 46. In another preferred aspect, the polypeptide comprises or consists of amino acids 22 to 368 of SEQ ID NO: 46, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 22 to 368 of SEQ ID NO: 46.
[0163] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 48 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 48. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 48. In another preferred aspect, the polypeptide comprises or consists of amino acids 25 to 330 of SEQ ID NO: 48, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 25 to 330 of SEQ ID NO: 48.
[0164] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 50 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 50. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 50. In another preferred aspect, the polypeptide comprises or consists of amino acids 17 to 236 of SEQ ID NO: 50, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 17 to 236 of SEQ ID NO: 50.
[0165] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 52 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 52. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 52. In another preferred aspect, the polypeptide comprises or consists of amino acids 19 to 250 of SEQ ID NO: 52, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 19 to 250 of SEQ ID NO: 52.
[0166] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 54 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 54. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 54. In another preferred aspect, the polypeptide comprises or consists of amino acids 23 to 478 of SEQ ID NO: 54, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 23 to 478 of SEQ ID NO: 54.
[0167] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 56 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 56. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 56. In another preferred aspect, the polypeptide comprises or consists of amino acids 17 to 230 of SEQ ID NO: 56, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 17 to 230 of SEQ ID NO: 56.
[0168] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 58 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 58. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 58. In another preferred aspect, the polypeptide comprises or consists of amino acids 20 to 257 of SEQ ID NO: 58, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 20 to 257 of SEQ ID NO: 58.
[0169] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 60 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 60. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 60. In another preferred aspect, the polypeptide comprises or consists of amino acids 23 to 251 of SEQ ID NO: 60, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 23 to 251 of SEQ ID NO: 60.
[0170] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 62 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 62. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 62. In another preferred aspect, the polypeptide comprises or consists of amino acids 19 to 349 of SEQ ID NO: 62, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 19 to 349 of SEQ ID NO: 62.
[0171] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 64 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 64. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 64. In another preferred aspect, the polypeptide comprises or consists of amino acids 24 to 436 of SEQ ID NO: 64, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 24 to 436 of SEQ ID NO: 64.
[0172] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 144 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 144. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 144. In another preferred aspect, the polypeptide comprises or consists of amino acids 21 to 344 of SEQ ID NO: 144, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 21 to 344 of SEQ ID NO: 144.
[0173] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 146 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 146. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 146. In another preferred aspect, the polypeptide comprises or consists of amino acids 21 to 389 of SEQ ID NO: 146, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 21 to 389 of SEQ ID NO: 146.
[0174] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 148 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 148. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 148. In another preferred aspect, the polypeptide comprises or consists of amino acids 22 to 406 of SEQ ID NO: 148, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 22 to 406 of SEQ ID NO: 148.
[0175] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 150 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 150. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 150. In another preferred aspect, the polypeptide comprises or consists of amino acids 20 to 427 of SEQ ID NO: 150, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 20 to 427 of SEQ ID NO: 150.
[0176] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 152 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 152. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 152. In another preferred aspect, the polypeptide comprises or consists of amino acids 18 to 267 of SEQ ID NO: 152, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 18 to 267 of SEQ ID NO: 152.
[0177] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 154 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 154. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 154. In another preferred aspect, the polypeptide comprises or consists of amino acids 21 to 273 of SEQ ID NO: 154, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 21 to 273 of SEQ ID NO: 154.
[0178] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 156 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 156. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 156. In another preferred aspect, the polypeptide comprises or consists of amino acids 21 to 322 of SEQ ID NO: 156, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 21 to 322 of SEQ ID NO: 156.
[0179] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 158 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 158. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 158. In another preferred aspect, the polypeptide comprises or consists of amino acids 18 to 234 of SEQ ID NO: 158, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 18 to 234 of SEQ ID NO: 158.
[0180] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 160 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 160. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 160. In another preferred aspect, the polypeptide comprises or consists of amino acids 24 to 233 of SEQ ID NO: 160, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 24 to 233 of SEQ ID NO: 160.
[0181] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 162 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 162. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 162. In another preferred aspect, the polypeptide comprises or consists of amino acids 17 to 237 of SEQ ID NO: 162, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 17 to 237 of SEQ ID NO: 162.
[0182] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 164 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 164. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 164. In another preferred aspect, the polypeptide comprises or consists of amino acids 20 to 484 of SEQ ID NO: 164, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 20 to 484 of SEQ ID NO: 164.
[0183] A polypeptide having cellulolytic enhancing activity preferably comprises or consists of the amino acid sequence of SEQ ID NO: 166 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 166. In another preferred aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 166. In another preferred aspect, the polypeptide comprises or consists of amino acids 22 to 320 of SEQ ID NO: 166, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises or consists of amino acids 22 to 320 of SEQ ID NO: 166.
[0184] Preferably, a fragment of the mature polypeptide of SEQ ID NO: 2 contains at least 277 amino acid residues, more preferably at least 287 amino acid residues, and most preferably at least 297 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 4 contains at least 185 amino acid residues, more preferably at least 195 amino acid residues, and most preferably at least 205 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 6 contains at least 200 amino acid residues, more preferably at least 212 amino acid residues, and most preferably at least 224 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 8 contains at least 175 amino acid residues, more preferably at least 185 amino acid residues, and most preferably at least 195 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 10 contains at least 240 amino acid residues, more preferably at least 255 amino acid residues, and most preferably at least 270 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 12 contains at least 255 amino acid residues, more preferably at least 270 amino acid residues, and most preferably at least 285 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 14 contains at least 175 amino acid residues, more preferably at least 190 amino acid residues, and most preferably at least 205 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 16 contains at least 200 amino acid residues, more preferably at least 210 amino acid residues, and most preferably at least 220 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 18 contains at least 185 amino acid residues, more preferably at least 195 amino acid residues, and most preferably at least 205 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 20 contains at least 190 amino acid residues, more preferably at least 200 amino acid residues, and most preferably at least 210 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 22 contains at least 260 amino acid residues, more preferably at least 275 amino acid residues, and most preferably at least 290 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 24 contains at least 250 amino acid residues, more preferably at least 265 amino acid residues, and most preferably at least 280 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 26 contains at least 195 amino acid residues, more preferably at least 205 amino acid residues, and most preferably at least 214 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 28 contains at least 285 amino acid residues, more preferably at least 300 amino acid residues, and most preferably at least 315 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 30 contains at least 200 amino acid residues, more preferably at least 210 amino acid residues, and most preferably at least 220 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 32 contains at least 255 amino acid residues, more preferably at least 270 amino acid residues, and most preferably at least 285 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 34 contains at least 360 amino acid residues, more preferably at least 380 amino acid residues, and most preferably at least 400 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 36 contains at least 200 amino acid residues, more preferably at least 210 amino acid residues, and most preferably at least 220 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 38 contains at least 170 amino acid residues, more preferably at least 180 amino acid residues, and most preferably at least 190 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 40 contains at least 190 amino acid residues, more preferably at least 200 amino acid residues, and most preferably at least 210 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 42 contains at least 265 amino acid residues, more preferably at least 280 amino acid residues, and most preferably at least 295 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 44 contains at least 180 amino acid residues, more preferably at least 190 amino acid residues, and most preferably at least 200 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 46 contains at least 320 amino acid residues, more preferably at least 335 amino acid residues, and most preferably at least 350 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 48 contains at least 255 amino acid residues, more preferably at least 270 amino acid residues, and most preferably at least 285 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 50 contains at least 190 amino acid residues, more preferably at least 200 amino acid residues, and most preferably at least 210 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 52 contains at least 200 amino acid residues, more preferably at least 210 amino acid residues, and most preferably at least 220 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 54 contains at least 380 amino acid residues, more preferably at least 400 amino acid residues, and most preferably at least 420 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 56 contains at least 180 amino acid residues, more preferably at least 190 amino acid residues, and most preferably at least 200 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 58 contains at least 210 amino acid residues, more preferably at least 220 amino acid residues, and most preferably at least 230 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 60 contains at least 190 amino acid residues, more preferably at least 200 amino acid residues, and most preferably at least 210 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 62 contains at least 270 amino acid residues, more preferably at least 290 amino acid residues, and most preferably at least 310 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 64 contains at least 340 amino acid residues, more preferably at least 360 amino acid residues, and most preferably at least 380 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 144 contains at least 280 amino acid residues, more preferably at least 295 amino acid residues, and most preferably at least 310 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 146 contains at least 310 amino acid residues, more preferably at least 330 amino acid residues, and most preferably at least 350 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 148 contains at least 320 amino acid residues, more preferably at least 340 amino acid residues, and most preferably at least 360 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 150 contains at least 350 amino acid residues, more preferably at least 370 amino acid residues, and most preferably at least 390 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 152 contains at least 220 amino acid residues, more preferably at least 230 amino acid residues, and most preferably at least 240 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 154 contains at least 220 amino acid residues, more preferably at least 230 amino acid residues, and most preferably at least 240 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 156 contains at least 255 amino acid residues, more preferably at least 270 amino acid residues, and most preferably at least 285 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 158 contains at least 185 amino acid residues, more preferably at least 195 amino acid residues, and most preferably at least 205 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 160 contains at least 180 amino acid residues, more preferably at least 190 amino acid residues, and most preferably at least 200 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 162 contains at least 190 amino acid residues, more preferably at least 200 amino acid residues, and most preferably at least 210 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 164 contains at least 385 amino acid residues, more preferably at least 410 amino acid residues, and most preferably at least 435 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 166 contains at least 255 amino acid residues, more preferably at least 270 amino acid residues, and most preferably at least 285 amino acid residues.
[0185] Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 1 contains at least 831 nucleotides, more preferably at least 861 nucleotides, and most preferably at least 891 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 3 contains at least 555 nucleotides, more preferably at least 585 nucleotides, and most preferably at least 615 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 5 contains at least 600 nucleotides, more preferably at least 636 nucleotides, and most preferably at least 672 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 7 contains at least 525 nucleotides, more preferably at least 555 nucleotides, and most preferably at least 585 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 9 contains at least 720 nucleotides, more preferably at least 765 nucleotides, and most preferably at least 810 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 11 contains at least 765 nucleotides, more preferably at least 810 nucleotides, and most preferably at least 855 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of nucleotides 67 to 796 of SEQ ID NO: 13 contains at least 525 nucleotides, more preferably at least 570 nucleotides, and most preferably at least 615 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 15 contains at least 600 nucleotides, more preferably at least 630 nucleotides, and most preferably at least 660 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 17 contains at least 555 nucleotides, more preferably at least 585 nucleotides, and most preferably at least 615 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 19 contains at least 570 nucleotides, more preferably at least 600 nucleotides, and most preferably at least 630 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 21 contains at least 780 nucleotides, more preferably at least 825 nucleotides, and most preferably at least 870 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 23 contains at least 750 nucleotides, more preferably at least 795 nucleotides, and most preferably at least 840 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 25 contains at least 585 nucleotides, more preferably at least 615 nucleotides, and most preferably at least 645 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 27 contains at least 855 nucleotides, more preferably at least 900 nucleotides, and most preferably at least 945 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 29 contains at least 600 nucleotides, more preferably at least 630 nucleotides, and most preferably at least 660 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 31 contains at least 765 nucleotides, more preferably at least 810 nucleotides, and most preferably at least 855 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 33 contains at least 1180 nucleotides, more preferably at least 1140 nucleotides, and most preferably at least 1200 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 35 contains at least 600 nucleotides, more preferably at least 630 nucleotides, and most preferably at least 660 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 37 contains at least 170 amino acid residues, more preferably at least 180 amino acid residues, and most preferably at least 190 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 39 contains at least 570 nucleotides, more preferably at least 600 nucleotides, and most preferably at least 630 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 41 contains at least 795 nucleotides, more preferably at least 840 nucleotides, and most preferably at least 885 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 43 contains at least 540 nucleotides, more preferably at least 570 nucleotides, and most preferably at least 600 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 45 contains at least 960 nucleotides, more preferably at least 1005 nucleotides, and most preferably at least 1050 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 47 contains at least 765 nucleotides, more preferably at least 810 nucleotides, and most preferably at least 855 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 49 contains at least 570 nucleotides, more preferably at least 600 nucleotides, and most preferably at least 630 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 51 contains at least 600 nucleotides, more preferably at least 630 nucleotides, and most preferably at least 660 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 53 contains at least 1140 nucleotides, more preferably at least 1200 nucleotides, and most preferably at least 1260 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 55 contains at least 540 nucleotides, more preferably at least 570 nucleotides, and most preferably at least 600 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 57 contains at least 630 nucleotides, more preferably at least 690 nucleotides, and most preferably at least 720 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 59 contains at least 570 nucleotides, more preferably at least 600 nucleotides, and most preferably at least 630 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 61 contains at least 810 nucleotides, more preferably at least 870 nucleotides, and most preferably at least 930 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 63 contains at least 1020 nucleotides, more preferably at least 1080 nucleotides, and most preferably at least 1140 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 143 contains at least 840 nucleotides, more preferably at least 885 nucleotides, and most preferably at least 930 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 145 contains at least 930 nucleotides, more preferably at least 960 nucleotides, and most preferably at least 1050 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 147 contains at least 960 nucleotides, more preferably at least 1020 nucleotides, and most preferably at least 1080 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 149 contains at least 1050 nucleotides, more preferably at least 1110 nucleotides, and most preferably at least 1170 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 151 contains at least 660 nucleotides, more preferably at least 690 nucleotides, and most preferably at least 720 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 153 contains at least 660 nucleotides, more preferably at least 690 nucleotides, and most preferably at least 720 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 155 contains at least 765 nucleotides, more preferably at least 810 nucleotides, and most preferably at least 855 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 157 contains at least 555 nucleotides, more preferably at least 585 nucleotides, and most preferably at least 615 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 159 contains at least 540 nucleotides, more preferably at least 570 nucleotides, and most preferably at least 600 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 161 contains at least 570 nucleotides, more preferably at least 600 nucleotides, and most preferably at least 630 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 163 contains at least 1155 nucleotides, more preferably at least 1230 nucleotides, and most preferably at least 1305 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 165 contains at least 765 nucleotides, more preferably at least 810 nucleotides, and most preferably at least 855 nucleotides.
[0186] In a fourth aspect, the polypeptide having cellulolytic enhancing activity is encoded by a polynucleotide that hybridizes under at least very low stringency conditions, preferably at least low stringency conditions, more preferably at least medium stringency conditions, more preferably at least medium-high stringency conditions, even more preferably at least high stringency conditions, and most preferably at least very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, or SEQ ID NO: 163, (ii) the genomic DNA sequence of the mature polypeptide coding sequence of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 155, SEQ ID NO: 157, or SEQ ID NO: 159, or the cDNA sequence of the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 161, SEQ ID NO: 163, or SEQ ID NO: 165, (iii) a subsequence of (i) or (ii), or (iv) a full-length complementary strand of (i), (ii), or (iii) (J. Sambrook, E. F. Fritsch, and T. Maniatus, 1989, supra). A subsequence of the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, or SEQ ID NO: 163 contains at least 100 contiguous nucleotides or preferably at least 200 contiguous nucleotides. Moreover, the subsequence may encode a polypeptide fragment that has cellulolytic enhancing activity. In a preferred aspect, the mature polypeptide coding sequence is nucleotides 388 to 1332 of SEQ ID NO: 1, nucleotides 98 to 821 of SEQ ID NO: 3, nucleotides 126 to 978 of SEQ ID NO: 5, nucleotides 55 to 678 of SEQ ID NO: 7, nucleotides 58 to 912 of SEQ ID NO: 9, nucleotides 46 to 951 of SEQ ID NO: 11, nucleotides 67 to 796 of SEQ ID NO: 13, nucleotides 77 to 766 of SEQ ID NO: 15, nucleotides 52 to 921 of SEQ ID NO: 17, nucleotides 46 to 851 of SEQ ID NO: 19, nucleotides 55 to 1239 of SEQ ID NO: 21, nucleotides 46 to 1250 of SEQ ID NO: 23, nucleotides 58 to 811 of SEQ ID NO: 25, nucleotides 64 to 1112 of SEQ ID NO: 27, nucleotides 64 to 859 of SEQ ID NO: 29, nucleotides 64 to 1018 of SEQ ID NO: 31, nucleotides 70 to 1483 of SEQ ID NO: 33, nucleotides 76 to 832 of SEQ ID NO: 35, nucleotides 58 to 974 of SEQ ID NO: 37, nucleotides 52 to 875 of SEQ ID NO: 39, nucleotides 58 to 1250 of SEQ ID NO: 41, nucleotides 52 to 795 of SEQ ID NO: 43, nucleotides 64 to 1104 of SEQ ID NO: 45, nucleotides 73 to 990 of SEQ ID NO: 47, nucleotides 49 to 1218 of SEQ ID NO: 49, nucleotides 55 to 930 of SEQ ID NO: 51, nucleotides 67 to 1581 of SEQ ID NO: 53, nucleotides 49 to 865 of SEQ ID NO: 55, nucleotides 58 to 1065 of SEQ ID NO: 57, nucleotides 67 to 868 of SEQ ID NO: 59, nucleotides 55 to 1099 of SEQ ID NO: 61, nucleotides 70 to 1483 of SEQ ID NO: 63, nucleotides 61 to 1032 of SEQ ID NO: 143, nucleotides 61 to 1167 of SEQ ID NO: 145, nucleotides 64 to 1218 of SEQ ID NO: 147, nucleotides 58 to 1281 of SEQ ID NO: 149, nucleotides 52 to 801 of SEQ ID NO: 151, nucleotides 61 to 819 of SEQ ID NO: 153, nucleotides 61 to 966 of SEQ ID NO: 155, nucleotides 52 to 702 of SEQ ID NO: 157, nucleotides 70 to 699 of SEQ ID NO: 159, nucleotides 49 to 711 of SEQ ID NO: 161, nucleotides 76 to 1452 of SEQ ID NO: 163, or nucleotides 64 to 1018 of SEQ ID NO: 165.
[0187] The nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, or SEQ ID NO: 163, or a subsequence thereof; as well as the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, or SEQ ID NO: 166, or a fragment thereof, may be used to design a nucleic acid probe to identify and clone DNA encoding polypeptides having cellulolytic enhancing activity from strains of different genera or species according to methods well known in the art. In particular, such probes can be used for hybridization with the genomic DNA or cDNA of the genus or species of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein. Such probes can be considerably shorter than the entire sequence, but should be at least 14, preferably at least 25, more preferably at least 35, and most preferably at least 70 nucleotides in length. It is, however, preferred that the nucleic acid probe is at least 100 nucleotides in length. For example, the nucleic acid probe may be at least 200 nucleotides, preferably at least 300 nucleotides, more preferably at least 400 nucleotides, or most preferably at least 500 nucleotides in length. Even longer probes may be used, e.g., nucleic acid probes that are preferably at least 600 nucleotides, more preferably at least 700 nucleotides, even more preferably at least 800 nucleotides, or most preferably at least 900 nucleotides in length. Both DNA and RNA probes can be used. The probes are typically labeled for detecting the corresponding gene (for example, with .sup.32P, .sup.3H, .sup.35S, biotin, or avidin). Such probes are encompassed by the present invention.
[0188] A genomic DNA or cDNA library prepared from such other strains may, therefore, be screened for DNA that hybridizes with the probes described above and encodes a polypeptide having cellulolytic enhancing activity. Genomic or other DNA from such other strains may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material. In order to identify a clone or DNA that is homologous with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, or SEQ ID NO: 163, or a subsequence thereof, the carrier material is preferably used in a Southern blot.
[0189] For purposes of the present invention, hybridization indicates that the nucleotide sequence hybridizes to a labeled nucleic acid probe corresponding to the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, or SEQ ID NO: 163; the genomic DNA sequence of the mature polypeptide coding sequence of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 155, SEQ ID NO: 157, or SEQ ID NO: 159, or the cDNA sequence of the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 161, SEQ ID NO: 163, or SEQ ID NO: 165; the full-length complementary strand thereof; or a subsequence thereof, under very low to very high stringency conditions, as described supra.
[0190] In a preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 1. In another preferred aspect, the nucleic acid probe is nucleotides 388 to 1332 of SEQ ID NO: 1. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 2, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 1. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid pEJG120 which is contained in E. coli NRRL B-30699, wherein the polynucleotide sequence thereof encodes a polypeptide having cellulolytic enhancing activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence contained in plasmid pEJG120 which is contained in E. coli NRRL B-30699.
[0191] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 3. In another preferred aspect, the nucleic acid probe is nucleotides 98 to 821 of SEQ ID NO: 3. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 4, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 3. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid pTter61C which is contained in E. coli NRRL B-30813, wherein the polynucleotide sequence thereof encodes a polypeptide having cellulolytic enhancing activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence contained in plasmid pTter61C which is contained in E. coli NRRL B-30813.
[0192] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 5. In another preferred aspect, the nucleic acid probe is nucleotides 126 to 978 of SEQ ID NO: 5. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 6, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 5. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid pTter61D which is contained in E. coli NRRL B-30812, wherein the polynucleotide sequence thereof encodes a polypeptide having cellulolytic enhancing activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence contained in plasmid pTter61D which is contained in E. coli NRRL B-30812.
[0193] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 7. In another preferred aspect, the nucleic acid probe is nucleotides 55 to 678 of SEQ ID NO: 7. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 8, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 7. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid pTter61E which is contained in E. coli NRRL B-30814, wherein the polynucleotide sequence thereof encodes a polypeptide having cellulolytic enhancing activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence contained in plasmid pTter61E which is contained in E. coli NRRL B-30814.
[0194] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 9. In another preferred aspect, the nucleic acid probe is nucleotides 58 to 912 of SEQ ID NO: 9 In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 10, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 9. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid pTter61G which is contained in E. coli NRRL B-30811, wherein the polynucleotide sequence thereof encodes a polypeptide having cellulolytic enhancing activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence contained in plasmid pTter61G which is contained in E. coli NRRL B-30811.
[0195] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 11. In another preferred aspect, the nucleic acid probe is nucleotides 46 to 951 of SEQ ID NO: 11. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 12, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 11. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid pTter61 F which is contained in E. coli NRRL B-50044, wherein the polynucleotide sequence thereof encodes a polypeptide having cellulolytic enhancing activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding region contained in plasmid pTter61 F which is contained in E. coli NRRL B-50044.
[0196] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 13. In another preferred aspect, the nucleic acid probe is nucleotides 67 to 796 of SEQ ID NO: 13. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 14, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 13. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid pDZA2-7 which is contained in E. coli NRRL B-30704, wherein the polynucleotide sequence thereof encodes a polypeptide having cellulolytic enhancing activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence contained in plasmid pDZA2-7 which is contained in E. coli NRRL B-30704.
[0197] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 15. In another preferred aspect, the nucleic acid probe is nucleotides 77 to 766 of SEQ ID NO: 15. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 16, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 15. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid pTr3337 which is contained in E. coli NRRL B-30878, wherein the polynucleotide sequence thereof encodes a polypeptide having cellulolytic enhancing activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence contained in plasmid pTr3337 which is contained in E. coli NRRL B-30878.
[0198] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 17. In another preferred aspect, the nucleic acid probe is nucleotides 52 to 921 of SEQ ID NO: 17. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 18, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 17. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid pSMai190 which is contained in E. coli NRRL B-50084, wherein the polynucleotide sequence thereof encodes a polypeptide having cellulolytic enhancing activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence contained in plasmid pSMai190 which is contained in E. coli NRRL B-50084.
[0199] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 19. In another preferred aspect, the nucleic acid probe is nucleotides 46 to 851 of SEQ ID NO: 19. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 20, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 19. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid pSMai192 which is contained in E. coli NRRL B-50086, wherein the polynucleotide sequence thereof encodes a polypeptide having cellulolytic enhancing activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence contained in plasmid pSMai192 which is contained in E. coli NRRL B-50086.
[0200] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 21. In another preferred aspect, the nucleic acid probe is nucleotides 55 to 1239 of SEQ ID NO: 21. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 22, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 21. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid pSMai191 which is contained in E. coli NRRL B-50085, wherein the polynucleotide sequence thereof encodes a polypeptide having cellulolytic enhancing activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence contained in plasmid pSMai191 which is contained in E. coli NRRL B-50085.
[0201] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 23. In another preferred aspect, the nucleic acid probe is nucleotides 46 to 1250 of SEQ ID NO: 23. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 24, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 23. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid pSMai193 which is contained in E. coli NRRL B-50087, wherein the polynucleotide sequence thereof encodes a polypeptide having cellulolytic enhancing activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence contained in plasmid pSMai193 which is contained in E. coli NRRL B-50087.
[0202] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 25. In another preferred aspect, the nucleic acid probe is nucleotides 58 to 811 of SEQ ID NO: 25. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 26, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 25. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid pSMai187 which is contained in E. coli NRRL B-50083, wherein the polynucleotide sequence thereof encodes a polypeptide having cellulolytic enhancing activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence contained in plasmid pSMai187 which is contained in E. coli NRRL B-50083.
[0203] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 27. In another preferred aspect, the nucleic acid probe is nucleotides 64 to 1112 of SEQ ID NO: 27. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 28, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 27. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid pXYZ1473 which is contained in E. coli DSM 22075, wherein the polynucleotide sequence thereof encodes a polypeptide having cellulolytic enhancing activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence contained in plasmid pXYZ1473 which is contained in E. coli DSM 22075.
[0204] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 29. In another preferred aspect, the nucleic acid probe is nucleotides 64 to 859 of SEQ ID NO: 29. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 30, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 29.
[0205] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 31. In another preferred aspect, the nucleic acid probe is nucleotides 64 to 1018 of SEQ ID NO: 31. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 32, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 31. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid pGEM-T-Ppin7 which is contained in E. coli DSM 22711, wherein the polynucleotide sequence thereof encodes a polypeptide having cellulolytic enhancing activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence contained in plasmid pGEM-T-Ppin7 which is contained in E. coli DSM 22711.
[0206] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 33. In another preferred aspect, the nucleic acid probe is nucleotides 70 to 1483 of SEQ ID NO: 33. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 34, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 33. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid pXYZ1483 which is contained in E. coli DSM 22600, wherein the polynucleotide sequence thereof encodes a polypeptide having cellulolytic enhancing activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence contained in plasmid pXYZ1483 which is contained in E. coli DSM 22600.
[0207] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 35. In another preferred aspect, the nucleic acid probe is nucleotides 76 to 832 of SEQ ID NO: 35. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 36, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 35. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid pGEM-T-GH61D23Y4 which is contained in E. coli DSM 22882, wherein the polynucleotide sequence thereof encodes a polypeptide having cellulolytic enhancing activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence contained in plasmid pGEM-T-GH61D23Y4 which is contained in E. coli DSM 22882.
[0208] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 37. In another preferred aspect, the nucleic acid probe is nucleotides 58 to 974 of SEQ ID NO: 37. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 38, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 37. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid pSMai213 which is contained in E. coli NRRL B-50300, wherein the polynucleotide sequence thereof encodes a polypeptide having cellulolytic enhancing activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence contained in plasmid pSMai213 which is contained in E. coli NRRL B-50300.
[0209] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 39. In another preferred aspect, the nucleic acid probe is nucleotides 52 to 875 of SEQ ID NO: 39. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 40, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 39. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid pSMai216 which is contained in E. coli NRRL B-50301, wherein the polynucleotide sequence thereof encodes a polypeptide having cellulolytic enhancing activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence contained in plasmid pSMai216 which is contained in E. coli NRRL B-50301.
[0210] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 41. In another preferred aspect, the nucleic acid probe is nucleotides 58 to 1250 of SEQ ID NO: 41. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 42, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 41. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid p pSMai217 which is contained in E. coli NRRL B-50302, wherein the polynucleotide sequence thereof encodes a polypeptide having cellulolytic enhancing activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence contained in plasmid pSMai217 which is contained in E. coli NRRL B-50302.
[0211] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 43. In another preferred aspect, the nucleic acid probe is nucleotides 52 to 795 of SEQ ID NO: 43. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 44, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 43. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid pSMai218 which is contained in E. coli NRRL B-50303, wherein the polynucleotide sequence thereof encodes a polypeptide having cellulolytic enhancing activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence contained in plasmid pSMai218 which is contained in E. coli NRRL B-50303.
[0212] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 45. In another preferred aspect, the nucleic acid probe is nucleotides 64 to 1104 of SEQ ID NO: 45. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 46, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 45. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid pAG68 which is contained in E. coli NRRL B-50320, wherein the polynucleotide sequence thereof encodes a polypeptide having cellulolytic enhancing activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence contained in plasmid pAG68 which is contained in E. coli NRRL B-50320.
[0213] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 47. In another preferred aspect, the nucleic acid probe is nucleotides 73 to 990 of SEQ ID NO: 47. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 48, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 47. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid pAG69 which is contained in E. coli NRRL B-50321, wherein the polynucleotide sequence thereof encodes a polypeptide having cellulolytic enhancing activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence contained in plasmid pAG69 which is contained in E. coli NRRL B-50321.
[0214] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 49. In another preferred aspect, the nucleic acid probe is nucleotides 49 to 1218 of SEQ ID NO: 49. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 50, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 49. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid pAG75 which is contained in E. coli NRRL B-50322, wherein the polynucleotide sequence thereof encodes a polypeptide having cellulolytic enhancing activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence contained in plasmid pAG75 which is contained in E. coli NRRL B-50322.
[0215] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 51. In another preferred aspect, the nucleic acid probe is nucleotides 55 to 930 of SEQ ID NO: 51. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 52, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 51. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid pAG76 which is contained in E. coli NRRL B-50323, wherein the polynucleotide sequence thereof encodes a polypeptide having cellulolytic enhancing activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence contained in plasmid pAG76 which is contained in E. coli NRRL B-50323.
[0216] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 53. In another preferred aspect, the nucleic acid probe is nucleotides 67 to 1581 of SEQ ID NO: 53. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 54, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 53. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid pAG77 which is contained in E. coli NRRL B-50324, wherein the polynucleotide sequence thereof encodes a polypeptide having cellulolytic enhancing activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence contained in plasmid pAG77 which is contained in E. coli NRRL B-50324.
[0217] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 55. In another preferred aspect, the nucleic acid probe is nucleotides 49 to 865 of SEQ ID NO: 55. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 56, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 55. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid pAG78 which is contained in E. coli NRRL B-50325, wherein the polynucleotide sequence thereof encodes a polypeptide having cellulolytic enhancing activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence contained in plasmid pAG78 which is contained in E. coli NRRL B-50325.
[0218] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 57. In another preferred aspect, the nucleic acid probe is nucleotides 58 to 1065 of SEQ ID NO: 57. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 58, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 57. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid p pAG79 which is contained in E. coli NRRL B-50326, wherein the polynucleotide sequence thereof encodes a polypeptide having cellulolytic enhancing activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence contained in plasmid pAG79 which is contained in E. coli NRRL B-50326.
[0219] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 59. In another preferred aspect, the nucleic acid probe is nucleotides 67 to 868 of SEQ ID NO: 59. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 60, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 59. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid plasmid pGEM-T-GH61a51486 which is contained in E. coli DSM 22656, wherein the polynucleotide sequence thereof encodes a polypeptide having cellulolytic enhancing activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence contained in plasmid plasmid pGEM-T-GH61a51486 which is contained in E. coli DSM 22656.
[0220] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 61. In another preferred aspect, the nucleic acid probe is nucleotides 55 to 1099 of SEQ ID NO: 61. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 62, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 61. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid pGEM-T-GH61DYF which is contained in E. coli DSM 22654, wherein the polynucleotide sequence thereof encodes a polypeptide having cellulolytic enhancing activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence contained in plasmid pGEM-T-GH61DYF which is contained in E. coli DSM 22654.
[0221] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 63. In another preferred aspect, the nucleic acid probe is nucleotides 70 to 1483 of SEQ ID NO: 63. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 64, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 63. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid pGEM-T-GH61D14YH which is contained in E. coli DSM 22657, wherein the polynucleotide sequence thereof encodes a polypeptide having cellulolytic enhancing activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence contained in plasmid pGEM-T-GH61D14YH which is contained in E. coli DSM 22657.
[0222] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 143. In another preferred aspect, the nucleic acid probe is nucleotides 61 to 1032 of SEQ ID NO: 143. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 143, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 143.
[0223] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 145. In another preferred aspect, the nucleic acid probe is nucleotides 61 to 1167 of SEQ ID NO: 145. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 145, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 145.
[0224] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 147. In another preferred aspect, the nucleic acid probe is nucleotides 64 to 1218 of SEQ ID NO: 147. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 147, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 147.
[0225] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 149. In another preferred aspect, the nucleic acid probe is nucleotides 58 to 1281 of SEQ ID NO: 149. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 149, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 149.
[0226] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 151. In another preferred aspect, the nucleic acid probe is nucleotides 52 to 801 of SEQ ID NO: 151. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 151, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 151.
[0227] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 153. In another preferred aspect, the nucleic acid probe is nucleotides 61 to 819 of SEQ ID NO: 153. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 153, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 153.
[0228] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 155. In another preferred aspect, the nucleic acid probe is nucleotides 61 to 966 of SEQ ID NO: 155. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 155, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 155.
[0229] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 157. In another preferred aspect, the nucleic acid probe is nucleotides 52 to 702 of SEQ ID NO: 157. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 157, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 157.
[0230] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 159. In another preferred aspect, the nucleic acid probe is nucleotides 70 to 699 of SEQ ID NO: 159. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 159, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 159.
[0231] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 161. In another preferred aspect, the nucleic acid probe is nucleotides 49 to 711 of SEQ ID NO: 161. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 161, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 161.
[0232] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 163. In another preferred aspect, the nucleic acid probe is nucleotides 76 to 1452 of SEQ ID NO: 163. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 163, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 163.
[0233] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 165. In another preferred aspect, the nucleic acid probe is nucleotides 64 to 1018 of SEQ ID NO: 165. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 165, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 165.
[0234] For long probes of at least 100 nucleotides in length, very low to very high stringency conditions are defined as prehybridization and hybridization at 42.degree. C. in 5.times.SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and either 25% formamide for very low and low stringencies, 35% formamide for medium and medium-high stringencies, or 50% formamide for high and very high stringencies, following standard Southern blotting procedures for 12 to 24 hours optimally. The carrier material is finally washed three times each for 15 minutes using 2.times.SSC, 0.2% SDS at 45.degree. C. (very low stringency), at 50.degree. C. (low stringency), at 55.degree. C. (medium stringency), at 60.degree. C. (medium-high stringency), at 65.degree. C. (high stringency), and at 70.degree. C. (very high stringency).
[0235] For short probes of about 15 nucleotides to about 70 nucleotides in length, stringency conditions are defined as prehybridization and hybridization at about 5.degree. C. to about 10.degree. C. below the calculated T.sub.m using the calculation according to Bolton and McCarthy (1962, Proc. Natl. Acad. Sci. USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40, 1.times.Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml following standard Southern blotting procedures for 12 to 24 hours optimally. The carrier material is finally washed once in 6.times.SCC plus 0.1% SDS for 15 minutes and twice each for 15 minutes using 6.times.SSC at 5.degree. C. to 10.degree. C. below the calculated T.sub.m.
[0236] In a fifth aspect, the polypeptide having cellulolytic enhancing activity is encoded by a polynucleotide comprising or consisting of a nucleotide sequence that has a degree of identity to the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, or SEQ ID NO: 163 of preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%, and even most preferably at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%.
[0237] In a sixth aspect, the polypeptide having cellulolytic enhancing activity is an artificial variant comprising a substitution, deletion, and/or insertion of one or more (e.g., several) amino acids of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, or SEQ ID NO: 166; or a homologous sequence thereof. Preferably, amino acid changes are of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of one to about 30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to about 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.
[0238] Examples of conservative substitutions are within the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R. L. Hill, 1979, In, The Proteins, Academic Press, New York. The most commonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.
[0239] Alternatively, the amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered. For example, amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like.
[0240] Essential amino acids in a parent polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for cellulolytic enhancing activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identities of essential amino acids can also be inferred from analysis of identities with polypeptides that are related to the parent polypeptide.
[0241] Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).
[0242] Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.
[0243] The total number of amino acid substitutions, deletions and/or insertions of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, or SEQ ID NO: 166, is not more than 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9.
[0244] A polypeptide having cellulolytic enhancing activity may be obtained from microorganisms of any genus. For purposes of the present invention, the term "obtained from" as used herein in connection with a given source shall mean that the polypeptide encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted. In one aspect, the polypeptide obtained from a given source is secreted extracellularly.
[0245] A polypeptide having cellulolytic enhancing activity may be a bacterial polypeptide. For example, the polypeptide may be a gram positive bacterial polypeptide such as a Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus, or Oceanobacillus polypeptide having cellulolytic enhancing activity, or a Gram negative bacterial polypeptide such as an E. coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, or Ureaplasma polypeptide having cellulolytic enhancing activity.
[0246] In one aspect, the polypeptide is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis polypeptide having cellulolytic enhancing activity.
[0247] In another aspect, the polypeptide is a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus polypeptide having cellulolytic enhancing activity.
[0248] In another aspect, the polypeptide is a Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans polypeptide having cellulolytic enhancing activity.
[0249] The polypeptide having cellulolytic enhancing activity may also be a fungal polypeptide, and more preferably a yeast polypeptide such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia polypeptide having cellulolytic enhancing activity; or more preferably a filamentous fungal polypeptide such as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryosphaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, lrpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylaria polypeptide having cellulolytic enhancing activity.
[0250] In another aspect, the polypeptide is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis polypeptide having cellulolytic enhancing activity.
[0251] In another aspect, the polypeptide is an Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum, Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium suiphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa, lrpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium pinophilum, Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia spededonium, Thielavia setosa, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma viride, or Trichophaea saccata polypeptide having cellulolytic enhancing activity.
[0252] It will be understood that for the aforementioned species the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.
[0253] Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).
[0254] Furthermore, polypeptides having cellulolytic enhancing activity may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms from natural habitats are well known in the art. The polynucleotide may then be obtained by similarly screening a genomic DNA or cDNA library of such a microorganism. Once a polynucleotide encoding a polypeptide has been detected with the probe(s), the polynucleotide can be isolated or cloned by utilizing techniques that are well known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra)
[0255] Polynucleotides comprising nucleotide sequences that encode polypeptide having cellulolytic enhancing activity can be isolated and utilized to express the polypeptide having cellulolytic enhancing activity for evaluation in the methods of the present invention.
[0256] The techniques used to isolate or clone a polynucleotide encoding a polypeptide are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. The cloning of the polynucleotides from such genomic DNA can be effected, e.g., by using the well known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g., Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligation activated transcription (LAT) and polynucleotide-based amplification (NASBA) may be used. The polynucleotides may be cloned from a strain or a related organism and thus, for example, may be an allelic or species variant of the polypeptide encoding region of the polynucleotide.
[0257] The polynucleotides comprise nucleotide sequences that have a degree of identity to the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, or SEQ ID NO: 163 of preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%, and even most preferably at least 96%, at least 97%, at least 98%, or at least 99%, which encode a polypeptide having cellulolytic enhancing activity.
[0258] The polynucleotide may also be a polynucleotide encoding a polypeptide having cellulolytic enhancing activity that hybridizes under at least very low stringency conditions, preferably at least low stringency conditions, more preferably at least medium stringency conditions, more preferably at least medium-high stringency conditions, even more preferably at least high stringency conditions, and most preferably at least very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, or SEQ ID NO: 163, (ii) the genomic DNA sequence of the mature polypeptide coding sequence of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 155, SEQ ID NO: 157, or SEQ ID NO: 159 or the cDNA sequence of the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 161, SEQ ID NO: 163, or SEQ ID NO: 165, or (iii) a full-length complementary strand of (i) or (ii); or allelic variants and subsequences thereof (Sambrook et al., 1989, supra), as defined herein.
[0259] As described earlier, the techniques used to isolate or clone a polynucleotide encoding a polypeptide are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof.
Enzyme Compositions
[0260] The enzyme compositions can comprise any protein that is useful in degrading or converting a cellulosic material.
[0261] In one aspect, the enzyme composition comprises or further comprises one or more (e.g., several) proteins selected from the group consisting of a cellulase, a hemicellulase, an esterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin. In another aspect, the cellulase is preferably one or more (e.g., several) enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase. In another aspect, the hemicellulase is preferably one or more (e.g., several) enzymes selected from the group consisting of an acetylmannan esterase, an acetylxylan esterase, an arabinanase, an arabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, a galactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and a xylosidase.
[0262] In another aspect, the enzyme composition comprises one or more (e.g., several) cellulolytic enzymes. In another aspect, the enzyme composition comprises or further comprises one or more (e.g., several) hemicellulolytic enzymes. In another aspect, the enzyme composition comprises one or more (e.g., several) cellulolytic enzymes and one or more (e.g., several) hemicellulolytic enzymes. In another aspect, the enzyme composition comprises one or more (e.g., several) enzymes selected from the group of cellulolytic enzymes and hemicellulolytic enzymes. In another aspect, the enzyme composition comprises an endoglucanase. In another aspect, the enzyme composition comprises a cellobiohydrolase. In another aspect, the enzyme composition comprises a beta-glucosidase. In another aspect, the enzyme composition comprises an endoglucanase and a cellobiohydrolase. In another aspect, the enzyme composition comprises an endoglucanase and a beta-glucosidase. In another aspect, the enzyme composition comprises a cellobiohydrolase and a beta-glucosidase. In another aspect, the enzyme composition comprises an endoglucanase, a cellobiohydrolase, and a beta-glucosidase.
[0263] In another aspect, the enzyme composition comprises an acetylmannan esterase. In another aspect, the enzyme composition comprises an acetylxylan esterase. In another aspect, the enzyme composition comprises an arabinanase (e.g., alpha-L-arabinanase). In another aspect, the enzyme composition comprises an arabinofuranosidase (e.g., alpha-L-arabinofuranosidase). In another aspect, the enzyme composition comprises a coumaric acid esterase. In another aspect, the enzyme composition comprises a feruloyl esterase. In another aspect, the enzyme composition comprises a galactosidase (e.g., alpha-galactosidase and/or beta-galactosidase). In another aspect, the enzyme composition comprises a glucuronidase (e.g., alpha-D-glucuronidase). In another aspect, the enzyme composition comprises a glucuronoyl esterase. In another aspect, the enzyme composition comprises a mannanase. In another aspect, the enzyme composition comprises a mannosidase (e.g., beta-mannosidase). In another aspect, the enzyme composition comprises a xylanase. In a preferred aspect, the xylanase is a Family 10 xylanase. In another aspect, the enzyme composition comprises a xylosidase (e.g., beta-xylosidase).
[0264] In another aspect, the enzyme composition comprises an esterase. In another aspect, the enzyme composition comprises an expansin. In another aspect, the enzyme composition comprises a laccase. In another aspect, the enzyme composition comprises a ligninolytic enzyme. In a preferred aspect, the ligninolytic enzyme is a manganese peroxidase. In another preferred aspect, the ligninolytic enzyme is a lignin peroxidase. In another preferred aspect, the ligninolytic enzyme is a H.sub.2O.sub.2-producing enzyme. In another aspect, the enzyme composition comprises a pectinase. In another aspect, the enzyme composition comprises a peroxidase. In another aspect, the enzyme composition comprises a protease. In another aspect, the enzyme composition comprises a swollenin.
[0265] In the methods of the present invention, the enzyme(s) can be added prior to or during fermentation, e.g., during saccharification or during or after propagation of the fermenting microorganism(s).
[0266] One or more (e.g., several) components of the enzyme composition may be wild-type proteins, recombinant proteins, or a combination of wild-type proteins and recombinant proteins. For example, one or more (e.g., several) components may be native proteins of a cell, which is used as a host cell to express recombinantly one or more (e.g., several) other components of the enzyme composition. One or more (e.g., several) components of the enzyme composition may be produced as monocomponents, which are then combined to form the enzyme composition. The enzyme composition may be a combination of multicomponent and monocomponent protein preparations.
[0267] The enzymes used in the methods of the present invention may be in any form suitable for use, such as, for example, a crude fermentation broth with or without cells removed, a cell lysate with or without cellular debris, a semi-purified or purified enzyme preparation, or a host cell as a source of the enzymes. The enzyme composition may be a dry powder or granulate, a non-dusting granulate, a liquid, a stabilized liquid, or a stabilized protected enzyme. Liquid enzyme preparations may, for instance, be stabilized by adding stabilizers such as a sugar, a sugar alcohol or another polyol, and/or lactic acid or another organic acid according to established processes.
[0268] The enzymes can be derived or obtained from any suitable origin, including, bacterial, fungal, yeast, plant, or mammalian origin. The term "obtained" means herein that the enzyme may have been isolated from an organism that naturally produces the enzyme as a native enzyme. The term "obtained" also means herein that the enzyme may have been produced recombinantly in a host organism employing methods described herein, wherein the recombinantly produced enzyme is either native or foreign to the host organism or has a modified amino acid sequence, e.g., having one or more (e.g., several) amino acids that are deleted, inserted and/or substituted, i.e., a recombinantly produced enzyme that is a mutant and/or a fragment of a native amino acid sequence or an enzyme produced by nucleic acid shuffling processes known in the art. Encompassed within the meaning of a native enzyme are natural variants and within the meaning of a foreign enzyme are variants obtained recombinantly, such as by site-directed mutagenesis or shuffling.
[0269] The polypeptide having enzyme activity may be a bacterial polypeptide. For example, the polypeptide may be a gram positive bacterial polypeptide such as a Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus, or Oceanobacillus polypeptide having enzyme activity, or a Gram negative bacterial polypeptide such as an E. coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, or Ureaplasma polypeptide having enzyme activity.
[0270] In a preferred aspect, the polypeptide is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis polypeptide having enzyme activity.
[0271] In another preferred aspect, the polypeptide is a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus polypeptide having enzyme activity.
[0272] In another preferred aspect, the polypeptide is a Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans polypeptide having enzyme activity.
[0273] The polypeptide having enzyme activity may also be a fungal polypeptide, and more preferably a yeast polypeptide such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia polypeptide having enzyme activity; or more preferably a filamentous fungal polypeptide such as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryosphaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, lrpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylaria polypeptide having enzyme activity.
[0274] In a preferred aspect, the polypeptide is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis polypeptide having enzyme activity.
[0275] In another preferred aspect, the polypeptide is an Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum, Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia spededonium, Thielavia setosa, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma viride, or Trichophaea saccata polypeptide having enzyme activity.
[0276] Chemically modified or protein engineered mutants of the polypeptides having enzyme activity may also be used.
[0277] One or more (e.g., several) components of the enzyme composition may be a recombinant component, i.e., produced by cloning of a DNA sequence encoding the single component and subsequent cell transformed with the DNA sequence and expressed in a host (see, for example, WO 91/17243 and WO 91/17244). The host is preferably a heterologous host (enzyme is foreign to host), but the host may under certain conditions also be a homologous host (enzyme is native to host). Monocomponent cellulolytic enzymes may also be prepared by purifying such a protein from a fermentation broth.
[0278] In one aspect, the one or more (e.g., several) cellulolytic enzymes comprise a commercial cellulolytic enzyme preparation. Examples of commercial cellulolytic enzyme preparations suitable for use in the present invention include, for example, CELLIC.TM. CTec (Novozymes A/S), CELLIC.TM. CTec2 (Novozymes A/S), CELLUCLAST.TM. (Novozymes A/S), NOVOZYM.TM. 188 (Novozymes A/S), CELLUZYME.TM. (Novozymes A/S), CEREFLO.TM. (Novozymes A/S), and ULTRAFLO.TM. (Novozymes A/S), ACCELERASE.TM. (Genencor Int.), LAMINEX.TM. (Genencor Int.), SPEZYME.TM. CP (Genencor Int.), FILTRASE.RTM. NL (DSM); METHAPLUS.RTM. S/L 100 (DSM), ROHAMENT.TM. 7069 W (Rohm GmbH), FIBREZYME.RTM. LDI (Dyadic International, Inc.), FIBREZYME.RTM. LBR (Dyadic International, Inc.), or VISCOSTAR.RTM. 150L (Dyadic International, Inc.). The cellulase enzymes are added in amounts effective from about 0.001 to about 5.0 wt % of solids, more preferably from about 0.025 to about 4.0 wt % of solids, and most preferably from about 0.005 to about 2.0 wt % of solids. The cellulase enzymes are added in amounts effective from about 0.001 to about 5.0 wt % of solids, more preferably from about 0.025 to about 4.0 wt % of solids, and most preferably from about 0.005 to about 2.0 wt % of solids.
[0279] Examples of bacterial endoglucanases that can be used in the methods of the present invention, include, but are not limited to, an Acidothermus cellulolyticus endoglucanase (WO 91/05039; WO 93/15186; U.S. Pat. No. 5,275,944; WO 96/02551; U.S. Pat. No. 5,536,655, WO 00/70031, WO 05/093050); Thermobifida fusca endoglucanase III (WO 05/093050); and Thermobifida fusca endoglucanase V (WO 05/093050).
[0280] Examples of fungal endoglucanases that can be used in the present invention include, but are not limited to, a Trichoderma reesei endoglucanase I (Penttila et al., 1986, Gene 45: 253-263; Trichoderma reesei Cel7B endoglucanase I; GENBANK.TM. accession no. M15665; SEQ ID NO: 66); Trichoderma reesei endoglucanase II (Saloheimo, et al., 1988, Gene 63:11-22; Trichoderma reesei Cel5A endoglucanase II; GENBANK.TM. accession no. M19373; SEQ ID NO: 68); Trichoderma reesei endoglucanase III (Okada et al., 1988, Appl. Environ. Microbiol. 64: 555-563; GENBANK.TM. accession no. AB003694; SEQ ID NO: 70); Trichoderma reesei endoglucanase V (Saloheimo et al., 1994, Molecular Microbiology 13: 219-228; GENBANK.TM. accession no. Z33381; SEQ ID NO: 72); Aspergillus aculeatus endoglucanase (Ooi et al., 1990, Nucleic Acids Research 18: 5884); Aspergillus kawachii endoglucanase (Sakamoto et al., 1995, Current Genetics 27: 435-439); Erwinia carotovara endoglucanase (Saarilahti et al., 1990, Gene 90: 9-14); Fusarium oxysporum endoglucanase (GENBANK.TM. accession no. L29381); Humicola grisea var. thermoidea endoglucanase (GENBANK.TM. accession no. AB003107); Melanocarpus albomyces endoglucanase (GENBANK.TM. accession no. MAL515703); Neurospora crassa endoglucanase (GENBANK.TM. accession no. XM_324477); Humicola insolens endoglucanase V (SEQ ID NO: 74); Myceliophthora thermophila CBS 117.65 endoglucanase (SEQ ID NO: 76); basidiomycete CBS 495.95 endoglucanase (SEQ ID NO: 78); basidiomycete CBS 494.95 endoglucanase (SEQ ID NO: 80); Thielavia terrestris NRRL 8126 CEL6B endoglucanase (SEQ ID NO: 82); Thielavia terrestris NRRL 8126 CEL6C endoglucanase (SEQ ID NO: 84); Thielavia terrestris NRRL 8126 CEL7C endoglucanase (SEQ ID NO: 86); Thielavia terrestris NRRL 8126 CEL7E endoglucanase (SEQ ID NO: 88); Thielavia terrestris NRRL 8126 CEL7F endoglucanase (SEQ ID NO: 90); Cladorrhinum foecundissimum ATCC 62373 CEL7A endoglucanase (SEQ ID NO: 92); and Trichoderma reesei strain No. VTT-D-80133 endoglucanase (SEQ ID NO: 94; GENBANK.TM. accession no. M15665). The endoglucanases of SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, and SEQ ID NO: 94 described above are encoded by the mature polypeptide coding sequence of SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, and SEQ ID NO: 93, respectively.
[0281] Examples of cellobiohydrolases useful in the present invention include, but are not limited to, Trichoderma reesei cellobiohydrolase I (SEQ ID NO: 96); Trichoderma reesei cellobiohydrolase II (SEQ ID NO: 98); Humicola insolens cellobiohydrolase I (SEQ ID NO: 100); Myceliophthora thermophila cellobiohydrolase II (SEQ ID NO: 102 and SEQ ID NO: 104); Thielavia terrestris cellobiohydrolase II (CEL6A) (SEQ ID NO: 106); Chaetomium thermophilum cellobiohydrolase I (SEQ ID NO: 108); and Chaetomium thermophilum cellobiohydrolase II (SEQ ID NO: 110). The cellobiohydrolases of SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, and SEQ ID NO: 112 described above are encoded by the mature polypeptide coding sequence of SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, and SEQ ID NO: 109, respectively.
[0282] Examples of beta-glucosidases useful in the present invention include, but are not limited to, Aspergillus oryzae beta-glucosidase (SEQ ID NO: 112); Aspergillus fumigatus beta-glucosidase (SEQ ID NO: 114); Penicillium brasilianum IBT 20888 beta-glucosidase (SEQ ID NO: 116); Aspergillus niger beta-glucosidase (SEQ ID NO: 118); and Aspergillus aculeatus beta-glucosidase (SEQ ID NO: 120). The beta-glucosidases of SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, and SEQ ID NO: 120 described above are encoded by the mature polypeptide coding sequence of SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, and SEQ ID NO: 119, respectively.
[0283] Examples of other beta-glucosidases useful in the present invention include a Aspergillus oryzae beta-glucosidase variant fusion protein of SEQ ID NO: 122 or the Aspergillus oryzae beta-glucosidase fusion protein of SEQ ID NO: 124. The beta-glucosidase fusion proteins of SEQ ID NO: 122 and SEQ ID NO: 124 are encoded by SEQ ID NO: 121 and SEQ ID NO: 123, respectively.
[0284] The Aspergillus oryzae polypeptide having beta-glucosidase activity can be obtained according to WO 2002/095014. The Aspergillus fumigatus polypeptide having beta-glucosidase activity can be obtained according to WO 2005/047499. The Penicillium brasilianum polypeptide having beta-glucosidase activity can be obtained according to WO 2007/019442. The Aspergillus niger polypeptide having beta-glucosidase activity can be obtained according to Dan et al., 2000, J. Biol. Chem. 275: 4973-4980. The Aspergillus aculeatus polypeptide having beta-glucosidase activity can be obtained according to Kawaguchi et al., 1996, Gene 173: 287-288.
[0285] Other useful endoglucanases, cellobiohydrolases, and beta-glucosidases are disclosed in numerous Glycosyl Hydrolase families using the classification according to Henrissat B., 1991, A classification of glycosyl hydrolases based on amino-acid sequence similarities, Biochem. J. 280: 309-316, and Henrissat B., and Bairoch A., 1996, Updating the sequence-based classification of glycosyl hydrolases, Biochem. J. 316: 695-696.
[0286] Other cellulolytic enzymes that may be useful in the present invention are described in EP 495,257, EP 531,315, EP 531,372, WO 89/09259, WO 94/07998, WO 95/24471, WO 96/11262, WO 96/29397, WO 96/034108, WO 97/14804, WO 98/08940, WO 98/012307, WO 98/13465, WO 98/015619, WO 98/015633, WO 98/028411, WO 99/06574, WO 99/10481, WO 99/025846, WO 99/025847, WO 99/031255, WO 2000/009707, WO 2002/050245, WO 2002/0076792, WO 2002/101078, WO 2003/027306, WO 2003/052054, WO 2003/052055, WO 2003/052056, WO 2003/052057, WO 2003/052118, WO 2004/016760, WO 2004/043980, WO 2004/048592, WO 2005/001065, WO 2005/028636, WO 2005/093050, WO 2005/093073, WO 2006/074005, WO 2006/117432, WO 2007/071818, WO 2007/071820, WO 2008/008070, WO 2008/008793, U.S. Pat. Nos. 4,435,307, 5,457,046, 5,648,263, 5,686,593, 5,691,178, 5,763,254, and 5,776,757.
[0287] In one aspect, the one or more (e.g., several) hemicellulolytic enzymes comprise a commercial hemicellulolytic enzyme preparation. Examples of commercial hemicellulolytic enzyme preparations suitable for use in the present invention include, for example, SHEARZYME.TM. (Novozymes A/S), CELLIC.TM. HTec (Novozymes A/S), CELLIC.TM. HTec2 (Novozymes A/S), VISCOZYME.RTM. (Novozymes A/S), ULTRAFLO.RTM. (Novozymes A/S), PULPZYME.RTM. HC (Novozymes A/S), MULTIFECT.RTM. Xylanase (Genencor), ACCELLERASE.RTM. XY (Genencor), ACCELLERASE.RTM. XC (Genencor), ECOPULP.RTM. TX-200A (AB Enzymes), HSP 6000 Xylanase (DSM), DEPOL.TM. 333P (Biocatalysts Limit, Wales, UK), DEPOL.TM. 740L. (Biocatalysts Limit, Wales, UK), and DEPOL.TM. 762P (Biocatalysts Limit, Wales, UK).
[0288] Examples of xylanases useful in the methods of the present invention include, but are not limited to, Aspergillus aculeatus xylanase (GeneSeqP:AAR63790; WO 94/21785), Aspergillus fumigatus xylanases (WO 2006/078256), and Thielavia terrestris NRRL 8126 xylanases (WO 2009/079210).
[0289] Examples of beta-xylosidases useful in the methods of the present invention include, but are not limited to, Trichoderma reesei beta-xylosidase (UniProtKB/TrEMBL accession number Q92458), Talaromyces emersonii (SwissProt accession number Q8X212), and Neurospora crassa (SwissProt accession number Q7SOW4).
[0290] Examples of acetylxylan esterases useful in the methods of the present invention include, but are not limited to, Hypocrea jecorina acetylxylan esterase (WO 2005/001036), Neurospora crassa acetylxylan esterase (UniProt accession number q7s259), Thielavia terrestris NRRL 8126 acetylxylan esterase (WO 2009/042846), Chaetomium globosum acetylxylan esterase (Uniprot accession number Q2GWX4), Chaetomium gracile acetylxylan esterase (GeneSeqP accession number AAB82124), Phaeosphaeria nodorum acetylxylan esterase (Uniprot accession number Q0UHJ1), and Humicola insolens DSM 1800 acetylxylan esterase (WO 2009/073709).
[0291] Examples of ferulic acid esterases useful in the methods of the present invention include, but are not limited to, Humicola insolens DSM 1800 feruloyl esterase (WO 2009/076122), Neurospora crassa feruloyl esterase (UniProt accession number Q9HGR3), and Neosartorya fischeri feruloyl esterase (UniProt Accession number A1D9T4).
[0292] Examples of arabinofuranosidases useful in the methods of the present invention include, but are not limited to, Humicola insolens DSM 1800 arabinofuranosidase (WO 2009/073383) and Aspergillus niger arabinofuranosidase (GeneSeqP accession number AAR94170).
[0293] Examples of alpha-glucuronidases useful in the methods of the present invention include, but are not limited to, Aspergillus clavatus alpha-glucuronidase (UniProt accession number alcc12), Trichoderma reesei alpha-glucuronidase (Uniprot accession number Q99024), Talaromyces emersonii alpha-glucuronidase (UniProt accession number Q8X211), Aspergillus niger alpha-glucuronidase (Uniprot accession number Q96WX9), Aspergillus terreus alpha-glucuronidase (SwissProt accession number Q0CJP9), and Aspergillus fumigatus alpha-glucuronidase (SwissProt accession number Q4WW45).
[0294] The enzymes and proteins used in the methods of the present invention may be produced by fermentation of the above-noted microbial strains on a nutrient medium containing suitable carbon and nitrogen sources and inorganic salts, using procedures known in the art (see, e.g., Bennett, J. W. and LaSure, L. (eds.), More Gene Manipulations in Fungi, Academic Press, C A, 1991). Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). Temperature ranges and other conditions suitable for growth and enzyme production are known in the art (see, e.g., Bailey, J. E., and Ollis, D. F., Biochemical Engineering Fundamentals, McGraw-Hill Book Company, N Y, 1986).
[0295] The fermentation can be any method of cultivation of a cell resulting in the expression or isolation of an enzyme. Fermentation may, therefore, be understood as comprising shake flask cultivation, or small- or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the enzyme to be expressed or isolated. The resulting enzymes produced by the methods described above may be recovered from the fermentation medium and purified by conventional procedures.
Nucleic Acid Constructs
[0296] An isolated polynucleotide encoding a polypeptide, e.g., a polypeptide having cellulolytic enhancing activity, a cellulolytic enzyme, a hemicellulolytic enzyme, etc., may be manipulated in a variety of ways to provide for expression of the polypeptide by constructing a nucleic acid construct comprising an isolated polynucleotide encoding the polypeptide operably linked to one or more (e.g., several) control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences. Manipulation of the polynucleotide's sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotide sequences utilizing recombinant DNA methods are well known in the art.
[0297] The control sequence may be a promoter sequence, a polynucleotide that is recognized by a host cell for expression of a polynucleotide encoding a polypeptide. The promoter sequence contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any polynucleotide that shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
[0298] Examples of suitable promoters for directing the transcription of the nucleic acid constructs in the present invention in a bacterial host cell are the promoters obtained from the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus subtilis levansucrase gene (sacB), Bacillus subtilis xylA and xylB genes, E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80: 21-25). Further promoters are described in "Useful proteins from recombinant bacteria" in Gilbert et al., 1980, Scientific American, 242: 74-94; and in Sambrook et al., 1989, supra.
[0299] Examples of suitable promoters for directing the transcription of the nucleic acid constructs in the present invention in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus nidulans acetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Dania (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor miehei lipase, Rhizomucor miehei aspartic proteinase, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpi promoter (a modified promoter from a gene encoding a neutral alpha-amylase in Aspergilli in which the untranslated leader has been replaced by an untranslated leader from a gene encoding triose phosphate isomerase in Aspergilli; non-limiting examples include modified promoters from the gene encoding neutral alpha-amylase in Aspergillus niger in which the untranslated leader has been replaced by an untranslated leader from the gene encoding triose phosphate isomerase in Aspergillus nidulans or Aspergillus oryzae); and mutant, truncated, and hybrid promoters thereof.
[0300] In a yeast host, useful promoters are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP), Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8: 423-488.
[0301] The control sequence may also be a suitable transcription terminator sequence, which is recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3'-terminus of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell of choice may be used in the present invention.
[0302] Preferred terminators for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.
[0303] Preferred terminators for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, supra.
[0304] The control sequence may also be a suitable leader sequence, when transcribed is a nontranslated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5'-terminus of the polynucleotide encoding the polypeptide. Any leader sequence that is functional in the host cell of choice may be used.
[0305] Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.
[0306] Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).
[0307] The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3'-terminus of the polynucleotide and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell of choice may be used.
[0308] Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase.
[0309] Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.
[0310] The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a polypeptide and directs the polypeptide into the cell's secretory pathway. The 5'-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the polypeptide. Alternatively, the 5'-end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. The foreign signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, the foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence in order to enhance secretion of the polypeptide. However, any signal peptide coding sequence that directs the expressed polypeptide into the secretory pathway of a host cell of choice may be used.
[0311] Effective signal peptide coding sequences for bacterial host cells are the signal peptide coding sequences obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus alpha-amylase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.
[0312] Effective signal peptide coding sequences for filamentous fungal host cells are the signal peptide coding sequences obtained from the genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase.
[0313] Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al., 1992, supra.
[0314] The control sequence may also be a propeptide coding sequence that encodes a propeptide positioned at the N-terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.
[0315] Where both signal peptide and propeptide sequences are present at the N-terminus of a polypeptide, the propeptide sequence is positioned next to the N-terminus of a polypeptide and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence.
[0316] It may also be desirable to add regulatory sequences that allow the regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those that cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzae glucoamylase promoter may be used. Other examples of regulatory sequences are those that allow for gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals. In these cases, the polynucleotide encoding the polypeptide would be operably linked with the regulatory sequence.
Expression Vectors
[0317] The various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more (e.g., several) convenient restriction sites to allow for insertion or substitution of a polynucleotide encoding a polypeptide, e.g., a polypeptide having cellulolytic enhancing activity, a cellulolytic enzyme, a hemicellulolytic enzyme, etc., at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.
[0318] The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.
[0319] The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.
[0320] The vector preferably contains one or more (e.g., several) selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
[0321] Examples of bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers that confer antibiotic resistance such as ampicillin, chloramphenicol, kanamycin, or tetracycline resistance. Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Preferred for use in an Aspergillus cell are the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the bar gene of Streptomyces hygroscopicus.
[0322] The vector preferably contains an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.
[0323] For integration into the host cell genome, the vector may rely on the polynucleotide's sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides for directing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, which have a high degree of sequence identity to the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding polynucleotides. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.
[0324] For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term "origin of replication" or "plasmid replicator" means a polynucleotide that enables a plasmid or vector to replicate in vivo.
[0325] Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and pUB110, pE194, pTA1060, and pAM.beta.1 permitting replication in Bacillus.
[0326] Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.
[0327] Examples of origins of replication useful in a filamentous fungal cell are AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al., 1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of the AMA1 gene and construction of plasmids or vectors comprising the gene can be accomplished according to the methods disclosed in WO 00/24883.
[0328] More than one copy of a polynucleotide may be inserted into a host cell to increase production of a polypeptide. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.
[0329] The procedures used to ligate the elements described above to construct the recombinant expression vectors are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra).
Host Cells
[0330] Recombinant host cells comprising a polynucleotide encoding a polypeptide, e.g., a polypeptide having cellulolytic enhancing activity, a cellulolytic enzyme, a hemicellulolytic enzyme, etc., can be advantageously used in the recombinant production of the polypeptide. A construct or vector comprising such a polynucleotide is introduced into a host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source.
[0331] The host cell may be any cell useful in the recombinant production of a polypeptide, e.g., a prokaryote or a eukaryote.
[0332] The prokaryotic host cell may be any gram-positive or gram-negative bacterium. Gram-positive bacteria include, but not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.
[0333] The bacterial host cell may be any Bacillus cell including, but not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.
[0334] The bacterial host cell may also be any Streptococcus cell including, but not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.
[0335] The bacterial host cell may also be any Streptomyces cell including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.
[0336] The introduction of DNA into a Bacillus cell may, for instance, be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen. Genet. 168: 111-115), by using competent cells (see, e.g., Young and Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), by electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or by conjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169: 5271-5278). The introduction of DNA into an E. coli cell may, for instance, be effected by protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166: 557-580) or electroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res. 16: 6127-6145). The introduction of DNA into a Streptomyces cell may, for instance, be effected by protoplast transformation and electroporation (see, e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405), by conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171: 3583-3585), or by transduction (see, e.g., Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98: 6289-6294). The introduction of DNA into a Pseudomonas cell may, for instance, be effected by electroporation (see, e.g., Choi et al., 2006, J. Microbiol. Methods 64: 391-397) or by conjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). The introduction of DNA into a Streptococcus cell may, for instance, be effected by natural competence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), by protoplast transformation (see, e.g., Catt and Jollick, 1991, Microbios 68: 189-207), by electroporation (see, e.g., Buckley et al., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or by conjugation (see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any method known in the art for introducing DNA into a host cell can be used.
[0337] The host cell may also be a eukaryote, such as a mammalian, insect, plant, or fungal cell.
[0338] The host cell may be a fungal cell. "Fungi" as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al., 1995, supra, page 171) and all mitosporic fungi (Hawksworth et al., 1995, supra).
[0339] The fungal host cell may be a yeast cell. "Yeast" as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F. A., Passmore, S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980).
[0340] The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell.
[0341] The fungal host cell may be a filamentous fungal cell. "Filamentous fungi" include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). The filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.
[0342] The filamentous fungal host cell may be an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.
[0343] For example, the filamentous fungal host cell may be an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium suiphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.
[0344] Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus and Trichoderma host cells are described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81: 1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153: 163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.
Methods of Production
[0345] Methods for producing a polypeptide, e.g., a polypeptide having cellulolytic enhancing activity, a cellulolytic enzyme, a hemicellulolytic enzyme, etc., comprise (a) cultivating a cell, which in its wild-type form is capable of producing the polypeptide, under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.
[0346] Alternatively, methods for producing a polypeptide, e.g., a polypeptide having cellulolytic enhancing activity, a cellulolytic enzyme, a hemicellulolytic enzyme, etc., comprise (a) cultivating a recombinant host cell under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.
[0347] In the production methods, the cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods well known in the art. For example, the cell may be cultivated by shake flask cultivation, and small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.
[0348] The polypeptide may be detected using methods known in the art that are specific for the polypeptides. These detection methods may include use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the polypeptide. The polypeptides having cellulolytic enhancing activity are detected using the methods described herein.
[0349] The resulting broth may be used as is or the polypeptide may be recovered using methods known in the art. For example, the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.
[0350] The polypeptides may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure polypeptides.
[0351] In an alternative aspect, the polypeptide is not recovered, but rather a host cell expressing a polypeptide is used as a source of the polypeptide.
Methods for Processing Cellulosic Material
[0352] The compositions and methods of the present invention can be used to saccharify a cellulosic material to fermentable sugars and convert the fermentable sugars to many useful substances, e.g., fuel, potable ethanol, and/or fermentation products (e.g., acids, alcohols, ketones, gases, and the like). The production of a desired fermentation product from cellulosic material typically involves pretreatment, enzymatic hydrolysis (saccharification), and fermentation.
[0353] The present invention also relates to methods for degrading or converting a cellulosic material, comprising: treating the cellulosic material with an enzyme composition in the presence of a polypeptide having cellulolytic enhancing activity and a liquor. In one aspect, the method above further comprises recovering the degraded or converted cellulosic material. Soluble products of degradation or conversion of the cellulosic material can be separated from the insoluble cellulosic material using technology well known in the art such as, for example, centrifugation, filtration, and gravity settling.
[0354] The present invention also relates to methods for producing a fermentation product, comprising: (a) saccharifying a cellulosic material with an enzyme composition in the presence of a polypeptide having cellulolytic enhancing activity and a liquor; (b) fermenting the saccharified cellulosic material with one or more (e.g., several) fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation.
[0355] The present invention also relates to methods of fermenting a cellulosic material, comprising: fermenting the cellulosic material with one or more (e.g., several) fermenting microorganisms, wherein the cellulosic material is saccharified with an enzyme composition in the presence of a polypeptide having cellulolytic enhancing activity and a liquor. In one aspect, the fermenting of the cellulosic material produces a fermentation product. In another aspect, the method further comprises recovering the fermentation product from the fermentation.
[0356] In one aspect, the liquor is recovered following saccharification or fermentation and recycled back to a new saccharification reaction. Recycling of the liquor can be accomplished using processes conventional in the art.
[0357] The processing of cellulosic material according to the present invention can be accomplished using processes conventional in the art. Moreover, the methods of the present invention can be implemented using any conventional biomass processing apparatus configured to operate in accordance with the invention.
[0358] Hydrolysis (saccharification) and fermentation, separate or simultaneous, include, but are not limited to, separate hydrolysis and fermentation (SHF); simultaneous saccharification and fermentation (SSF); simultaneous saccharification and cofermentation (SSCF); hybrid hydrolysis and fermentation (HHF); separate hydrolysis and co-fermentation (SHCF); hybrid hydrolysis and co-fermentation (HHCF); and direct microbial conversion (DMC), also sometimes called consolidated bioprocessing (CBP). SHF uses separate process steps to first enzymatically hydrolyze cellulosic material to fermentable sugars, e.g., glucose, cellobiose, cellotriose, and pentose monomers, and then ferment the fermentable sugars to ethanol. In SSF, the enzymatic hydrolysis of cellulosic material and the fermentation of sugars to ethanol are combined in one step (Philippidis, G. P., 1996, Cellulose bioconversion technology, in Handbook on Bioethanol: Production and Utilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C., 179-212). SSCF involves the cofermentation of multiple sugars (Sheehan, J., and Himmel, M., 1999, Enzymes, energy and the environment: A strategic perspective on the U.S. Department of Energy's research and development activities for bioethanol, Biotechnol. Prog. 15: 817-827). HHF involves a separate hydrolysis step, and in addition a simultaneous saccharification and hydrolysis step, which can be carried out in the same reactor. The steps in an HHF process can be carried out at different temperatures, i.e., high temperature enzymatic saccharification followed by SSF at a lower temperature that the fermentation strain can tolerate. DMC combines all three processes (enzyme production, hydrolysis, and fermentation) in one or more (e.g., several) steps where the same organism is used to produce the enzymes for conversion of the cellulosic material to fermentable sugars and to convert the fermentable sugars into a final product (Lynd, L. R., Weimer, P. J., van Zyl, W. H., and Pretorius, I. S., 2002, Microbial cellulose utilization: Fundamentals and biotechnology, Microbiol. Mol. Biol. Reviews 66: 506-577). It is understood herein that any method known in the art comprising pretreatment, enzymatic hydrolysis (saccharification), fermentation, or a combination thereof, can be used in the practicing the methods of the present invention.
[0359] A conventional apparatus can include a fed-batch stirred reactor, a batch stirred reactor, a continuous flow stirred reactor with ultrafiltration, and/or a continuous plug-flow column reactor (Fernanda de Castilhos Corazza, Flavio Faria de Moraes, Gisella Maria Zanin and Ivo Neitzel, 2003, Optimal control in fed-batch reactor for the cellobiose hydrolysis, Acta Scientiarum. Technology 25: 33-38; Gusakov, A. V., and Sinitsyn, A. P., 1985, Kinetics of the enzymatic hydrolysis of cellulose: 1. A mathematical model for a batch reactor process, Enz. Microb. Technol. 7: 346-352), an attrition reactor (Ryu, S. K., and Lee, J. M., 1983, Bioconversion of waste cellulose by using an attrition bioreactor, Biotechnol. Bioeng. 25: 53-65), or a reactor with intensive stirring induced by an electromagnetic field (Gusakov, A. V., Sinitsyn, A. P., Davydkin, I. Y., Davydkin, V. Y., Protas, O. V., 1996, Enhancement of enzymatic cellulose hydrolysis using a novel type of bioreactor with intensive stirring induced by electromagnetic field, Appl. Biochem. Biotechnol. 56: 141-153). Additional reactor types include: fluidized bed, upflow blanket, immobilized, and extruder type reactors for hydrolysis and/or fermentation.
[0360] Pretreatment. In practicing the methods of the present invention, any pretreatment process known in the art can be used to disrupt plant cell wall components of cellulosic material (Chandra et al., 2007, Substrate pretreatment: The key to effective enzymatic hydrolysis of lignocellulosics? Adv. Biochem. Engin./Biotechnol. 108: 67-93; Galbe and Zacchi, 2007, Pretreatment of lignocellulosic materials for efficient bioethanol production, Adv. Biochem. Engin./Biotechnol. 108: 41-65; Hendriks and Zeeman, 2009, Pretreatments to enhance the digestibility of lignocellulosic biomass, Bioresource Technol. 100: 10-18; Mosier et al., 2005, Features of promising technologies for pretreatment of lignocellulosic biomass, Bioresource Technol. 96: 673-686; Taherzadeh and Karimi, 2008, Pretreatment of lignocellulosic wastes to improve ethanol and biogas production: A review, Int. J. of Mol. Sci. 9: 1621-1651; Yang and Wyman, 2008, Pretreatment: the key to unlocking low-cost cellulosic ethanol, Biofuels Bioproducts and Biorefining-Biofpr. 2: 26-40).
[0361] The cellulosic material can also be subjected to particle size reduction, pre-soaking, wetting, washing, or conditioning prior to pretreatment using methods known in the art.
[0362] Conventional pretreatments include, but are not limited to, steam pretreatment (with or without explosion), dilute acid pretreatment, hot water pretreatment, alkaline pretreatment, lime pretreatment, wet oxidation, wet explosion, ammonia fiber explosion, organosolv pretreatment, and biological pretreatment. Additional pretreatments include ammonia percolation, ultrasound, electroporation, microwave, supercritical CO.sub.2, supercritical H.sub.2O, ozone, and gamma irradiation pretreatments.
[0363] The cellulosic material can be pretreated before hydrolysis and/or fermentation. Pretreatment is preferably performed prior to the hydrolysis. Alternatively, the pretreatment can be carried out simultaneously with enzyme hydrolysis to release fermentable sugars, such as glucose, xylose, and/or cellobiose. In most cases the pretreatment step itself results in some conversion of biomass to fermentable sugars (even in absence of enzymes).
[0364] Steam Pretreatment: In steam pretreatment, cellulosic material is heated to disrupt the plant cell wall components, including lignin, hemicellulose, and cellulose to make the cellulose and other fractions, e.g., hemicellulose, accessible to enzymes. Cellulosic material is passed to or through a reaction vessel where steam is injected to increase the temperature to the required temperature and pressure and is retained therein for the desired reaction time. Steam pretreatment is preferably done at 140-230.degree. C., more preferably 160-200.degree. C., and most preferably 170-190.degree. C., where the optimal temperature range depends on any addition of a chemical catalyst. Residence time for the steam pretreatment is preferably 1-15 minutes, more preferably 3-12 minutes, and most preferably 4-10 minutes, where the optimal residence time depends on temperature range and any addition of a chemical catalyst. Steam pretreatment allows for relatively high solids loadings, so that cellulosic material is generally only moist during the pretreatment. The steam pretreatment is often combined with an explosive discharge of the material after the pretreatment, which is known as steam explosion, that is, rapid flashing to atmospheric pressure and turbulent flow of the material to increase the accessible surface area by fragmentation (Duff and Murray, 1996, Bioresource Technology 855: 1-33; Galbe and Zacchi, 2002, Appl. Microbiol. Biotechnol. 59: 618-628; U.S. Patent Application No. 20020164730). During steam pretreatment, hemicellulose acetyl groups are cleaved and the resulting acid autocatalyzes partial hydrolysis of the hemicellulose to monosaccharides and oligosaccharides. Lignin is removed to only a limited extent.
[0365] A catalyst such as H.sub.2SO.sub.4 or SO.sub.2 (typically 0.3 to 3% w/w) is often added prior to steam pretreatment, which decreases the time and temperature, increases the recovery, and improves enzymatic hydrolysis (Ballesteros et al., 2006, Appl. Biochem. Biotechnol. 129-132: 496-508; Varga et al., 2004, Appl. Biochem. Biotechnol. 113-116: 509-523; Sassner et al., 2006, Enzyme Microb. Technol. 39: 756-762).
[0366] Chemical Pretreatment: The term "chemical treatment" refers to any chemical pretreatment that promotes the separation and/or release of cellulose, hemicellulose, and/or lignin. Examples of suitable chemical pretreatment processes include, for example, dilute acid pretreatment, lime pretreatment, wet oxidation, ammonia fiber/freeze explosion (AFEX), ammonia percolation (APR), and organosolv pretreatments.
[0367] In dilute acid pretreatment, cellulosic material is mixed with dilute acid, typically H.sub.2SO.sub.4, and water to form a slurry, heated by steam to the desired temperature, and after a residence time flashed to atmospheric pressure. The dilute acid pretreatment can be performed with a number of reactor designs, e.g., plug-flow reactors, counter-current reactors, or continuous counter-current shrinking bed reactors (Duff and Murray, 1996, supra; Schell et al., 2004, Bioresource Technol. 91: 179-188; Lee et al., 1999, Adv. Biochem. Eng. Biotechnol. 65: 93-115).
[0368] Several methods of pretreatment under alkaline conditions can also be used. These alkaline pretreatments include, but are not limited to, lime pretreatment, wet oxidation, ammonia percolation (APR), and ammonia fiber/freeze explosion (AFEX).
[0369] Lime pretreatment is performed with calcium carbonate, sodium hydroxide, or ammonia at low temperatures of 85-150.degree. C. and residence times from 1 hour to several days (Wyman et al., 2005, Bioresource Technol. 96: 1959-1966; Mosier et al., 2005, Bioresource Technol. 96: 673-686). WO 2006/110891, WO 2006/11899, WO 2006/11900, and WO 2006/110901 disclose pretreatment methods using ammonia.
[0370] Wet oxidation is a thermal pretreatment performed typically at 180-200.degree. C. for 5-15 minutes with addition of an oxidative agent such as hydrogen peroxide or over-pressure of oxygen (Schmidt and Thomsen, 1998, Bioresource Technol. 64: 139-151; Palonen et al., 2004, Appl. Biochem. Biotechnol. 117: 1-17; Varga et al., 2004, Biotechnol. Bioeng. 88: 567-574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81: 1669-1677). The pretreatment is performed at preferably 1-40% dry matter, more preferably 2-30% dry matter, and most preferably 5-20% dry matter, and often the initial pH is increased by the addition of alkali such as sodium carbonate.
[0371] A modification of the wet oxidation pretreatment method, known as wet explosion (combination of wet oxidation and steam explosion), can handle dry matter up to 30%. In wet explosion, the oxidizing agent is introduced during pretreatment after a certain residence time. The pretreatment is then ended by flashing to atmospheric pressure (WO 2006/032282).
[0372] Ammonia fiber explosion (AFEX) involves treating cellulosic material with liquid or gaseous ammonia at moderate temperatures such as 90-100.degree. C. and high pressure such as 17-20 bar for 5-10 minutes, where the dry matter content can be as high as 60% (Gollapalli et al., 2002, Appl. Biochem. Biotechnol. 98: 23-35; Chundawat et al., 2007, Biotechnol. Bioeng. 96: 219-231; Alizadeh et al., 2005, Appl. Biochem. Biotechnol. 121: 1133-1141; Teymouri et al., 2005, Bioresource Technol. 96: 2014-2018). AFEX pretreatment results in the depolymerization of cellulose and partial hydrolysis of hemicellulose. Lignin-carbohydrate complexes are cleaved.
[0373] Organosolv pretreatment delignifies cellulosic material by extraction using aqueous ethanol (40-60% ethanol) at 160-200.degree. C. for 30-60 minutes (Pan et al., 2005, Biotechnol. Bioeng. 90: 473-481; Pan et al., 2006, Biotechnol. Bioeng. 94: 851-861; Kurabi et al., 2005, Appl. Biochem. Biotechnol. 121: 219-230). Sulphuric acid is usually added as a catalyst. In organosolv pretreatment, the majority of hemicellulose is removed.
[0374] Other examples of suitable pretreatment methods are described by Schell et al., 2003, Appl. Biochem. and Biotechnol. Vol. 105-108, p. 69-85, and Mosier et al., 2005, Bioresource Technology 96: 673-686, and U.S. Published Application 2002/0164730.
[0375] In one aspect, the chemical pretreatment is preferably carried out as an acid treatment, and more preferably as a continuous dilute and/or mild acid treatment. The acid is typically sulfuric acid, but other acids can also be used, such as acetic acid, citric acid, nitric acid, phosphoric acid, tartaric acid, succinic acid, hydrogen chloride, or mixtures thereof. Mild acid treatment is conducted in the pH range of preferably 1-5, more preferably 1-4, and most preferably 1-3. In one aspect, the acid concentration is in the range from preferably 0.01 to 20 wt % acid, more preferably 0.05 to 10 wt % acid, even more preferably 0.1 to 5 wt % acid, and most preferably 0.2 to 2.0 wt % acid. The acid is contacted with cellulosic material and held at a temperature in the range of preferably 160-220.degree. C., and more preferably 165-195.degree. C., for periods ranging from seconds to minutes to, e.g., 1 second to 60 minutes.
[0376] In another aspect, pretreatment is carried out as an ammonia fiber explosion step (AFEX pretreatment step).
[0377] In another aspect, pretreatment takes place in an aqueous slurry. In preferred aspects, cellulosic material is present during pretreatment in amounts preferably between 10-80 wt %, more preferably between 20-70 wt %, and most preferably between 30-60 wt %, such as around 50 wt %. The pretreated cellulosic material can be unwashed or washed using any method known in the art, e.g., washed with water.
[0378] Mechanical Pretreatment: The term "mechanical pretreatment" refers to various types of grinding or milling (e.g., dry milling, wet milling, or vibratory ball milling).
[0379] Physical Pretreatment: The term "physical pretreatment" refers to any pretreatment that promotes the separation and/or release of cellulose, hemicellulose, and/or lignin from cellulosic material. For example, physical pretreatment can involve irradiation (e.g., microwave irradiation), steaming/steam explosion, hydrothermolysis, and combinations thereof.
[0380] Physical pretreatment can involve high pressure and/or high temperature (steam explosion). In one aspect, high pressure means pressure in the range of preferably about 300 to about 600 psi, more preferably about 350 to about 550 psi, and most preferably about 400 to about 500 psi, such as around 450 psi. In another aspect, high temperature means temperatures in the range of about 100 to about 300.degree. C., preferably about 140 to about 235.degree. C. In a preferred aspect, mechanical pretreatment is performed in a batch-process, steam gun hydrolyzer system that uses high pressure and high temperature as defined above, e.g., a Sunds Hydrolyzer available from Sunds Defibrator AB, Sweden.
[0381] Combined Physical and Chemical Pretreatment: Cellulosic material can be pretreated both physically and chemically. For instance, the pretreatment step can involve dilute or mild acid treatment and high temperature and/or pressure treatment. The physical and chemical pretreatments can be carried out sequentially or simultaneously, as desired. A mechanical pretreatment can also be included.
[0382] Accordingly, in a preferred aspect, cellulosic material is subjected to mechanical, chemical, or physical pretreatment, or any combination thereof, to promote the separation and/or release of cellulose, hemicellulose, and/or lignin.
[0383] Biological Pretreatment: The term "biological pretreatment" refers to any biological pretreatment that promotes the separation and/or release of cellulose, hemicellulose, and/or lignin from cellulosic material. Biological pretreatment techniques can involve applying lignin-solubilizing microorganisms (see, for example, Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol: Production and Utilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C., 179-212; Ghosh and Singh, 1993, Physicochemical and biological treatments for enzymatic/microbial conversion of cellulosic biomass, Adv. Appl. Microbiol. 39: 295-333; McMillan, J. D., 1994, Pretreating lignocellulosic biomass: a review, in Enzymatic Conversion of Biomass for Fuels Production, Himmel, M. E., Baker, J. O., and Overend, R. P., eds., ACS Symposium Series 566, American Chemical Society, Washington, D.C., chapter 15; Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T., 1999, Ethanol production from renewable resources, in Advances in Biochemical Engineering/Biotechnology, Scheper, T., ed., Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Olsson and Hahn-Hagerdal, 1996, Fermentation of lignocellulosic hydrolysates for ethanol production, Enz. Microb. Tech. 18: 312-331; and Vallander and Eriksson, 1990, Production of ethanol from lignocellulosic materials: State of the art, Adv. Biochem. Eng./Biotechnol. 42: 63-95).
[0384] Saccharification. In the hydrolysis step, also known as saccharification, the cellulosic material, e.g., pretreated, is hydrolyzed to break down cellulose and alternatively also hemicellulose to fermentable sugars, such as glucose, cellobiose, xylose, xylulose, arabinose, mannose, galactose, and/or soluble oligosaccharides. The hydrolysis is performed enzymatically by an enzyme composition in the presence of a polypeptide having cellulolytic enhancing activity and a liquor. The enzyme and protein components of the compositions can be added sequentially.
[0385] Enzymatic hydrolysis is preferably carried out in a suitable aqueous environment under conditions that can be readily determined by one skilled in the art. In a preferred aspect, hydrolysis is performed under conditions suitable for the activity of the enzyme(s), i.e., optimal for the enzyme(s). The hydrolysis can be carried out as a fed batch or continuous process where the pretreated cellulosic material (substrate) is fed gradually to, for example, an enzyme containing hydrolysis solution.
[0386] The saccharification is generally performed in stirred-tank reactors or fermentors under controlled pH, temperature, and mixing conditions. Suitable process time, temperature and pH conditions can readily be determined by one skilled in the art. For example, the saccharification can last up to 200 hours, but is typically performed for preferably about 12 to about 96 hours, more preferably about 16 to about 72 hours, and most preferably about 24 to about 48 hours. The temperature is in the range of preferably about 25.degree. C. to about 70.degree. C., more preferably about 30.degree. C. to about 65.degree. C., and more preferably about 40.degree. C. to 60.degree. C., in particular about 50.degree. C. The pH is in the range of preferably about 3 to about 8, more preferably about 3.5 to about 7, and most preferably about 4 to about 6, in particular about pH 5. The dry solids content is in the range of preferably about 5 to about 50 wt %, more preferably about 10 to about 40 wt %, and most preferably about 20 to about 30 wt %.
[0387] The optimum amounts of the enzymes and polypeptides having cellulolytic enhancing activity depend on several factors including, but not limited to, the mixture of component cellulolytic enzymes, the cellulosic substrate, the concentration of cellulosic substrate, the pretreatment(s) of the cellulosic substrate, temperature, time, pH, and inclusion of fermenting organism (e.g., yeast for Simultaneous Saccharification and Fermentation).
[0388] In one aspect, an effective amount of cellulolytic or hemicellulolytic enzyme protein to cellulosic material is about 0.5 to about 50 mg, preferably at about 0.5 to about 40 mg, more preferably at about 0.5 to about 25 mg, more preferably at about 0.75 to about 20 mg, more preferably at about 0.75 to about 15 mg, even more preferably at about 0.5 to about 10 mg, and most preferably at about 2.5 to about 10 mg per g of cellulosic material.
[0389] In another aspect, an effective amount of a polypeptide having cellulolytic enhancing activity to cellulosic material is about 0.01 to about 50.0 mg, preferably about 0.01 to about 40 mg, more preferably about 0.01 to about 30 mg, more preferably about 0.01 to about 20 mg, more preferably about 0.01 to about 10 mg, more preferably about 0.01 to about 5 mg, more preferably at about 0.025 to about 1.5 mg, more preferably at about 0.05 to about 1.25 mg, more preferably at about 0.075 to about 1.25 mg, more preferably at about 0.1 to about 1.25 mg, even more preferably at about 0.15 to about 1.25 mg, and most preferably at about 0.25 to about 1.0 mg per g of cellulosic material.
[0390] In another aspect, an effective amount of a polypeptide having cellulolytic enhancing activity to cellulolytic enzyme protein is about 0.005 to about 1.0 g, preferably at about 0.01 to about 1.0 g, more preferably at about 0.15 to about 0.75 g, more preferably at about 0.15 to about 0.5 g, more preferably at about 0.1 to about 0.5 g, even more preferably at about 0.1 to about 0.5 g, and most preferably at about 0.05 to about 0.2 g per g of cellulolytic enzyme protein.
[0391] Fermentation. The fermentable sugars obtained from the hydrolyzed cellulosic material can be fermented by one or more (e.g., several) fermenting microorganisms capable of fermenting the sugars directly or indirectly into a desired fermentation product. "Fermentation" or "fermentation process" refers to any fermentation process or any process comprising a fermentation step. Fermentation processes also include fermentation processes used in the consumable alcohol industry (e.g., beer and wine), dairy industry (e.g., fermented dairy products), leather industry, and tobacco industry. The fermentation conditions depend on the desired fermentation product and fermenting organism and can easily be determined by one skilled in the art.
[0392] In the fermentation step, sugars, released from cellulosic material as a result of the pretreatment and enzymatic hydrolysis steps, are fermented to a product, e.g., ethanol, by a fermenting organism, such as yeast. Hydrolysis (saccharification) and fermentation can be separate or simultaneous, as described herein.
[0393] Any suitable hydrolyzed cellulosic material can be used in the fermentation step in practicing the present invention. The material is generally selected based on the desired fermentation product, i.e., the substance to be obtained from the fermentation, and the process employed, as is well known in the art.
[0394] The term "fermentation medium" is understood herein to refer to a medium before the fermenting microorganism(s) is(are) added, such as, a medium resulting from a saccharification process, as well as a medium used in a simultaneous saccharification and fermentation process (SSF).
[0395] "Fermenting microorganism" refers to any microorganism, including bacterial and fungal organisms, suitable for use in a desired fermentation process to produce a fermentation product. The fermenting organism can be C.sub.6 and/or C.sub.5 fermenting organisms, or a combination thereof. Both C.sub.6 and C.sub.5 fermenting organisms are well known in the art. Suitable fermenting microorganisms are able to ferment, i.e., convert, sugars, such as glucose, xylose, xylulose, arabinose, maltose, mannose, galactose, or oligosaccharides, directly or indirectly into the desired fermentation product.
[0396] Examples of bacterial and fungal fermenting organisms producing ethanol are described by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69: 627-642.
[0397] Examples of fermenting microorganisms that can ferment C.sub.6 sugars include bacterial and fungal organisms, such as yeast. Preferred yeast includes strains of the Saccharomyces spp., preferably Saccharomyces cerevisiae.
[0398] Examples of fermenting organisms that can ferment C.sub.5 sugars include bacterial and fungal organisms, such as some yeast. Preferred C.sub.5 fermenting yeast include strains of Pichia, preferably Pichia stipitis, such as Pichia stipitis CBS 5773; strains of Candida, preferably Candida boidinii, Candida brassicae, Candida sheatae, Candida diddensii, Candida pseudotropicalis, or Candida utilis.
[0399] Other fermenting organisms include strains of Zymomonas, such as Zymomonas mobilis; Hansenula, such as Hansenula anomala; Kluyveromyces, such as K. fragilis; Schizosaccharomyces, such as S. pombe; E. coli, especially E. coli strains that have been genetically modified to improve the yield of ethanol; Clostridium, such as Clostridium acetobutylicum, Chlostridium thermocellum, and Chlostridium phytofermentans; Geobacillus sp.; Thermoanaerobacter, such as Thermoanaerobacter saccharolyticum; and Bacillus, such as Bacillus coagulans.
[0400] In a preferred aspect, the yeast is a Saccharomyces spp. In a more preferred aspect, the yeast is Saccharomyces cerevisiae. In another more preferred aspect, the yeast is Saccharomyces distaticus. In another more preferred aspect, the yeast is Saccharomyces uvarum. In another preferred aspect, the yeast is a Kluyveromyces. In another more preferred aspect, the yeast is Kluyveromyces marxianus. In another more preferred aspect, the yeast is Kluyveromyces fragilis. In another preferred aspect, the yeast is a Candida. In another more preferred aspect, the yeast is Candida boidinii. In another more preferred aspect, the yeast is Candida brassicae. In another more preferred aspect, the yeast is Candida diddensii. In another more preferred aspect, the yeast is Candida pseudotropicalis. In another more preferred aspect, the yeast is Candida utilis. In another preferred aspect, the yeast is a Clavispora. In another more preferred aspect, the yeast is Clavispora lusitaniae. In another more preferred aspect, the yeast is Clavispora opuntiae. In another preferred aspect, the yeast is a Pachysolen. In another more preferred aspect, the yeast is Pachysolen tannophilus. In another preferred aspect, the yeast is a Pichia. In another more preferred aspect, the yeast is a Pichia stipitis. In another preferred aspect, the yeast is a Bretannomyces. In another more preferred aspect, the yeast is Bretannomyces clausenii (Philippidis, G. P., 1996, Cellulose bioconversion technology, in Handbook on Bioethanol: Production and Utilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C., 179-212).
[0401] Bacteria that can efficiently ferment hexose and pentose to ethanol include, for example, Zymomonas mobilis, Clostridium acetobutylicum, Clostridium thermocellum, Chlostridium phytofermentans, Geobacillus sp., Thermoanaerobacter saccharolyticum, and Bacillus coagulans (Philippidis, 1996, supra).
[0402] In a preferred aspect, the bacterium is a Zymomonas. In a more preferred aspect, the bacterium is Zymomonas mobilis. In another preferred aspect, the bacterium is a Clostridium. In another more preferred aspect, the bacterium is Clostridium thermocellum.
[0403] Commercially available yeast suitable for ethanol production includes, e.g., ETHANOL RED.TM. yeast (available from Fermentis/Lesaffre, USA), FALI.TM. (available from Fleischmann's Yeast, USA), SUPERSTART.TM. and THERMOSACC.TM. fresh yeast (available from Ethanol Technology, WI, USA), BIOFERM.TM. AFT and XR (available from NABC--North American Bioproducts Corporation, GA, USA), GERT STRAND.TM. (available from Gert Strand AB, Sweden), and FERMIOL.TM. (available from DSM Specialties).
[0404] In a preferred aspect, the fermenting microorganism has been genetically modified to provide the ability to ferment pentose sugars, such as xylose utilizing, arabinose utilizing, and xylose and arabinose co-utilizing microorganisms.
[0405] The cloning of heterologous genes into various fermenting microorganisms has led to the construction of organisms capable of converting hexoses and pentoses to ethanol (cofermentation) (Chen and Ho, 1993, Cloning and improving the expression of Pichia stipitis xylose reductase gene in Saccharomyces cerevisiae, Appl. Biochem. Biotechnol. 39-40: 135-147; Ho et al., 1998, Genetically engineered Saccharomyces yeast capable of effectively cofermenting glucose and xylose, Appl. Environ. Microbiol. 64: 1852-1859; Kotter and Ciriacy, 1993, Xylose fermentation by Saccharomyces cerevisiae, Appl. Microbiol. Biotechnol. 38: 776-783; Walfridsson et al., 1995, Xylose-metabolizing Saccharomyces cerevisiae strains overexpressing the TKL1 and TAL1 genes encoding the pentose phosphate pathway enzymes transketolase and transaldolase, Appl. Environ. Microbiol. 61: 4184-4190; Kuyper et al., 2004, Minimal metabolic engineering of Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: a proof of principle, FEMS Yeast Research 4: 655-664; Beall et al., 1991, Parametric studies of ethanol production from xylose and other sugars by recombinant Escherichia coli, Biotech. Bioeng. 38: 296-303; Ingram et al., 1998, Metabolic engineering of bacteria for ethanol production, Biotechnol. Bioeng. 58: 204-214; Zhang et al., 1995, Metabolic engineering of a pentose metabolism pathway in ethanologenic Zymomonas mobilis, Science 267: 240-243; Deanda et al., 1996, Development of an arabinose-fermenting Zymomonas mobilis strain by metabolic pathway engineering, Appl. Environ. Microbiol. 62: 4465-4470; WO 2003/062430, xylose isomerase).
[0406] In a preferred aspect, the genetically modified fermenting microorganism is Saccharomyces cerevisiae. In another preferred aspect, the genetically modified fermenting microorganism is Zymomonas mobilis. In another preferred aspect, the genetically modified fermenting microorganism is Escherichia coli. In another preferred aspect, the genetically modified fermenting microorganism is Klebsiella oxytoca. In another preferred aspect, the genetically modified fermenting microorganism is Kluyveromyces sp.
[0407] It is well known in the art that the organisms described above can also be used to produce other substances, as described herein.
[0408] The fermenting microorganism is typically added to the degraded lignocellulose or hydrolysate and the fermentation is performed for about 8 to about 96 hours, such as about 24 to about 60 hours. The temperature is typically between about 26.degree. C. to about 60.degree. C., in particular about 32.degree. C. or 50.degree. C., and at about pH 3 to about pH 8, such as around pH 4-5, 6, or 7.
[0409] In a preferred aspect, the yeast and/or another microorganism is applied to the degraded cellulosic material and the fermentation is performed for about 12 to about 96 hours, such as typically 24-60 hours. In a preferred aspect, the temperature is preferably between about 20.degree. C. to about 60.degree. C., more preferably about 25.degree. C. to about 50.degree. C., and most preferably about 32.degree. C. to about 50.degree. C., in particular about 32.degree. C. or 50.degree. C., and the pH is generally from about pH 3 to about pH 7, preferably around pH 4-7. However, some fermenting organisms, e.g., bacteria, have higher fermentation temperature optima. Yeast or another microorganism is preferably applied in amounts of approximately 10.sup.5 to 10.sup.12, preferably from approximately 10.sup.7 to 10.sup.10, especially approximately 2.times.10.sup.8 viable cell count per ml of fermentation broth. Further guidance in respect of using yeast for fermentation can be found in, e.g., "The Alcohol Textbook" (Editors K. Jacques, T. P. Lyons and D. R. Kelsall, Nottingham University Press, United Kingdom 1999), which is hereby incorporated by reference.
[0410] For ethanol production, following the fermentation the fermented slurry is distilled to extract the ethanol. The ethanol obtained according to the methods of the invention can be used as, e.g., fuel ethanol, drinking ethanol, i.e., potable neutral spirits, or industrial ethanol.
[0411] A fermentation stimulator can be used in combination with any of the processes described herein to further improve the fermentation process, and in particular, the performance of the fermenting microorganism, such as, rate enhancement and ethanol yield. A "fermentation stimulator" refers to stimulators for growth of the fermenting microorganisms, in particular, yeast. Preferred fermentation stimulators for growth include vitamins and minerals. Examples of vitamins include multivitamins, biotin, pantothenate, nicotinic acid, meso-inositol, thiamine, pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and Vitamins A, B, C, D, and E. See, for example, Alfenore et al., Improving ethanol production and viability of Saccharomyces cerevisiae by a vitamin feeding strategy during fed-batch process, Springer-Verlag (2002), which is hereby incorporated by reference. Examples of minerals include minerals and mineral salts that can supply nutrients comprising P, K, Mg, S, Ca, Fe, Zn, Mn, and Cu.
[0412] Fermentation products: A fermentation product can be any substance derived from the fermentation. The fermentation product can be, without limitation, an alcohol (e.g., arabinitol, n-butanol, isobutanol, ethanol, glycerol, methanol, ethylene glycol, 1,3-propanediol [propylene glycol], butanediol, glycerin, sorbitol, and xylitol); an alkane (e.g., pentane, hexane, heptane, octane, nonane, decane, undecane, and dodecane), a cycloalkane (e.g., cyclopentane, cyclohexane, cycloheptane, and cyclooctane), an alkene (e.g. pentene, hexene, heptene, and octene); an amino acid (e.g., aspartic acid, glutamic acid, glycine, lysine, serine, and threonine); a gas (e.g., methane, hydrogen (H.sub.2), carbon dioxide (CO.sub.2), and carbon monoxide (CO)); isoprene; a ketone (e.g., acetone); an organic acid (e.g., acetic acid, acetonic acid, adipic acid, ascorbic acid, citric acid, 2,5-diketo-D-gluconic acid, formic acid, fumaric acid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid, oxaloacetic acid, propionic acid, succinic acid, and xylonic acid); and polyketide. The fermentation product can also be protein as a high value product.
[0413] In a preferred aspect, the fermentation product is an alcohol. It will be understood that the term "alcohol" encompasses a substance that contains one or more hydroxyl moieties. In a more preferred aspect, the alcohol is n-butanol. In another more preferred aspect, the alcohol is isobutanol. In another more preferred aspect, the alcohol is ethanol. In another more preferred aspect, the alcohol is methanol. In another more preferred aspect, the alcohol is arabinitol. In another more preferred aspect, the alcohol is butanediol. In another more preferred aspect, the alcohol is ethylene glycol. In another more preferred aspect, the alcohol is glycerin. In another more preferred aspect, the alcohol is glycerol. In another more preferred aspect, the alcohol is 1,3-propanediol. In another more preferred aspect, the alcohol is sorbitol. In another more preferred aspect, the alcohol is xylitol. See, for example, Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T., 1999, Ethanol production from renewable resources, in Advances in Biochemical Engineering/Biotechnology, Scheper, T., ed., Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Silveira, M. M., and Jonas, R., 2002, The biotechnological production of sorbitol, Appl. Microbiol. Biotechnol. 59: 400-408; Nigam, P., and Singh, D., 1995, Processes for fermentative production of xylitol--a sugar substitute, Process Biochemistry 30 (2): 117-124; Ezeji, T. C., Qureshi, N. and Blaschek, H. P., 2003, Production of acetone, butanol and ethanol by Clostridium beijerinckii BA101 and in situ recovery by gas stripping, World Journal of Microbiology and Biotechnology 19 (6): 595-603.
[0414] In another preferred aspect, the fermentation product is an alkane. The alkane can be an unbranched or a branched alkane. In another more preferred aspect, the alkane is pentane. In another more preferred aspect, the alkane is hexane. In another more preferred aspect, the alkane is heptane. In another more preferred aspect, the alkane is octane. In another more preferred aspect, the alkane is nonane. In another more preferred aspect, the alkane is decane. In another more preferred aspect, the alkane is undecane. In another more preferred aspect, the alkane is dodecane.
[0415] In another preferred aspect, the fermentation product is a cycloalkane. In another more preferred aspect, the cycloalkane is cyclopentane. In another more preferred aspect, the cycloalkane is cyclohexane. In another more preferred aspect, the cycloalkane is cycloheptane. In another more preferred aspect, the cycloalkane is cyclooctane.
[0416] In another preferred aspect, the fermentation product is an alkene. The alkene can be an unbranched or a branched alkene. In another more preferred aspect, the alkene is pentene. In another more preferred aspect, the alkene is hexene. In another more preferred aspect, the alkene is heptene. In another more preferred aspect, the alkene is octene.
[0417] In another preferred aspect, the fermentation product is an amino acid. In another more preferred aspect, the organic acid is aspartic acid. In another more preferred aspect, the amino acid is glutamic acid. In another more preferred aspect, the amino acid is glycine. In another more preferred aspect, the amino acid is lysine. In another more preferred aspect, the amino acid is serine. In another more preferred aspect, the amino acid is threonine. See, for example, Richard, A., and Margaritis, A., 2004, Empirical modeling of batch fermentation kinetics for poly(glutamic acid) production and other microbial biopolymers, Biotechnology and Bioengineering 87 (4): 501-515.
[0418] In another preferred aspect, the fermentation product is a gas. In another more preferred aspect, the gas is methane. In another more preferred aspect, the gas is H.sub.2. In another more preferred aspect, the gas is CO.sub.2. In another more preferred aspect, the gas is CO. See, for example, Kataoka, N., A. Miya, and K. Kiriyama, 1997, Studies on hydrogen production by continuous culture system of hydrogen-producing anaerobic bacteria, Water Science and Technology 36 (6-7): 41-47; and Gunaseelan V. N. in Biomass and Bioenergy, Vol. 13 (1-2), pp. 83-114, 1997, Anaerobic digestion of biomass for methane production: A review.
[0419] In another preferred aspect, the fermentation product is isoprene.
[0420] In another preferred aspect, the fermentation product is a ketone. It will be understood that the term "ketone" encompasses a substance that contains one or more ketone moieties. In another more preferred aspect, the ketone is acetone. See, for example, Qureshi and Blaschek, 2003, supra.
[0421] In another preferred aspect, the fermentation product is an organic acid. In another more preferred aspect, the organic acid is acetic acid. In another more preferred aspect, the organic acid is acetonic acid. In another more preferred aspect, the organic acid is adipic acid. In another more preferred aspect, the organic acid is ascorbic acid. In another more preferred aspect, the organic acid is citric acid. In another more preferred aspect, the organic acid is 2,5-diketo-D-gluconic acid. In another more preferred aspect, the organic acid is formic acid. In another more preferred aspect, the organic acid is fumaric acid. In another more preferred aspect, the organic acid is glucaric acid. In another more preferred aspect, the organic acid is gluconic acid. In another more preferred aspect, the organic acid is glucuronic acid. In another more preferred aspect, the organic acid is glutaric acid. In another preferred aspect, the organic acid is 3-hydroxypropionic acid. In another more preferred aspect, the organic acid is itaconic acid. In another more preferred aspect, the organic acid is lactic acid. In another more preferred aspect, the organic acid is malic acid. In another more preferred aspect, the organic acid is malonic acid. In another more preferred aspect, the organic acid is oxalic acid. In another more preferred aspect, the organic acid is propionic acid. In another more preferred aspect, the organic acid is succinic acid. In another more preferred aspect, the organic acid is xylonic acid. See, for example, Chen, R., and Lee, Y. Y., 1997, Membrane-mediated extractive fermentation for lactic acid production from cellulosic biomass, Appl. Biochem. Biotechnol. 63-65: 435-448.
[0422] In another preferred aspect, the fermentation product is polyketide.
[0423] Recovery. The fermentation product(s) can be optionally recovered from the fermentation medium using any method known in the art including, but not limited to, chromatography, electrophoretic procedures, differential solubility, distillation, or extraction. For example, alcohol is separated from the fermented cellulosic material and purified by conventional methods of distillation. Ethanol with a purity of up to about 96 vol. % can be obtained, which can be used as, for example, fuel ethanol, drinking ethanol, i.e., potable neutral spirits, or industrial ethanol.
[0424] The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.
EXAMPLES
Media
[0425] 2X YT plates were composed of 16 g of tryptone, 10 g of yeast extract, 5 g of NaCl, 15 g of Noble agar, and deionized water to 1 liter.
[0426] PDA plates were composed of 39 grams of potato dextrose agar and deionized water to 1 liter.
[0427] MDU2BP medium was composed of 45 g of maltose, 1 g of MgSO.sub.4.7H.sub.2O, 1 g of NaCl, 2 g of K.sub.2SO.sub.4, 12 g of KH.sub.2PO.sub.4, 7 g of yeast extract, 2 g of urea, 0.5 ml of AMG trace metals solution, and deionized water to 1 liter; pH 5.0.
[0428] AMG trace metals solution was composed of 14.3 g of ZnSO.sub.4.7H.sub.2O, 2.5 g of CuSO.sub.4.5H.sub.2O, 0.5 g of NiCl.sub.2.6H.sub.2O, 13.8 g of FeSO.sub.4.7H.sub.2O, 8.5 g of MnSO.sub.4.7H.sub.2O, 3 g of citric acid, and deionized water to 1 liter.
Example 1: Pretreatment of Corn Stover
[0429] Corn stover was pretreated at the U.S. Department of Energy National Renewable Energy Laboratory (NREL) using 1.4% (w/v) sulfuric acid for 8 minutes at 165.degree. C. and 107 psi. The water-insoluble solids in the pretreated corn stover contained 57.5% cellulose, 4.6% hemicelluloses, and 28.4% lignin. Cellulose and hemicellulose were determined by a two-stage sulfuric acid hydrolysis with subsequent analysis of sugars by high performance liquid chromatography using NREL Standard Analytical Procedure #002. Lignin was determined gravimetrically after hydrolyzing the cellulose and hemicellulose fractions with sulfuric acid using NREL Standard Analytical Procedure #003.
[0430] The pretreated corn stover was adjusted to pH 5.0 by repeated addition of 10 N NaOH in aliquots of a few milliliters, followed by thorough mixing and incubation at room temperature for approximately 1 hour. The pH was confirmed after overnight incubation at 4.degree. C., and the pH-adjusted corn stover was autoclaved for 20 minutes at approximately 120.degree. C., and then stored at 4.degree. C. to minimize the risk of microbial contamination. The dry weight of the pretreated corn stover was 33% TS (total solids), which was confirmed before each use.
[0431] The pretreated corn stover was milled prior to use. Milled pretreated corn stover (initial dry weight 32.35% TS) was prepared by milling in a Cosmos ICMG 40 wet multi-utility grinder (EssEmm Corporation, Tamil Nadu, India). Milled pretreated corn stover was also, in some cases, subsequently washed repeatedly with deionized water followed by decanting off the supernatant fraction. The dry weight of the milled, water-washed pretreated corn stover was 7.114% TS.
[0432] Alternatively, milled, water-washed pretreated corn stover was washed extensively with water at 50.degree. C. Approximately 600 ml of water washed pretreated corn stover was diluted with approximately 500 ml of distilled, deionized water and incubated at 50.degree. C. with shaking for 7 days. Three to four times per day, the diluted pretreated corn stover was permitted to settle, and the supernatant water was decanted and replaced with 500 ml of fresh deionized water. The dry weight of the milled, hot-water washed pretreated corn stover was 6.74% TS.
Example 2: Separation of Pretreated Corn Stover Liquor
[0433] Acid pretreated corn stover liquor was obtained by vacuum-filtration of the pH-adjusted NREL pretreated corn stover (Example 1) using Whatman #3 filter paper in a Buchner funnel, or through a 0.22 .mu.m STERICUP.RTM. sterile vacuum-filter (Millipore, Bedford, Mass., USA). For the Whatman-filtered liquor, the liquor was additionally sterile-filtered using a 0.22 .mu.m STERICUP.RTM. sterile vacuum-filter to minimize the risk of microbial contamination.
[0434] In later experiments, pretreated corn stover liquor was obtained by squeezing acid-pretreated corn stover in the following manner. Approximately 20 kg of dilute acid pretreated corn stover was loaded into the cotton sheet lining of a SRL Water Press Model BP40-S/S (Zambelli Enotech, Camisano Vicentino, Italy). Municipal water pressure (approximately 35 psi) was applied for 20 minutes, and the resulting liquid pressed out was captured as liquor. This acidic liquor was stored at 4.degree. C., and was subsequently pH-adjusted to 5.0 by addition of 10 N NaOH, and sterile-filtered using a 0.22 .mu.m STERICUP.RTM. sterile vacuum-filter.
Example 3: Hydrolysis of Cellulose and Assay for GH61 Polypeptide Enhancement Thereof
[0435] The hydrolysis of pretreated corn stover was conducted using 2.2 ml, 96-deep well plates (Axygen, Union City, Calif., USA) containing a total reaction mass of 1 g. The hydrolysis was performed with 5% total solids of either washed milled pretreated corn stover, unwashed pretreated corn stover, equivalent to 28.75 or 14.75 mg of cellulose per ml, respectively, or with a concentration of microcrystalline cellulose (AVICEL.RTM., EM Science, Gibbstown, N.J., USA) equivalent to 28.75 mg of cellulose per ml. Later hydrolysis reactions were performed with milled, washed or milled, unwashed pretreated corn stover with a cellulose content of 59%, equivalent to 29.5 mg of cellulose per ml, or an equivalent concentration of microcrystalline cellulose (AVICEL.RTM., Sigma-Aldrich, St. Louis, Mo., USA). Hydrolysis reactions were performed in 50 mM sodium acetate pH 5.0 containing 1 mM manganese sulfate using a Trichoderma reesei cellulase preparation (CELLUCLAST.RTM. supplemented with Aspergillus oryzae beta-glucosidase available from Novozymes A/S, Bagsvaerd, Denmark; the cellulase composition is designated herein in the Examples as "Trichoderma reesei cellulase composition") at 4 mg per g of cellulose. Thermoascus aurantiacus GH61A or Thelavia terrestris GH61E polypeptide having cellulolytic enhancing activity was added at concentrations between 0 and 50% (w/w) of total protein. Pretreated corn stover liquors, enzymatically- or chemically-treated corn stover liquors, synthetic mixtures containing mono- and disaccharides at equivalent concentrations to corn stover liquors, pretreated biomass component liquors, and post-fermentation residual liquors were added between 0 and 20% (v/v) as indicated. Plates were sealed using an ALPS-300.TM. plate heat sealer (Abgene, Epsom, United Kingdom) and incubated at 50.degree. C. for 0-168 hours with mixing at 150 rpm. All experiments were performed in duplicate or triplicate. Other hydrolysis reactions were performed similarly, with the following differences: plates were sealed using an ALPS-3000.TM. plate heat sealer (Abgene, Epsom, United Kingdom), and incubated at 50.degree. C. with vigorous initial mixing at each sampling time, but mixing was not continuous.
[0436] At various time points between 24 and 168 hours of incubation, 100 .mu.l aliquots were removed and the extent of hydrolysis was assayed by high-performance liquid chromatography (HPLC) using the protocol described below.
[0437] For HPLC analysis, samples were filtered using a 0.45 .mu.m MULTISCREEN.RTM. 96-well filter plate (Millipore, Bedford, Mass., USA) and filtrates were analyzed for sugar content as described below. The sugar concentrations of samples diluted in 0.005 M H.sub.2SO.sub.4 were measured using a 4.6.times.250 mm AMINEX.RTM. HPX-87H column (Bio-Rad Laboratories, Inc., Hercules, Calif., USA) by elution with 0.5% w/w benzoic acid-5 mM H.sub.2SO.sub.4 at a flow rate of 0.6 ml per minute at 65.degree. C. for 11 minutes, and quantification by integration of glucose and cellobiose signals from refractive index detection (CHEMSTATION.RTM., AGILENT.RTM. 1100 HPLC, Agilent Technologies, Santa Clara, Calif., USA) calibrated by pure sugar samples. The resultant equivalents were used to calculate the fraction or percentage of cellulose conversion for each reaction. The extent of each hydrolysis was determined as the fraction of total cellulose converted to cellobiose+glucose, and was not corrected for soluble sugars present in pretreated corn stover liquor, or was corrected for soluble sugars present in liquor as indicated.
[0438] All HPLC data processing was performed using KALEIDAGRAPH.RTM. software (Synergy software, Reading, Pa., USA) or MICROSOFT EXCEL.RTM. (Microsoft, Seattle, Wash., USA). Measured sugar concentrations were adjusted for the appropriate dilution factor. Glucose and cellobiose were chromatographically separated and integrated and their respective concentrations determined independently. To calculate fractional conversion the glucose and cellobiose values were combined. Fractional hydrolysis is reported as the ratio of the mass corrected concentrations of glucose and cellobiose to the initial concentration of cellulose as given by Equation 1. Triplicate data points were averaged and standard deviation was calculated.
fractional .times. .times. hydrolysis = ( ( [ cellobiose ] .times. ( mg / ml ) .times. 1.053 ) + ( [ glucose ] .times. ( mg / ml ) ) / 1.111 ) [ cellulose ] .times. ( mg / ml ) ( Equation .times. .times. 1 ) ##EQU00001##
[0439] The concentration-dependence of GH61 polypeptide-dependent enhancement of cellulose hydrolysis by the T. reesei cellulase composition was determined by titration of the GH61 polypeptide between 0 and 50% (w/w) total protein added to a constant T. reesei cellulase concentration of 4 mg per g cellulose, plotting fractional hydrolysis against GH61 polypeptide concentration, and fitting using a modified saturation-binding model as given by Equation 2.
fractional .times. .times. hydrolysis = .DELTA. .times. fractional .times. .times. hydrolysis .times. [ G .times. H .times. 6 .times. 1 ] + fractional .times. .times. hydrolysis ( 0 ) .function. ( K 1 2 .times. apparent + [ GH .times. .times. 61 ] ) K 1 2 .times. apparent + [ GH .times. .times. 61 ] ( Equation .times. .times. 2 ) ##EQU00002##
In Equation 2 "fractional hydrolysis.sub.(0)" was the hydrolysis in the absence of a GH61 polypeptide; .DELTA.fractional hydrolysis was the total GH61 polypeptide-dependent enhancement; i.e., the difference between the fractional hydrolysis at apparent "saturating" GH61 polypeptide concentration and the fractional hydrolysis in the absence of the GH61 polypeptide; and K.sub.1/2 apparent was the GH61 polypeptide concentration necessary to observe a half-maximal enhancement of hydrolysis.
Example 4: Preparation of Thermoascus aurantiacus GH61A Polypeptide Having Cellulolytic Enhancing Activity
[0440] Thermoascus aurantiacus GH61A polypeptide having cellulolytic enhancing activity (SEQ ID NO: 13 [DNA sequence] and SEQ ID NO: 14 [deduced amino acid sequence]) was recombinantly produced in Aspergillus oryzae JaL250 according to WO 2005/074656. The recombinantly produced Thermoascus aurantiacus GH61A polypeptide was first concentrated by ultrafiltration using a 10 kDa membrane, buffer exchanged into 20 mM Tris-HCl pH 8.0, and then purified using a 100 ml Q-SEPHAROSE.RTM. Big Beads column (GE Healthcare, Piscataway, N.J., USA) with a 600 ml 0-600 mM NaCl linear gradient in the same buffer. Fractions of 10 ml were collected and pooled based on SDS-PAGE.
[0441] The pooled fractions (90 ml) were then further purified using a 20 ml MONO Q.RTM. column (GE Healthcare, Piscataway, N.J., USA) with a 500 ml 0-500 mM NaCl linear gradient in the same buffer. Fractions of 6 ml were collected and pooled based on SDS-PAGE. The pooled fractions (24 ml) were concentrated by ultrafiltration using a 10 kDa membrane, and chromatographed using a 320 ml SUPERDEX.RTM. 200 SEC column (GE Healthcare, Piscataway, N.J., USA) with isocratic elution of approximately 1.3 liter of 150 mM NaCl-20 mM Tris-HCl pH 8.0. Fractions of 5 ml were collected and pooled based on SDS-PAGE. Protein concentration was determined using a Microplate BCA.TM. Protein Assay Kit (Thermo Fisher Scientific Inc., Rockford, Ill., USA) in which bovine serum albumin was used as a protein standard.
Example 5: Preparation of Thielavia terrestris GH61E Polypeptide Having Cellulolytic Enhancing Activity
[0442] Thielavia terrestris GH61E polypeptide having cellulolytic enhancing activity (SEQ ID NO: 7 [DNA sequence] and SEQ ID NO: 8 [deduced amino acid sequence]) was recombinantly produced in Aspergillus oryzae JaL250 according to U.S. Pat. No. 7,361,495. The Thielavia terrestris GH61E polypeptide was desalted and buffer-exchanged into 20 mM sodium acetate-150 mM NaCl pH 5.0 using a HIPREP.RTM. 26/10 desalting column according to the manufacturer's instructions. Protein concentration was determined using a Microplate BCA.TM. Protein Assay Kit in which bovine serum albumin was used as a protein standard.
Example 6: Preparation of Aspergillus fumigatus GH61B Polypeptide Having Cellulolytic Enhancing Activity
[0443] Aspergillus fumigatus GH61B polypeptide having cellulolytic enhancing activity (SEQ ID NO: 29 [DNA sequence] and SEQ ID NO: 30 [deduced amino acid sequence]) was recombinantly produced using Aspergillus oryzae JaL355 as a host according to WO 2010/138754. The recombinantly produced A. fumigatus GH61B polypeptide was desalted and concentrated into 20 mM Tris pH 8.0 using a 10 kDa MWCO membrane and purified by size exclusion chromatography using SUPERDEX.RTM. S75 (GE Healthcare, Piscataway, N.J., USA). The purification buffer was 150 mM NaCl, 20 mM Tris 8.0. Homogeneity was confirmed by SDS-PAGE.
Example 7: Preparation of Penicillium pinophilum GH61A Polypeptide Having Cellulolytic Enhancing Activity
[0444] Penicillium pinophilum GH61A polypeptide having cellulolytic enhancing activity (SEQ ID NO: 31 [DNA sequence] and SEQ ID NO: 32 [deduced amino acid sequence]) was recombinantly produced using Aspergillus oryzae HowB101 as a host according to WO 2011/005867. The recombinantly produced P. pinophilum GH61A polypeptide was desalted and concentrated into 20 mM Tris pH 8.0 using a 10 kDa MWCO membrane and purified by size exclusion chromatography using SUPERDEX.RTM. S75. The purification buffer was 150 mM NaCl, 20 mM Tris 8.0. Homogeneity was confirmed by SDS-PAGE.
Example 8: Preparation of Trichoderma reesei CEL7B Endoglucanase I
[0445] Trichoderma reesei CEL7B endoglucanase I (EGI) (SEQ ID NO: 65 [DNA sequence] and SEQ ID NO: 66 [deduced amino acid sequence]) was cloned and expressed in Aspergillus oryzae JaL250 as described in WO 2005/067531. Filtered broth was concentrated and buffer exchanged using a tangential flow concentrator equipped with a 10 kDa polyethersulfone membrane with 20 mM Tris-HCl pH 8.5. The sample was loaded onto a Q SEPHAROSE.RTM. High Performance column (GE Healthcare, Piscataway, N.J., USA) equilibrated in 20 mM Tris pH 8.5, and bound proteins were eluted with a linear gradient from 0-600 mM sodium chloride. The fractions were concentrated and desalted into 20 mM Tris pH 8.0, 150 mM NaCl using VIVASPIN 20.RTM. 10 kDa MWCO centrifugal concentration devices (GE Healthcare UK limited, Little Chalfont, Buckinghamshire, UK). Protein concentration was determined using a Microplate BCA.TM. Protein Assay Kit in which bovine serum albumin was used as a protein standard.
Example 9: Preparation of Trichoderma reesei CEL5A Endoglucanase II
[0446] The Trichoderma reesei RutC30 Cel5A endoglucanase II gene (SEQ ID NO: 67 [DNA sequence] and SEQ ID NO: 68 [deduced amino acid sequence]) was cloned and expressed in Aspergillus oryzae as described below.
[0447] Two synthetic oligonucleotide primers, shown below, were designed to PCR amplify the endoglucanase II gene from Trichoderma reesei RutC30 genomic DNA. Genomic DNA was isolated using a DNEASY.RTM. Plant Maxi Kit (QIAGEN Inc., Valencia, Calif., USA). An IN-FUSION.TM. PCR Cloning Kit (BD Biosciences, Palo Alto, Calif., USA) was used to clone the fragment directly into pAILo2 (WO 2004/099228).
Forward Primer:
TABLE-US-00001
[0448] (SEQ ID NO: 125) 5'-ACTGGATTTACCATGAACAAGTCCGTGGCTCCATTGCT-3'
Reverse Primer:
TABLE-US-00002
[0449] (SEQ ID NO: 126) 5'-TCACCTCTAGTTAATTAACTACTTTCTTGCGAGACACG-3'
Bold letters represent coding sequence. The remaining sequence contains sequence identity to insertion sites of pAILo2.
[0450] Fifty picomoles of each of the primers above were used in a PCR reaction containing 200 ng of Trichoderma reesei genomic DNA, 1.times.Pfx Amplification Buffer (Invitrogen, Carlsbad, Calif., USA), 6 .mu.l of 10 mM blend of dATP, dTTP, dGTP, and dCTP, 2.5 units of PLATINUM.RTM. Pfx DNA polymerase (Invitrogen Corp., Carlsbad, Calif., USA), and 1 .mu.l of 50 mM MgSO.sub.4 in a final volume of 50 .mu.l. The amplification reaction was incubated in an EPPENDORF.RTM. MASTERCYCLER.RTM. 5333 (Eppendorf Scientific, Inc., Westbury, N.Y., USA) programmed for one cycle at 98.degree. C. for 2 minutes; and 35 cycles each at 94.degree. C. for 30 seconds, 61.degree. C. for 30 seconds, and 68.degree. C. for 1.5 minutes. After the 35 cycles, the reaction was incubated at 68.degree. C. for 10 minutes and then cooled at 10.degree. C. A 1.5 kb PCR reaction product was isolated on a 0.8% GTG.RTM. agarose gel (Cambrex Bioproducts, East Rutherford, N.J., USA) using 40 mM Tris base, 20 mM sodium acetate, 1 mM disodium EDTA (TAE) buffer and 0.1 .mu.g of ethidium bromide per ml. The DNA band was visualized with the aid of a DARKREADER.TM. Transilluminator (Clare Chemical Research, Dolores, Colo., USA). The 1.5 kb DNA band was excised with a disposable razor blade and purified using an ULTRAFREE.RTM. DA spin cup (Millipore, Billerica, Mass., USA) according to the manufacturer's instructions.
[0451] Plasmid pAILo2 was linearized by digestion with Nco I and Pac I. The plasmid fragment was purified by gel electrophoresis and ultrafiltration as described above. Cloning of the purified PCR fragment into the linearized and purified pAILo2 vector was performed using an IN-FUSION.TM. PCR Cloning Kit. The reaction (20 .mu.l) contained 1.times.IN-FUSION.TM. Buffer (BD Biosciences, Palo Alto, Calif., USA), 1.times.BSA, 1 .mu.l of IN-FUSION.TM. enzyme (diluted 1:10) (BD Biosciences, Palo Alto, Calif., USA), 100 ng of pAILo2 digested with Nco I and Pac I, and 100 ng of the Trichoderma reesei Cel5A endoglucanase II PCR product. The reaction was incubated at room temperature for 30 minutes. A 2 .mu.l sample of the reaction was used to transform E. coli XL10 SOLOPACK.RTM. Gold cells (Stratagene, La Jolla, Calif., USA) according to the manufacturer's instructions. After a recovery period, two 100 .mu.l aliquots from the transformation reaction were plated onto 150 mm 2X YT plates supplemented with 100 .mu.g of ampicillin per ml. The plates were incubated overnight at 37.degree. C. A set of 3 putative recombinant clones was recovered from the selection plates and plasmid DNA was prepared from each one using a BIOROBOT.RTM. 9600 (QIAGEN Inc., Valencia, Calif., USA). Clones were analyzed by Pci I/Bsp LU11 I restriction digestion. One clone with the expected restriction digestion pattern was then sequenced to confirm that there were no mutations in the cloned insert. Clone #3 was selected and designated pAILo27.
[0452] Aspergillus oryzae JaL250 protoplasts were prepared according to the method of Christensen et al., 1988, supra. Five micrograms of pAILo27 (as well as pAILo2 as a control) were used to transform Aspergillus oryzae JaL250 protoplasts. The transformation of Aspergillus oryzae JaL250 with pAILo27 yielded about 50 transformants. Eleven transformants were isolated to individual PDA plates and incubated for five days at 34.degree. C.
[0453] Confluent spore plates were washed with 3 ml of 0.01% TWEEN.RTM. 80 (a nonionic surfactant and emulsifier derived from polyethoxylated sorbitan and oleic acid) and the spore suspension was used to inoculate 25 ml of MDU2BP medium in 125 ml glass shake flasks. Transformant cultures were incubated at 34.degree. C. with constant shaking at 200 rpm. At day five post-inoculation, cultures were centrifuged at 6000.times.g and their supernatants collected. Five microliters of each supernatant were mixed with an equal volume of 2.times.loading buffer (10% beta-mercaptoethanol) and loaded onto a 1.5 mm 8%-16% Tris-glycine SDS-PAGE gel and stained with SIMPLYBLUE.TM. SafeStain (Invitrogen Corp., Carlsbad, Calif., USA). SDS-PAGE profiles of the culture broths showed that ten out of eleven transformants produced a new protein band of approximately 45 kDa. Transformant number 1, designated Aspergillus oryzae JaL250AlLo27, was cultivated in a fermentor.
[0454] One hundred ml of shake flask medium were added to a 500 ml shake flask. The shake flask medium was composed per liter of 50 g of sucrose, 10 g of KH.sub.2PO.sub.4, 0.5 g of CaCl.sub.2, 2 g of MgSO.sub.4.7H.sub.2O, 2 g of K.sub.2SO.sub.4, 2 g of urea, 10 g of yeast extract, 2 g of citric acid, and 0.5 ml of trace metals solution. The trace metals solution was composed per liter of 13.8 g of FeSO.sub.4.7H.sub.2O, 14.3 g of ZnSO.sub.4.7H.sub.2O, 8.5 g of MnSO.sub.4.H.sub.2O, 2.5 g of CuSO.sub.4.5H.sub.2O, and 3 g of citric acid. The shake flask was inoculated with two plugs of Aspergillus oryzae JaL250AlLo27 from a PDA plate and incubated at 34.degree. C. on an orbital shaker at 200 rpm for 24 hours. Fifty ml of the shake flask broth was used to inoculate a 3 liter fermentation vessel.
[0455] A total of 1.8 liters of the fermentation batch medium was added to a three liter glass jacketed fermentor (Applikon Biotechnology, Schiedam, Netherlands). The fermentation batch medium was composed per liter of 10 g of yeast extract, 24 g of sucrose, 5 g of (NH.sub.4).sub.2SO.sub.4, 2 g of KH.sub.2PO.sub.4, 0.5 g of CaCl.sub.2.2H.sub.2O, 2 g of MgSO.sub.4.7H.sub.2O, 1 g of citric acid, 2 g of K.sub.2SO.sub.4, 0.5 ml of anti-foam, and 0.5 ml of trace metals solution. The trace metals solution was composed per liter of 13.8 g of FeSO.sub.4.7H.sub.2O, 14.3 g of ZnSO.sub.4.7H.sub.2O, 8.5 g of MnSO.sub.4.H.sub.2O, 2.5 g of CuSO.sub.4.5H.sub.2O, and 3 g of citric acid. Fermentation feed medium was composed of maltose, which was. dosed at a rate of 0 to 4.4 g/l/hr for a period of 185 hours. The fermentation vessel was maintained at a temperature of 34.degree. C. and pH was controlled using an Applikon 1030 control system (Applikon Biotechnology, Schiedam, Netherlands) to a set-point of 6.1+/-0.1. Air was added to the vessel at a rate of 1 vvm and the broth was agitated by a Rushton impeller rotating at 1100 to 1300 rpm. At the end of the fermentation, whole broth was harvested from the vessel and centrifuged at 3000.times.g to remove the biomass. The supernatant was sterile filtered and stored at 5 to 10.degree. C.
[0456] The supernatant was desalted and buffer-exchanged into 20 mM Bis-Tris pH 6.0 using a HIPREP.RTM. 26/10 desalting column (GE Healthcare, Piscataway, N.J., USA) according to the manufacturer's instructions. The buffer exchanged sample was loaded onto a MonoQ.RTM. column (GE Healthcare, Piscataway, N.J., USA) equilibrated with 20 mM Bis-Tris pH 6.0, and the bound protein was eluted with a linear gradient from 0 to 1000 mM sodium chloride. Protein fractions were pooled and buffer exchanged into 1.2 M (NH.sub.4).sub.2SO.sub.4-20 mM Tris-HCl pH 8.5. The sample was loaded onto a Phenyl SUPEROSE.TM. column (HR 16/10) equilibrated with 1.2 M (NH.sub.4).sub.2SO.sub.4-20 mM Tris-HCl pH 8.0. Bound proteins were eluted with a linear gradient over 20 column volumes from 1.2 to 0 M (NH.sub.4).sub.2SO.sub.4 in 20 mM Tris-HCl pH 8.5. The fractions were pooled, concentrated, and loaded onto a SUPERDEX.RTM. 75 HR 26/60 column (GE Healthcare, Piscataway, N.J., USA) equilibrated with 20 mM Tris-150 mM sodium chloride pH 8.5. Fractions were pooled and concentrated in 20 mM Tris-150 mM sodium chloride pH 8.5. Protein concentration was determined using a Microplate BCA.TM. Protein Assay Kit in which bovine serum albumin was used as a protein standard.
Example 10: Preparation of Trichoderma reesei CEL7A Cellobiohydrolase I
[0457] Trichoderma reesei CEL7A cellobiohydrolase I (SEQ ID NO: 95 [DNA sequence] and SEQ ID NO: 96 [deduced amino acid sequence]) was prepared as described by Ding and Xu, 2004, "Productive cellulase adsorption on cellulose" in Lignocellulose Biodegradation (Saha, B. C. ed.), Symposium Series 889, pp. 154-169, American Chemical Society, Washington, D.C. Protein concentration was determined using a Microplate BCA.TM. Protein Assay Kit in which bovine serum albumin was used as a protein standard.
Example 11: Preparation of Trichoderma reesei CEL6A Cellobiohydrolase II
[0458] The Trichoderma reesei RutC30 CEL6A cellobiohydrolase II gene (SEQ ID NO: 97 [DNA sequence] and SEQ ID NO: 98 [deduced amino acid sequence]) was isolated from Trichoderma reesei RutC30 as described in WO 2005/056772. The Trichoderma reesei CEL6A cellobiohydrolase II gene was expressed in Fusarium venenatum using pEJG61 as an expression vector according to the procedures described in U.S. Published Application No. 20060156437. Fermentation was performed as described in U.S. Published Application No. 20060156437. Filtered broth was desalted and buffer-exchanged into 20 mM sodium acetate-150 mM NaCl pH 5.0 using a HIPREP.RTM. 26/10 Desalting Column according to the manufacturer's instructions. Protein concentration was determined using a Microplate BCA.TM. Protein Assay Kit in which bovine serum albumin was used as a protein standard.
Example 12: Preparation of Aspergillus oryzae CEL3A Beta-Glucosidase
[0459] Aspergillus oryzae CEL3A beta-glucosidase (SEQ ID NO: 111 [DNA sequence] and SEQ ID NO: 112 [deduced amino acid sequence]) was recombinantly prepared as described in WO 2004/099228, and purified as described by Langston et al., 2006, Biochim. Biophys. Acta Proteins Proteomics 1764: 972-978. Protein concentration was determined using a Microplate BCA.TM. Protein Assay Kit.
Example 13: Effect of GH61 Polypeptides Having Cellulolytic Enhancing Activity on Hydrolysis of Microcrystalline Cellulose or PCS by the Trichoderma reesei Cellulase Composition
[0460] The effect of the Thermoascus aurantiacus GH61A polypeptide on the hydrolysis of AVICEL.RTM. or milled washed PCS by the Trichoderma reesei cellulase composition was determined using the same experimental conditions and procedures according to Example 3. In general, in experiments performed in subsequent examples, a control reaction was included in which increasing concentrations of GH61 polypeptide were added to the hydrolysis of either AVICEL.RTM. or pretreated corn stover with the T. reesei cellulase composition in the absence of other liquors, compounds, or variously treated liquors.
[0461] The presence of the T. aurantiacus GH61A polypeptide did not enhance the hydrolysis of AVICEL.RTM. by the T. reesei cellulase composition. Conversion of AVICEL.RTM. was 0.119.+-.0.00251 and 0.339.+-.0.00222 at 1 and 3 days, respectively, in the absence of the T. aurantiacus GH61A polypeptide compared to 0.112.+-.0.00376 and 0.333.+-.0.00328 at 1 and 3, respectively, in the presence of 24% (w/w) of the T. aurantiacus GH61A polypeptide.
[0462] The presence of the T. aurantiacus GH61A polypeptide enhanced the hydrolysis of milled washed PCS by the T. reesei cellulase composition. Conversion of milled washed PCS was 0.249.+-.0.00104 and 0.545.+-.0.00656 at 1 and 3 days, respectively, in the presence of the T. aurantiacus GH61A polypeptide compared to 0.222.+-.0.00464 and 0.412.+-.0.0237 at 1 and 3 days, respectively, in the absence of the T. aurantiacus GH61A polypeptide.
[0463] The presence of the T. aurantiacus GH61A polypeptide marginally enhanced the hydrolysis of milled hot-washed PCS by the T. reesei cellulase composition. Conversion of hot washed PCS was 0.315.+-.0.00267 and 0.383.+-.0.00498, at 1 and 3 days, respectively, in the absence of the T. aurantiacus GH61A polypeptide compared to 0.331.+-.0.0115 and 0.409.+-.0.0145 at 1 and 3, respectively, in the presence of 8% (w/w) of the T. aurantiacus GH61A polypeptide.
[0464] The presence of the Thelavia terrestris GH61E polypeptide did not enhance the hydrolysis of AVICEL.RTM. by the T. reesei cellulase composition. Conversion of AVICEL.RTM. was 0.122.+-.0.00426, 0.242.+-.0.00813 and 0.315.+-.0.00814, at 1, 3, and 5 days, respectively, in the absence of the T. terrestris GH61E polypeptide compared with 0.121.+-.0.000824, 0.228.+-.0.000978 and 0.307.+-.0.00348 at 1, 3, and 5 days respectively, in the presence of 24% (w/w) of the T. terrestris GH61E polypeptide.
[0465] The presence of the Aspergillus fumigatus GH61B polypeptide did not significantly enhance the hydrolysis of AVICEL.RTM. by the T. reesei cellulase composition. Conversion of AVICEL.RTM. was 0.150.+-.0.009, 0.31.+-.0.001, and 0.48.+-.0.001 at 1, 3, and 7 days, respectively, in the absence of the A. fumigatus GH61B polypeptide compared to 0.148.+-.0.002%, 0.311.+-.0.001%, and 0.54.+-.0.02% at 1, 3, and 7 days, respectively, in the presence of the A. fumigatus GH61B polypeptide.
Example 14: Effect of Thermoascus aurantiacus GH61A Polypeptide on Cellulolysis of Unwashed and Washed Acid-Pretreated Corn Stover
[0466] The effect of Thermoascus aurantiacus GH61A polypeptide on hydrolysis of both milled washed and milled unwashed NREL pretreated corn stover by the Trichoderma reesei cellulase composition were assayed for comparison. To an equivalent, fixed concentration of the T. reesei cellulase composition at 4 mg per gram of cellulose, increasing concentrations of T. aurantiacus GH61A polypeptide between 0 and 24% (w/w) were added to equivalent dry masses of milled unwashed pretreated corn stover or milled washed pretreated corn stover, and the fractional hydrolysis was assayed according to Example 3.
[0467] FIG. 1 shows the fractional hydrolysis of variously washed pretreated corn stover substrates. The total enhancement in total conversion from the T. aurantiacus GH61A polypeptide was larger in magnitude and was apparent at earlier stages of hydrolysis for unwashed corn stover in comparison to washed pretreated corn stover. Over the range of concentrations tested, there was no GH61 polypeptide concentration-dependence of hydrolysis after 1 day of hydrolysis for washed milled pretreated corn stover (open squares), whereas a slight but significant increase of hydrolysis as GH61 polypeptide concentration increased was observed for unwashed milled corn stover (open circles). After 3 days of hydrolysis, washed pretreated corn stover showed a sharp, saturable enhancement of hydrolysis with GH61 polypeptide addition, well-fitted by a square hyperbolic binding function (closed squares). Conversely, there was a linear increase in hydrolysis with GH61 polypeptide concentration for the unwashed pretreated corn stover that extrapolated to a higher overall conversion at GH61 polypeptide concentrations greater than 50% (w/w) (closed circles). For hot-water washed pretreated corn stover prepared according to Example 1, this trend was even more apparent; after 3 days of hydrolysis, no GH61 polypeptide enhancement was observed (open triangles), and GH61 polypeptide-dependent enhancement of cellulolysis was not apparent until 5 days of hydrolysis (closed triangles). The total magnitude of GH61 polypeptide-dependent enhancement for hot-water washed pretreated corn stover was only 5% at 7 days, whereas for the GH61 polypeptide enhancement for unwashed pretreated corn stover was at least 15% at 7 days of hydrolysis (data not shown). In each case, the conversion by the T. reesei cellulase composition in the absence of the GH61 polypeptide (y-intercept) was higher for the more extensively washed substrates, indicating the removal of soluble cellulase inhibitors by washing.
Example 15: The Effect of Addition of Acid-Pretreated Corn Stover Liquor to Washed Milled Pretreated Corn Stover
[0468] Acid-pretreated corn stover liquor, fractionated according to Example 2, was added at concentrations between 0 and 15% (v/v) to milled, water-washed acid-pretreated corn stover and hydrolyzed by 4 mg of the Trichoderma reesei cellulase composition per g cellulose plus increasing concentrations of the Thermoascus aurantiacus GH61A polypeptide according to Example 3.
[0469] FIG. 2A shows the extent of hydrolysis for the various additions of the T. aurantiacus GH61A polypeptide and NREL acid-pretreated corn stover liquor to milled, water-washed acid-pretreated corn stover at 1 day (white bars) and 3 days (gray bars) of hydrolysis.
[0470] FIG. 2B shows a replot of the data presented in FIG. 2A with non-linear least square fits to Equation 2 according to Example 3 or with linear least square fits. The Figure demonstrates that the functional dependence of the extent of hydrolysis was square hyperbolic, i.e., saturating, for concentrations of liquor <10% (v/v), and became linear or exponential at concentrations of liquor 10% (v/v) at 3 days of hydrolysis. The extent of hydrolysis at the highest added concentration of the T. aurantiacus GH61A polypeptide was greater than that observed in the absence of liquor, and extrapolation of the trends to predict fractional hydrolysis levels at higher GH61 polypeptide concentrations indicated a greater conversion at higher liquor concentrations. From fits of Equation 2, the total enhancement from the GH61 polypeptide, .DELTA.fractional hydrolysis, was 0.150.+-.0.000550 in the absence of added liquor, 0.166.+-.0.0155 with 2% liquor, 0.227.+-.0.0827 with 5% liquor, and 0.417.+-.0.240 with 10% liquor, and the increase was linear with GH61 polypeptide concentration at 15% liquor.
Example 16: Effect of Addition of Acid-Pretreated Corn Stover Liquor on Thermoascus aurantiacus GH61A Polypeptide During Hydrolysis of Microcrystalline Cellulose
[0471] Pretreated corn stover liquor, fractionated according to Example 2, was added to a saccharification reaction of microcrystalline cellulose by the Trichoderma reesei cellulase composition according to Example 3 at concentrations between 0 and 20% (v/v). The Thermoascus aurantiacus GH61A polypeptide was titrated between 0 and 24% (w/w) of total protein.
[0472] FIG. 3 shows that the T. aurantiacus GH61A polypeptide did not enhance on hydrolysis of microcrystalline cellulose by the T. reesei cellulase composition in the absence of liquor. However, in the presence of NREL pretreated corn stover liquor, the T. aurantiacus GH61A polypeptide enhanced cellulolysis. FIG. 3A shows fractional hydrolysis at various GH61 polypeptide concentrations for increasing concentrations of NREL pretreated corn stover liquor at 1 day (open circles) and 3 days of hydrolysis (closed circles). As pretreated corn stover liquor was added from 0% v/v (circles) to 5% (diamonds), 10% (triangles), and 15% (inverted triangles), in the absence of the T. aurantiacus GH61A polypeptide, there was increasing inhibition of the T. reesei cellulase composition, as was apparent from the reduction in fractional hydrolysis. As the concentration of the T. aurantiacus GH61A polypeptide was increased, for those samples containing acid-pretreated corn stover liquor, the extent of hydrolysis increased to an extent beyond the level observed for hydrolysis in the absence of liquor. These data were corrected for saccharides present in the added liquor, and the possibility that either the T. aurantiacus GH61A polypeptide or the T. reesei cellulase composition in combination with the T. aurantiacus GH61A polypeptide were converting some substrate in pretreated corn stover liquor to glucose was unlikely, as the extent of enhancement from the T. aurantiacus GH61A polypeptide at 5% liquor addition would correspond to a conversion of 55 g/L glucose equivalents from the added liquor.
[0473] FIG. 3B shows the results of addition of a synthetic mixture of the major sugar components of pretreated corn stover liquor; glucose, cellobiose, xylose, and arabinose with or without added phenol to mimic the phenolic lignin degradation compounds present in pretreated corn stover liquor, at the concentrations present in actual liquor. The synthetic sugar mixture was added between 0% and 15%, (symbols as in FIG. 3A), and the synthetic sugar mixture containing phenol was added at either 5% (squares) or 15% (right triangles). For this example, added liquor sugar concentrations were not subtracted from the overall apparent hydrolysis, as was evident from the upward shift in apparent hydrolysis with increasing liquor addition. From FIG. 3B, fractional hydrolysis was independent of GH61 polypeptide concentration, thus there was no apparent GH61 polypeptide cellulolytic enhancing activity in the presence of the synthetic liquor mixtures on microcrystalline cellulose. These data indicated that the GH61 polypeptide cellulolytic enhancing activity derives from the minor components of NREL pretreated corn stover liquor.
Example 17: Effect of Preconditioning Acid-Pretreated Corn Stover Liquor with Thelavia terrestris GH61E Polypeptide
[0474] Pretreated corn stover liquor, extracted according to Example 2, was added to a saccharification reaction of microcrystalline cellulose by the Trichoderma reesei cellulase composition according to Example 3 at concentrations between 0 and 20% (v/v). Alternatively, 10 ml of liquor were incubated at 50.degree. C. overnight with 0.43 mg protein of the T. reesei cellulase composition per ml, or with 20 .mu.g protein of the Thielavia terrestris GH61E polypeptide per ml, or both (pre-conditioned liquor). The enzyme was removed from these samples using a 3 kDa MWCO AMICON.RTM. centrifuge filter (Millipore, Bedford, Mass., USA), and the filtered flow-through liquor was added to saccharifications of microcrystalline cellulose at concentrations between 0 and 20% (v/v). The T. terrestris GH61E polypeptide was titrated between concentrations of 0 and 24% (w/w) of total protein.
[0475] FIG. 4 shows that, like the T. aurantiacus GH61A polypeptide demonstrated in Example 11, the Thelavia terrestris GH61E polypeptide had cellulolytic enhancing activity on microcrystalline cellulose in the presence of NREL pretreated corn stover liquor and did not enhance cellulolysis in the absence of liquor. FIG. 4 (all panels, circles) shows that increasing T. terrestris GH61E polypeptide concentration on microcrystalline cellulose in the absence of liquor did not enhance hydrolysis. As liquor was added from 0% (v/v) (circles) to 5% (squares), 10% (diamonds), and 15% (triangles) in the absence of the T. terrestris GH61E polypeptide, there was increasing inhibition of the T. reesei cellulase composition, as was apparent from the reduction in fractional hydrolysis (all panels, y-intercepts). As the T. terrestris GH61E polypeptide concentration was increased, for those samples containing either liquor or enzymatically pre-conditioned liquor, the extent of hydrolysis increased. In most cases, despite the inhibition of cellulolysis arising from the liquor addition, the increase in hydrolysis led to an extent of saccharification beyond the level observed for hydrolysis in the absence of liquor at high Thelavia terrestris GH61E polypeptide concentrations. FIG. 4A shows the effects of liquor that had not been enzymatically treated, FIG. 4B shows the effects of liquor that had been saccharified using the T. reesei cellulase composition, FIG. 4C shows the effects of liquor that had been incubated with both the T. reesei cellulase composition and the T. terrestris GH61E polypeptide. In each case, GH61 polypeptide cellulolytic enhancing activity was apparent in the presence of pretreated corn stover liquor. Control reactions containing identical concentrations of liquor or pretreated liquor and enzyme in identical concentrations to those present in the cellulose-containing samples were performed in parallel, and sugars produced from these control reactions were subtracted from the saccharification totals. In each case the conversion of pretreated corn stover liquor to glucose equivalents produced minimal total sugar, and far less than was necessary to account for the large cellulolytic enhancement observed in saccharifications containing both the Thielavia terrestris GH61E polypeptide and liquor.
Example 18: Effect of Acid-Pretreated Corn Stover Liquor or Steam-Pretreated Corn Stover Liquor on Thelavia terrestris GH61E Polypeptide Activity
[0476] Pretreated corn stover liquor was extracted according to Example 2 with the following exceptions: 0.6 kg of steam explosion-pretreated corn stover or NREL acid-pretreated corn stover were suspended in 900 ml of deionized water and mixed for 2 hours. The solids were filtered using filter paper and sterile filtered with a 0.45 .mu.m filter. Two hundred ml of each was ultracentrifuged using a 44.5 mM diameter 1 kDa MWCO centrifuge filter (Millipore, Bedford, Mass., USA). The concentrated retentate was restored to the original volume by addition of water. The original liquors and the molecular weight separated fractions of each were added to saccharification reactions of microcrystalline cellulose with the Trichoderma reesei cellulase composition according to Example 3 at concentrations of 5% and 15% (v/v). The Thelavia terrestris GH61E polypeptide was titrated at concentrations between 0 and 24% (w/w) total protein.
[0477] FIG. 5A shows that increasing concentrations of the Thelavia terrestris GH61E polypeptide increased the hydrolysis of microcrystalline cellulose by the T. reesei cellulase composition in the presence of the acid-pretreated corn stover liquor. Thus in the presence of the acid-pretreated corn stover liquor, the T. terrestris GH61E polypeptide significantly enhanced cellulolysis. FIG. 5B shows that increasing concentrations of the Thelavia terrestris GH61E polypeptide marginally increased hydrolysis in the presence of steam explosion-pretreated corn stover, thus demonstrating minimal enhancement of cellulolysis by Thelavia terrestris GH61E under similar conditions.
[0478] Overall, the results indicated that conditions of pretreatment are critical for the production of liquor components necessary for GH61 cellulolytic enhancing activity on microcrystalline cellulose. More severe pretreatments, exemplified herein by the acid-pretreated corn stover (e.g., NREL PCS), generate soluble components or higher concentrations of the soluble components than do milder pretreatments, as illustrated by steam-pretreated corn stover above.
Example 19: Separation of GH61 Polypeptide-Enhancing Liquor Components from Non-Enhancing Components and Inhibitory Components in NREL Pretreated Corn Stover Liquor
[0479] Pretreated corn stover liquor was extracted and separated by ultrafiltration into nominal molecular weight fractions above and below 1 kDa according to Example 18. The molecular weight separated fractions were added to a saccharification reaction of microcrystalline cellulose with the T. reesei cellulase composition at concentrations between 0 and 15% (v/v) according to Example 3. The Thelavia terrestris GH61E polypeptide was titrated at concentrations between 0 and 24% (w/w) total protein.
[0480] As shown in FIG. 5A, at zero T. terrestris GH61E polypeptide concentration, both the whole liquor and the lower molecular weight fractions showed inhibition at higher liquor concentrations (circles, squares). The inhibition from liquor has been discussed previously in Examples 10, 12 and 13. Conversely, there was no difference in hydrolysis at either 5 or 15% (v/v) of the higher molecular weight fraction (triangles). Increasing concentrations of the T. terrestris GH61E polypeptide with all these liquor molecular weight fractions increased cellulose hydrolysis. The unfractionated pretreated corn stover liquor yielded the highest GH61 polypeptide-dependent enhancement, however the high molecular weight pretreated corn stover liquor fraction also yielded substantial GH61 polypeptide-dependent enhancement.
[0481] The efficacy of molecular weight-based filtration was confirmed by fractionation of PCS liquor using AMICON.RTM. 30, 10, and 3 kDa MWCO centrifuge filters (Millipore, Bedford, Mass., USA) and assaying for GH61 cellulolytic enhancing activity. NREL acid-pretreated corn stover liquor was extracted according to Example 2, and then filtered through successively smaller MWCO filters. The retentates were repeatedly washed with 3 to 5 volumes of water. The retentates for each molecular weight filter were assayed for GH61 cellulolytic enhancing activity according to Example 3 with the following exceptions: 5% (v/v) of each retentate was added to saccharification reactions of microcrystalline cellulose (AVICEL.RTM.), and the extent of hydrolysis was determined at 1 and 6 days of saccharification. FIG. 6 shows the fractional hydrolysis of AVICEL.RTM. by the T. reesei cellulase composition with added T. aurantiacus GH61A polypeptide as indicated, in the presence of the filter rententates indicated. FIG. 6 shows that the GH61 polypeptide-enhancing factor was not retained, or was only marginally retained by a 3 kDa filter. Increasing concentrations of GH61 polypeptide increased the hydrolysis of AVICEL.RTM. in the presence of flow-through from the 3 kDa MWCO filter, whereas a decrease, or no change in hydrolysis with GH61 polypeptide concentration was observed when incubated in the presence of retentates from molecular weight filters of 3 kDa and greater. These data, combined with the data presented in FIG. 5A, indicated that the nominal molecular weight of the compounds facilitating GH61 polypeptide cellulolytic enhancing activity may lie between 1 and 3 kDa, and that molecular weight separation of the NREL acid-pretreated corn stover liquor can separate enhancing from inhibitory factors.
Example 20: Effect of Thermoascus aurantiacus GH61 Polypeptide and Pretreated Corn Stover Liquor Enhances Cellulolytic Activity of Individual Cellulases on AVICEL.RTM.
[0482] Individual monocomponent cellulases Cel5A endoglucanase II, Cel6A cellobiohydrolase, Cel7A cellobiohydrolase, and Cel7B endoglucanase I from Trichoderma reesei and beta-glucosidase from Aspergillus oryzae were assayed for enhancement of their ability to hydrolyze microcrystalline cellulose when incubated with the Thermoascus aurantiacus GH61A polypeptide with or without 5% NREL acid-pretreated corn stover liquor. AVICEL.RTM. hydrolyses were performed according to Example 3, with the following exceptions. Instead of the Trichoderma reesei cellulase composition, purified T. reesei monocomponents and purified Aspergillus oryzae beta-glucosidase were used at a concentration of 10 mg of enzyme protein per g cellulose. Alternatively, mixtures of monocomponent cellulases were used, with each cellulase dosed at 10 mg of enzyme protein per g cellulose. Saccharification reactions were performed with or without 1 mg of the T. aurantiacus GH61A polypeptide per g cellulose and with or without 5% NREL pretreated corn stover liquor at 50.degree. C. for 7 days.
[0483] FIG. 7A shows the fractional hydrolysis achieved by each monocomponent and monocomponent mixture at 3, 5, and 7 days of hydrolysis, with and without the T. aurantiacus GH61A polypeptide in the presence of acid-pretreated corn stover liquor. In each case, in the absence of liquor, no GH61 polypeptide-dependent enhancement was observed (data not shown). Conversely, when acid-pretreated corn stover liquor was present, the T. aurantiacus GH61A polypeptide enhanced the cellulase activity of each monocomponent or monocomponent mixture by 5% or more at 7 days of hydrolysis.
[0484] FIG. 7B shows the enhancement of cellulase activity for each monocomponent arising from the T. aurantiacus GH61A polypeptide in the presence of NREL acid-pretreated corn stover liquor at 3, 5, and 7 days of hydrolysis, which is given by the ratio of fractional hydrolysis in the presence to the absence of the GH61 polypeptide:
GH .times. .times. 61 .times. .times. effect = % .times. .times. conversion .times. ( + GH .times. .times. 61 + liquor ) % .times. .times. conversion .times. ( no .times. .times. GH .times. .times. 61 + liquor ) ( Equation .times. .times. 3 ) ##EQU00003##
Enhancement of hydrolysis by the GH61 polypeptide yields a ratio >1; inhibition of hydrolysis yields a ratio <1, and no effect on hydrolysis yields a ratio=1
[0485] In each case at 5 and 7 days of hydrolysis, every monocomponent and every monocomponent mixture had enhanced cellulolytic activity, indicated by a GH61 effect >1. The mixtures containing 3 to 5 cellulase components had the highest overall cellulase concentration in the reactions, thus had the highest fractional hydrolysis, and the lowest apparent GH61 effect. Despite this, the T. aurantiacus GH61A polypeptide still provided an enhancement of 1.05.+-.0.00604 for the 5-component cellulose mixture (Cel5A endoglucanase II, Cel6A cellobiohydrolase, Cel7A cellobiohydrolase, and Cel7B endoglucanase I from T. reesei and beta-glucosidase from Aspergillus oryzae) at 7 days of hydrolysis. The largest relative enhancements by the T. aurantiacus GH61A polypeptide to AVICEL.RTM. saccharification by cellulose monocomponents or mixtures thereof were observed for GH61 polypeptide enhancement of T. reesei Cel7A cellobiohydrolase (1.33.+-.0.127), or mixtures containing the T. reesei Cel7A cellobiohydrolase and T. reesei Cel5A endoglucanase II (1.33.+-.0.00719).
Example 21: Enrichment of NREL Acid-Pretreated Corn Stover Liquor Components
[0486] NREL acid-pretreated corn stover liquor was fractionated by adsorption to microcrystalline cellulose as described below. NREL acid-pretreated corn stover liquor (1.2 liters) was incubated with 10 g of AVICEL.RTM. overnight at room temperature. The supernatant fraction was removed by vacuum filtration through Whatman #3 filter paper. The AVICEL.RTM. and adsorbed liquor components were washed 6 times with 500 ml of acetonitrile (occasionally acetone was used), followed by elution with 2 liters of water in 300-400 ml fractions. The liquor components that in combination with a GH61 polypeptide demonstrated a GH61 polypeptide-dependent enhancement of cellulolysis by the Trichoderma reesei cellulase composition were largely eluted in the first 2 water elution fractions (FIG. 8B), though later elution fractions contained some small amount of residual liquor. Water-eluted liquor components were concentrated approximately 10-fold using a Macrosep 1 kD Omega centrifuge filter (Pall Corporation, East Hills, N.Y., USA) and applied to an 8.0 mm.times.300 mm Shodex Sugar SP0810 HPLC chromatography column (Showa Denka America, Inc., NY, USA) using an AGILENT.RTM. 1100 HPLC and CHEMSTATION.RTM. software (Agilent Technologies, Santa Clara, Calif., USA) and separated by isocratic elution with water at a flow rate of 0.5 ml per minute at 80.degree. C. for 50 minutes, and collecting 250 .mu.l fractions in 2.2 ml, 96-deep well plates (Axygen, Union City, Calif., USA). Repeated 50 .mu.l injections of the liquor followed by repeated fraction collection yielded approximately 8 ml of each fraction. Peaks were identified by diode array detection at 210, 280 and 340 nm using a CHEMSTATION.RTM., AGILENT.RTM. 1100 HPLC (Agilent Technologies, Santa Clara, Calif., USA) (FIG. 8, dashed lines).
[0487] Peak fractions were assayed according to Example 3 with the following exceptions. 400 .mu.l of each fraction were added to the hydrolysis reactions containing 4 mg of the T. reesei cellulase composition and 1 mg of Thermoascus aurantiacus GH61A polypeptide. Aliquots were removed and analyzed for sugar content at 1, 3, and 10 days of saccharification. Control reactions containing the original NREL acid-pretreated corn stover liquor and control reactions containing no liquor and no GH61 polypeptide were run in parallel. FIG. 8B shows that addition of the various fractions resulted in fluctuation between fractions around a mean fractional hydrolysis value of 0.617 (FIG. 8, solid lines). Several peaks of activity consisting of multiple sequential fractions with high cellulolytic activity in the presence of the T. aurantiacus GH61A polypeptide were observed, including one broad peak of activity centered at fraction 7C. This peak corresponded with a peak in A.sub.(280). The extent of the cellulolytic enhancement by the fraction appeared small, indicating that the amount fractionated was very small. Absorbance at 280 nm was consistent with contents of the fractions possessing aromatic characteristics.
Example 22: Effect of NREL Pretreated Corn Stover Liquor Fractions and the Thermoascus aurantiacus GH61A Polypeptide on Hydrolysis of Microcrystalline Cellulose by the Trichoderma reesei Cellulase Composition
[0488] Pooled, lyophilized fractions of the NREL acid-pretreated corn stover liquor were each added to approximately 25 mg per ml of AVICEL.RTM. in 700 .mu.l of 50 mM sodium acetate pH 5.0 in the presence of 3 mM calcium chloride with 37.5 .mu.g of the Thermoascus aurantiacus GH61A polypeptide per ml and were incubated in 1.7 ml microcentrifuge tubes at 50.degree. C. with shaking at 1500 rpm for 48 hours in a Thermomixer (Eppendorf, Hamburg, Germany). Following the incubation, the samples were centrifuged at 31,000 rpm in a microcentrifuge for 5 minutes. A series of AVICEL.RTM. masses were weighed out and suspended in 700 .mu.l of equivalent buffer, incubated for an equivalent length of time, and pelleted equivalently. The height of the AVICEL.RTM. was measured using a transparent ruler, and assuming the microcentrifuge tubes were roughly equivalent in volume, the volume of the conical portion of the tube was given by the following equation:
V = 1 3 .times. .pi. .times. .times. r 2 .times. h ( Equation .times. .times. 4 ) ##EQU00004##
Thus the volume of the AVICEL.RTM. scales proportionally with the height of the pellet, and a measurement of pellet height could therefore be used to approximate the volume.
[0489] FIG. 9 shows a standard curve of AVICEL.RTM. height vs. mass of AVICEL.RTM.. The measurement of the pellet heights for various masses of AVICEL.RTM. scaled linearly within a volume region spanning the conical portion of the tube.
[0490] FIG. 10 shows the height of the AVICEL.RTM. for several of the pooled NREL acid-pretreated corn stover liquor HPLC fractions incubated with the T. aurantiacus GH61A polypeptide. One of these pooled fractions (denoted 6H-7A) incubated with the T. aurantiacus GH61A polypeptide was notably higher, 9 mM compared with an average of all other samples of 5.2 mM, a 1.7-fold larger volume. The AVICEL.RTM. in this sample had swollen to a volume equivalent to an AVICEL.RTM. mass of approximately 90 mg. Pooled fractions 6H-7A along with the T. aurantiacus GH61A polypeptide clearly induced swelling of the AVICEL.RTM., whereas inclusion of other liquor fractions with the GH61 polypeptide did not induce swelling.
[0491] The supernatants from the AVICEL.RTM. incubations were then decanted off and the pellet dried using a GeneVac EZ-2 Plus.RTM. vacuum concentrator (Genevac Inc., Gardiner, N.Y., USA). The dried residues were dissolved in deuterated water and were analyzed by .sup.1H NMR using a VARIAN.RTM. MercuryVx 400 MHz NMR (Varian, Palo Alto, Calif., USA). While the components of the liquor could not be identified by NMR spectroscopy, the NREL liquor fractions that produced swelling of the AVICEL.RTM. on incubation with the T. aurantiacus GH61 polypeptide generated NMR peaks corresponding to saccharide chemical shifts.
[0492] The solid residual cellulose was then tested for reducing end content using a method modified from Zhang and Lynd, 2005, Biomacromolecules 6: 1510-151. The cellulose was washed in 1 ml of 1.1% sodium dodecyl sulfate (SDS) and incubated at 95.degree. C. for 10 minutes with shaking, and then the suspension was pelleted by centrifugation at 31,000 rpm in a microcentrifuge. The SDS solution was decanted off and the pellet was washed by repeated resuspension in 1.5 ml of 70% ethanol and pelleting. The pellets were finally incubated with a 1:1 solution of H.sub.2O and BCA working solution (1:1 mixtures of 0.624 g of CuSO.sub.4.5H.sub.2O, and 0.631 g of L-serine per 500 ml of H.sub.2O with 0.971 g of disodium 2,2'-bichinchoninate, 27.12 g of Na.sub.2CO.sub.3, and 12.1 g of NaHCO.sub.3 per 500 ml of H.sub.2O) at 65.degree. C. for 30 minutes. The concentration of reducing ends was determined by comparison to a standard curve generated by serial dilution of glucose in the same BCA working solution and measurement of A.sub.(600 nm) in a 96-well microtiter plate using a POWERWAVE X.TM. microplate spectrophotometer (Biotek Instruments, Winooski, Vt. USA).
[0493] FIG. 11A shows the glucose reducing end standard curve of A.sub.600 nm vs. glucose concentration. The concentration of reducing ends in the T. aurantiacus GH61A polypeptide incubated samples was calculated from this standard curve. FIG. 11B shows the number of reducing end equivalents for AVICEL.RTM. incubated with the GH61 polypeptide and the indicated NREL acid-pretreated corn stover HPLC fractions. The same NREL acid-pretreated corn stover fractions that induced GH61 polypeptide-dependent swelling of the AVICEL.RTM., showed a much higher apparent concentration of reducing end equivalents in the insoluble, washed residual cellulose suggesting that hydration of these reducing ends was the likely cause of the swelling of the cellulose.
Example 23: Enrichment of Acid-Pretreated Xylan Components
[0494] Xylan was acid-pretreated and then fractionated by HPLC chromatography according to Example 21 for NREL acid-pretreated corn stover liquor with the following exceptions. Acid pretreated xylan was generated by incubating 45 g of beechwood xylan with 400 g of 1.1% H.sub.2SO.sub.4 at 190.degree. C. for 2 minutes in a a 1 gallon, high-pressure horizontal stirred reactor (Parr Instrument Company, Moline, Ill., USA). No preincubation with cellulose and organic phase wash was performed. The acid-pretreated xylan was filtered with a 3 kDa MWCO VIVASPIN 20.RTM. centrifuge filter (GE Healthcare, Piscataway, N.J., USA), retained on a Macrosep 1 kD Omega centrifuge filter (Pall, Ann Arbor, Mich.), washed with 3-fold volumes of deionized water, and applied to a 8.0 mm.times.300 mm Shodex Sugar SP0810 chromatography column (Showa Denka America, Inc., NY, USA) using an AGILENT.RTM. 1100 HPLC and CHEMSTATION.RTM. software, and separated by isocratic elution with water at a flow rate of 0.5 ml per minute at 80.degree. C. for 50 minutes, collecting 250 .mu.l fractions in 2.0 ml, 96-deep well plates (Axygen, Union City, Calif., USA). Repeated 100 .mu.l injections of the liquor followed by repeated fraction collection yielded approximately 5 ml of each fraction, which were pooled and lyophilized to dryness. Later separations included an overnight incubation of the acid-pretreated xylan with endoxylanase (SHEARZYME.TM., Novozymes A/S, Bagsvaerd, Denmark) in 50 mM sodium acetate pH 5.0 at 50.degree. C. to hydrolyze xylo-oligomers, followed by removal of the enzyme with a 3 kDa MWCO VIVASPIN 20.RTM. centrifuge filter prior to chromatography. Thermoascus aurantiacus GH61A polypeptide titrations to AVICEL.RTM. hydrolysis reactions containing either the endoxylanase-treated or the untreated xylan liquor were performed according to Example 3 and confirmed that endoxylanase treatment had not altered the effects of acid-pretreated xylan on GH61 polypeptide enhancement of cellulolysis in these samples. Absorbance of the eluted fractions was not determined. Fractions were assayed by dissolution of the lyophilized fractions in 200 .mu.l of water, and addition of 100 .mu.l to saccharification reactions according to Example 3, containing 4 mg of the T. reesei cellulase composition and 1 mg of the T. aurantiacus GH61A polypeptide, or no GH61A.
[0495] FIG. 12 shows the hydrolysis of AVICEL.RTM. by the T. reesei cellulase composition with supplemented T. aurantiacus GH61A polypeptide and the HPLC fractions as indicated. This elution profile demonstrated a broad elution peak of fractions that in the presence of the GH61 polypeptide resulted in higher hydrolysis by the T. reesei cellulase composition. Thus, these fractions contained compounds that in the presence of GH61 polypeptide promoted GH61 polypeptide-dependent cellulolytic enhancement. Several inhibitory fractions were also observed, as is evident in FIG. 12, fraction 5D. The elution of the components that facilitate GH61 polypeptide cellulolytic enhancement centered on the same fractions that had previously been shown to facilitate GH61 polypeptide cellulolytic enhancement in chromatography of NREL pretreated corn stover liquor (Example 15). It is likely that the width of the elution was due to overloading of the HPLC column and concomitant loss of theoretical plate. This hypothesis was further supported by the magnitude of the enhancement in cellulolysis by the HPLC fractions examined, which was much higher than observed following NREL acid-pretreated corn stover liquor.
[0496] The fractions that had higher fractional hydrolysis in the presence of the Thermoascus aurantiacus GH61A polypeptide were analyzed by LC-MS in the following manner. Samples were diluted in 0.1% formic acid to 20 .mu.g per ml approximate concentrations. Samples were then either filtered using Ultrafree-MC centrifugal filter devices (Millipore Corporation, Billerica, Mass., USA) or were centrifuged for 10 minutes at 21,000.times.g and then stored at 4.degree. C. if analysis was not performed immediately. UPLC.RTM. tandem mass spectrometry, was performed using a Q-Tof micron.TM. hybrid orthogonal quadrupole time-of-flight mass spectrometer (Waters Micromass MS Technologies, Milford, Mass., USA) using MASSLYNX.TM. software version 4.1 (Waters Micromass MS Technologies, Milford, Mass., USA). The Q-TOF MICRO.TM. was fitted with an ACQUITY UPLC.RTM. using a 1.0.times.50 mm, C18, 1.7 .mu.m, BEH Acquity column (Waters Corp, Milford, Mass., USA) to permit chromatographic separation of analytes. The following elution gradient was applied over a 37 minute interval at a flow rate of 100 .mu.l per minute: 0-15 minutes from 1-15% acetonitrile with 0.1% formic acid, 15-20 minutes from 15-40% acetonitrile with 0.1% formic acid, 20-25 minutes from 40-80% acetonitrile with 0.1% formic acid, 25-30 minutes with 80% acetonitrile with 0.1% formic acid, 30-31 minutes from 80-1% acetonitrile with 0.1% formic acid, and 31-37 minutes with 1% acetonitrile with 0.1% formic acid. Elution was monitored at 280 nm through a diode array detector and eluents from the column were introduced directly into the Q-TOF MICRO.TM. via electrospray ion source. A cone voltage of 20 volts was typically used and the collision energy was varied in the range of 5-15 volts. Data were acquired in survey scan mode from a mass range of m/z 50 to 1000 with switching criteria for MS to MS/MS that included an ion intensity of greater than 50.0 counts per second. The acquired spectra were combined, smoothed, and centered in an automated fashion. Analytes were identified by comparison to spectra of standard compounds when possible. Standard compounds were analyzed before and after the samples to confirm the mass accuracy and retention time stability of the sample analyses.
[0497] FIG. 13 shows a representative LC-MS chromatogram of a T. aurantiacus GH61A polypeptide cellulolytic enhancing acid-pretreated xylan HPLC fraction. It is clear from the number of peaks that significant heterogeneity was observed. The bulk of the components were eluted from the LC in the first 6-minutes, suggesting a highly polar or charged composition.
Example 24: Using GH61 Polypeptide-Binding Affinity to Enrich NREL Acid-Pretreated Corn Stover Liquor for Components that are Functional in GH61 Polypeptide-Dependent Cellulolytic Enhancement
[0498] Thermoascus aurantiacus GH61A polypeptide affinity was used as a means to enrich NREL acid-pretreated corn stover liquor components that bind to the GH61 polypeptide. Seventy mg of the T. aurantiacus GH61A polypeptide were incubated overnight with 50 ml of liquor. The GH61 polypeptide-bound liquor components were separated from the free components by ultracentrifugation using a 10 kDa MWCO VIVASPIN 20.RTM. centrifuge filter, concentrating the protein and protein-bound fraction 10-fold, followed by washing with an equivalent volume of water to the starting material (50 ml total volume), and repeating 5-times. Finally, the protein was denatured by incubation at 90.degree. C. for 30 minutes, and centrifuged using a 10 kDa MWCO VIVASPIN 20.RTM. centrifuge filter. The flow-through from the filter was analyzed using liquid chromatography mass spectrometry according to Example 23, with electrospray mass spectrometry performed in both positive and negative ionization modes.
[0499] FIG. 14 shows the LC-MS chromatogram of the NREL acid-pretreated corn stover liquor components that bound to the T. aurantiacus GH61A polypeptide and were eluted by denaturation. Both positive and negative mode ion chromatographs were combined, yielding a small set of unique mass ions, many of which could be excluded as dimers, fragments of larger parent ions or contaminants derived from the buffer or from the centrifuge columns, leading to identification of approximately 30 unique mass ions of reasonable intensity. Based on the monoisotopic masses obtained, chemical formulae were determined and a database search of chemical compounds (ChemSpider, Royal Society of Chemistry) yielded a list of putative compounds for GH61 polypeptide assay. In the chromatograms, 3 broadly classified sets of compounds could be identified: the first set eluted in the first 2-minutes, had smaller mass and were likely small polar or charged compounds; a second lower abundance set, corresponding to a broadly varied set of molecular weights but likely to be less polar, eluted with intermediate retention times; and finally a series of larger molecular weight ions eluted between 12 and 20 minutes, corresponding to less polar or higher molecular weight compounds. It was observed that the search of monoisotopic masses frequently yielded compounds consistent with plant flavonols, flavanols, oxidized flavonoids, oxidized flavanols and similar compounds, thus a large set of these compounds were tested for GH61 cellulolytic enhancing activity.
Example 25: Effect of NREL Pretreated Corn Stover Liquor and the Thermoascus Aurantiacus GH61A Polypeptide on Generating Soluble Cellodextrin from Microcrystalline Cellulose or Phosphoric Acid-Swollen Cellulose
[0500] Monosaccharide, disaccharide, and aldonic and uronic acid standards (Sigma-Aldrich, St. Louis, Mo., USA) were dissolved in water at 1 mg per ml and were diluted to 50 .mu.g per ml in 10 mM NaOH. Cellooligosaccharides (Sigma-Aldrich, St, Louis, Mo., USA) were dissolved in water at 25 to 30 mg per ml and were diluted as above. Xylooligosaccharides (Megazyme Bray, Co. Wicklow, Ireland) were dissolved in water at 10 to 30 mg per ml and were diluted as above. Lactobionic acid was purchased from Sigma-Aldrich (St. Louis, Mo., USA), dissolved in water at 2.5 mg per ml. The aldonic acids of each cellooligosaccharide were generated by incubation of 17 .mu.g of Humicola insolens cellobiose dehydrogenase (WO 2010/080527) per ml with 2.5 mg per ml of each cellooligosaccharide in 50 mM sodium acetate pH 5.0 at 50.degree. C. and 10 mM dichloroindophenol (DCIP; Sigma-Aldrich, St. Louis, Mo., USA) was added incrementally. The equivalence point was achieved when DCIP was not reduced by the solution, and the color remained purple. Formation of the aldonate products was confirmed by LC-MS according to Example 25. Cellobiose dehydrogenase was then removed by ultrafiltration through a VIVASPIN 20.RTM. 10 kDa MWCO centrifuge filter.
[0501] The Thermoascus aurantiacus GH61A polypeptide was incubated at 37.5 .mu.g/ml with either 29.5 mg per ml microcrystalline cellulose (AVICEL.RTM..TM., Sigma Aldrich, St. Louis, Mo., USA) or 1.2% (w/w) phosphoric acid-swollen cellulose (PASC) in 50 mM sodium acetate, 1 mM MnSO.sub.4 for 5 days at 50.degree. C. with or without 10% (v/v) NREL pretreated corn stover liquor. NREL pretreated corn stover liquor was extracted according to Example 2. Phosphoric acid swollen cellulose was prepared from AVICEL.RTM. PH101 using the protocol described by Zhang et al., 2006, Biomacromolecules 7: 644-648. Control incubations containing AVICEL.RTM. alone, PASC alone, AVICEL.RTM. or PASC with NREL acid-pretreated corn stover liquor in the absence of the T. aurantiacus GH61A polypeptide were performed in the same manner. The samples were then filtered through a 0.22 .mu.m centrifuge filter, and the filtrates were diluted 1:10 in 1 ml final volume of 10 mM NaOH and analyzed by DIONEX.RTM. Ion Chromatography with pulsed amperometry detection (IC-PAD, Dionex Corporation, Sunnyvale, Calif.) using DIONEX.RTM. Chromeleon or PeakNet Software. Ten .mu.l of 2.5 mg per ml lactobionic acid was added as an external loading control. Chromatographic separation was obtained using a PA-20 column (Dionex Corporation, Sunnyvale, Calif., USA), with relevant guard, borate and amine-trap pre-columns, and elution was achieved with an isocratic gradient of 13 mM sodium hydroxide, 2.5 mM sodium acetate for 20 minutes, followed by a linear gradient from 13 to 50 mM NaOH, isocratic gradient for 10 minutes and linear gradient from 0.5 to 40 mM sodium acetate in 50 mM NaOH.
[0502] FIG. 15A shows the chromatograms of AVICEL.RTM. and PASC incubations with GH61 polypeptide, with GH61 polypeptide and pretreated corn stover liquor, and control incubations. The chromatograms indicated that soluble oligosaccharide products were evolved from the incubations containing the GH61 polypeptide, liquor, buffer, and cellulose in both PASC and microcrystalline forms. The arrows indicate unique elution peaks that were present only in the incubations containing the full set of components. FIG. 15B shows a comparison of chromatogram of AVICEL.RTM. incubated with the T. aurantiacus GH61A polypeptide, acid-pretreated corn stover liquor, and buffer to the chromatograms of standard compounds. FIG. 15B indicated that the retention times of the novel products were consistent with those of cellopentaose, cellotetraose, and cellotriose. Three additional elution peaks were also evident; a large peak with retention time of 49 minutes matched no available standard but was likely cellohexaose based on its retention time in comparison to the cellooligosaccharides and aldonic acid standards. The relative peak intensities indicated that the major product may be cellotetraose, though the sparing solubility of cellooligosaccharides of DP 6 may have understated the actual production of these oligomers.
Example 26: Effect of Water-Extracted and Alkaline Pretreated Corn Stover Liquors in Combination with the Thermoascus aurantiacus GH61A on Cellulolysis by the T. reesei Cellulase Composition
[0503] The Thermoascus aurantiacus GH61A polypeptide was assayed for cellulolytic enhancing activity according to Example 3 with 10% (v/v) added liquor from either alkaline pretreated corn stover or 10% (v/v) added liquor from water-extracted acid-pretreated corn stover according to Example 2. Alkaline pretreated corn stover was prepared in the following manner. Milled and washed, raw PCS was treated with 4%, 6%, 8% or 10% (w/w) NaOH for 1 hr at 90.degree. C. in a 2-L stirred tank reactor (IKA Works, Inc., Wilmington, N.C., USA). Water extracted corn stover liquors were generated in the following manner. Raw corn stover was milled in a WILEY.RTM. Mill (Model 4; Thomas Scientific, Swedesboro, N.J., USA) with a nominal screen size of 2 mm. The milled stover was sieved through a #40 mesh screen and the fines were discarded. A Dionex Accelerated Solvent Extractor (ASE) 350 (Dionex Corporation, Sunnyvale, Calif., USA) instrument was used for all pretreatments with two cycles for each pretreatment. The first cycle of each pretreatment was a pressurized hot water extraction run in standard flow mode of operation. The second cycle of the pretreatment was the same for each experimental sample, and was a mid-severity dilute acid pretreatment run in pressure solvent saver mode of operation. Approximately 20.0 g of the sieved corn stover were packed into a 100 ml stainless steel extraction cell. During the pressurized hot water extraction the stover was extracted at temperatures of 100-170.degree. C. in 10.degree. C. increments for a static reaction time of 7 minutes. After extraction the cell was purged with nitrogen for 60 seconds, and all extraction liquor collected. Static pretreatment time was defined from the time the internal temperature of the cell reached equilibrium with the heating chamber. Liquors from the first, water-extraction step were filtered using a 10 kDa MWCO VIVASPIN 20.RTM. centrifuge filter and were pH adjusted to 5.0 by addition of NaOH or HCl, prior to use.
[0504] As shown previously, GH61 polypeptide titrations in the absence of added liquors did not significantly enhance microcrystalline cellulose hydrolysis by the T. reesei cellulase composition (Examples 15, 16, and 18). FIG. 16A shows the fractional hydrolysis of AVICEL.RTM. by the T. reesei cellulase composition with increasing concentrations of the T. aurantiacus GH61A polypeptide in the presence of liquors from various severities of alkaline pretreated corn stover. Addition of alkaline corn stover liquors increasing in severity from 4% NaOH to 10% NaOH showed reduced fractional hydrolysis, thus increasing inhibition in the absence of the GH61 polypeptide. Unlike acid-pretreated corn stover liquors, however, addition of the GH61 polypeptide in the presence of alkaline liquors provided no substantial improvement over saccharifications without liquor.
[0505] The higher severity alkaline pretreated corn stover liquors in combination with the T. aurantiacus GH61A polypeptide did provide a small amount of cellulolytic enhancing activity at 7 days of hydrolysis. Comparing zero to 50% GH61 polypeptide additions, hydrolysis in the presence of 6% NaOH liquor was 0.473.+-.0.00483 and 0.488.+-.0.00724, 8% NaOH was 0.454.+-.0.00352 and 0.485.+-.0.000119, and 10% NaOH was 0.445.+-.0.00625 and 0.476.+-.0.00732 The addition of the T. aurantiacus GH61A polypeptide was sufficient to partially mitigate the inhibition arising from the liquor additions; as the hydrolysis in the absence of liquor was 0.502.+-.0.0109 and 0.499.+-.0.00642 at zero and 50% (w/w) GH61A polypeptide, respectively.
[0506] FIG. 16B shows the fractional hydrolysis of AVICEL.RTM. by the T. reesei cellulase composition with increasing concentrations of the T. aurantiacus GH61A polypeptide in the presence of corn stover liquors extracted with water at the indicated temperatures. Pressurized water-extracted corn stover liquors did not appreciably enhance fractional hydrolysis in the presence of the GH61 polypeptide. Water-extracted liquors generated at 150.degree. C. and above show some inhibition, both in the absence and presence of the GH61 polypeptide. These data indicated that pressurized water-extraction did not extract the liquor components required to observe GH61 polypeptide cellulolytic enhancing activity on microcrystalline cellulose.
Example 27: Effect of Acid-Pretreated Xylan, Acid-Pretreated Biomass Component Mixtures Containing Xylan, or Acid-Pretreated Monosaccharide Components of Xylan Including Xylose and Arabinose in Combination with Thermascus aurantiacus GH61A Polypeptide on Cellulose Hydrolysis by the Trichoderma reesei Cellulase Composition
[0507] The cellulolytic enhancing effect of the T. aurantiacus GH61A polypeptide on microcrystalline cellulose was assayed with components of biomass that had been acid-pretreated.
[0508] The Thermoascus aurantiacus GH61A polypeptide was assayed for cellulolytic enhancing activity according to the procedure described in Example 3 with the following exceptions: hydrolysis reactions were performed with 10% (v/v) added liquor from acid-pretreated corn stover (Example 2) or with 10% (v/v) added liquor from acid-pretreated biomass components including xylan, cellulose, protein, lipid, monosaccharide, and combinations thereof. AVICEL.RTM. (1.8 g), oatspelt xylan (1.1 g; TCI), lignin (0.9 g; MeadWestvaco), corn gluten (0.25 g; Sigma Aldrich, St. Louis, Mo., USA), gallic acid (0.25 g; Sigma Aldrich, St. Louis, Mo., USA), ferulic acid (0.25 g; Sigma Aldrich, St. Louis, Mo., USA), or corn oil (0.25 g; Sigma Aldrich, St. Louis, Mo., USA) were pretreated by dissolution in 25 ml of 1% H.sub.2SO.sub.4 and incubation in an SBL-2 fluidized sand bath reactor with TC-8D Temperature Controller (Techne Inc., Burlington, N.J., USA) for a 5 minutes temperature ramp followed by 5 minutes at 190.degree. C. Aliquots from saccharification reaction were removed for analysis at 1 and 5 days. Liquors were filtered using a 10 kDa MWCO VIVASPIN 20.RTM. centrifuge filter and pH adjusted to 5.0 by addition of NaOH or HCl, prior to use.
[0509] FIGS. 17A and 17B show fractional hydrolysis of AVICEL.RTM. by the T. reesei cellulase composition with various concentrations of the T. aurantiacus GH61A polypeptide in the presence of liquors derived from acid-pretreatment of various biomass components. Titration of the T. aurantiacus GH61A polypeptide from 0 to 50% (w/w) addition to the T. reesei cellulase composition yielded, in the absence of liquor, no enhancement of AVICEL.RTM. hydrolysis (Example 15, 16, 18, and FIG. 19B). Increasing concentrations of the T. aurantiacus GH61A polypeptide increases fractional hydrolysis in the presence of a subset of the liquors examined. Small GH61 polypeptide concentration-dependent hydrolysis enhancements were observed in the presence of liquors of acid-pretreated lignin, lipid, protein, cellulose, lignin model compounds such as gallic acid, and hemicellulase components such as ferulic acid. The extent of GH61 polypeptide enhancement of fractional hydrolysis in the presence of acid-liquors was from 0.390.+-.0.007 to 0.435.+-.0.010 in the presence of lignin liquor, from 0.305.+-.0.013 to 0.341.+-.0.016 in the presence of corn oil liquor, 0.493.+-.0.007 to 0.524.+-.0.004 in the presence of corn gluten liquor, 0.134.+-.0.021 to 0.184.+-.0.023 in the presence of gallic acid liquor, 0.352.+-.0.011 to 0.397.+-.0.029 in the presence of ferulic acid liquor at 7 days of hydrolysis. Conversely, liquor derived from acid-pretreatment of xylan yielded a substantial GH61 polypeptide concentration-dependent enhancement of cellulolysis, from 0.390.+-.0.009 to 0.540.+-.0.013 at 7 days of hydrolysis. Where possible, biomass components had been acid pretreated at their respective concentrations in NREL acid-pretreated corn stover, thus the observed GH61 cellulolytic enhancements should scale proportionally with their contributions to enhancements in pretreated corn stover. The bulk of the apparent GH61 polypeptide concentration-dependent cellulolytic enhancements on cellulose are thus derived from the xylan components.
Example 28: Effect of Residual Liquors Post-Fermentation in Combination with the Thermoascus aurantiacus GH61A Polypeptide on Cellulose Hydrolysis by the T. reesei Cellulase Composition
[0510] The Thermoascus aurantiacus GH61A polypeptide was assayed for cellulolytic-enhancing activity according to Example 3 with the following exceptions: 10% (v/v) added liquor from post-fermentation residues was added, and aliquots were removed at 1, 3, and 7 days of saccharification. Liquors were filtered using a 10 kDa MWCO VIVASPIN 20.RTM. centrifuge filter and pH adjusted to 5.0 by addition of NaOH or HCl, prior to use. Fermentation liquors were obtained as follows: 20% total solids NREL unwashed, unmilled pretreated corn stover was hydrolyzed by 5 mg per gram cellulose of a T. reesei cellulase composition for 5-days. The hydrolysate was fermented for 2 days using a Saccharomyces cerevisiae strain comprising a xylose isomerase gene (WO 2003/062430) at 3% cell density for 2 days. Fifty ml of the fermentation liquor was filtered as described and assayed for GH61 polypeptide concentration-dependent cellulolytic enhancement activity.
[0511] FIG. 18 shows the fractional hydrolysis of AVICEL.RTM. by the T. reesei cellulase composition with various concentrations of the T. aurantiacus GH61A polypeptide in the presence of post-fermentation liquors. The addition of increasing T. aurantiacus GH61A polypeptide concentrations significantly enhanced hydrolysis of AVICEL.RTM. in the presence of the post-fermentation liquors from 0.246.+-.0.007 to 0.265.+-.0.007 at 1 day of hydrolysis and from 0.616.+-.0.006 to 0.895.+-.0.002 at 7 days of hydrolysis.
Example 29: Effect of Corn Stover Liquors from Increasing Severity of Acid Pretreatments in Combination with the Thermoascus aurantiacus GH61A Polypeptide on Cellulose Hydrolysis by the T. reesei Cellulase Composition
[0512] The Thermoascus aurantiacus GH61A polypeptide was assayed for cellulolytic enhancing activity according to Example 3 with 10% (v/v) added liquor from acid-pretreated corn stover or microcrystalline cellulose (AVICEL.RTM.) of various severity pretreatments. Liquors were filtered using a 10 kDa MWCO VIVASPIN 20.RTM. centrifuge filter and pH adjusted to 5.0 by addition of NaOH or HCl, prior to use. Liquors were generated using an SBL-2 fluidized sand bath reactor with TC-8D Temperature Controller, incubating corn stover with 1% H.sub.2SO.sub.4 in 25 ml and incubating at temperatures between 140.degree. C. and 170.degree. C. for 1 to 5 minutes, or by incubating AVICEL.RTM. with 1% H.sub.2SO.sub.4 in 25 ml at temperatures between 110.degree. C. and 190.degree. C. for 5 minutes.
[0513] FIGS. 19A and 19B show the fractional hydrolysis of AVICEL.RTM. containing various severity acid-treatments of corn stover by the T. reesei cellulase composition and various concentrations of the T. aurantiacus GH61A polypeptide. Liquors were generated by systematically varying both temperature between 140.degree. C. and 170.degree. C. and varying time at each temperature between 1 and 5 minutes. For saccharification reactions containing liquors produced by acid-pretreatment at temperatures greater than 140.degree. C. and 5 minutes, increasing T. aurantiacus GH61A polypeptide concentrations increased fractional hydrolysis. The higher severity pretreatments, particularly those of 160.degree. C. and above, showed a greater increase in fractional hydrolysis from GH61 polypeptide addition than did the lower severity pretreatments of 150.degree. C. and less. Additionally, higher severity pretreatment liquors were increasingly inhibitory to cellulolysis, thus the greatest overall cellulose conversion was obtained by addition of high GH61 polypeptide concentrations (50% w/w) at intermediate pretreatment severity, specifically 150.degree. C. for 5 minutes, 160.degree. C. for 3-5 minutes, or 170.degree. C. for 1 minute. These data demonstrated that pretreatment of a biomass can be tailored to maximize hydrolysis for a given GH61 polypeptide concentration in a cellulase composition. There is an optimum pretreatment condition that balances GH61 polypeptide cellulolytic-enhancing activity with production of soluble inhibitor compounds.
[0514] FIG. 19C shows the fractional hydrolysis of AVICEL.RTM. containing various severity acid-treatments of AVICEL.RTM. by the T. reesei cellulase composition and various concentrations of the T. aurantiacus GH61A polypeptide. In calculating the fractional hydrolysis, sugars derived from liquor addition were not subtracted, and contributed up to 3% of conversion. From FIG. 19C it is clear that liquors derived from acid-treatment of microcrystalline cellulose in combination with the T. aurantiacus GH61A polypeptide enhanced hydrolysis only marginally.
Example 30: Effect of Xylan Liquors from Increasing Severity of Acid Pretreatments in Combination with the Thermoascus aurantiacus GH61A Polypeptide on Cellulose Hydrolysis by the T. reesei Cellulase Composition
[0515] The Thermoascus aurantiacus GH61A polypeptide was assayed for cellulolytic enhancing activity according to Example 3 with 10% (v/v) added liquor from xylan pretreated with varying severity. Liquors were generated by incubation of 1.1 g of either oatspelt xylan or wheat flour with 1% H.sub.2SO.sub.4 or 3% HCl in 25 ml total volume in an SBL-2 fluidized sand bath reactor with TC-8D Temperature Controller for 10 minutes total; a 5 minute ramp period followed by a 5 minute incubation at the indicated temperature. Incubation temperatures were varied between 60.degree. C. and 180.degree. C. Pretreatment liquors were filtered using a 10 kDa MWCO VIVASPIN 20.RTM. centrifuge filter and pH adjusted to 5.0 by addition of NaOH or HCl, prior to use.
[0516] FIGS. 20A and 20B show the effect of acid-pretreated xylan liquors added to the T. reesei cellulase compositions with increasing concentrations of the T. aurantiacus GH61A polypeptide. In the absence of the GH61 polypeptide, liquors of increasing pretreatment severity were increasingly inhibitory to the cellulolysis of AVICEL.RTM., particularly those generated with 3% HCl. Titration of increasing GH61 polypeptide concentration in the presence of xylan acid-pretreated at temperatures greater than 140.degree. C. led to increasing cellulolysis, and the net change in hydrolysis with GH61 polypeptide addition was higher for higher severity pretreatments. Hydrolysis was not improved by the GH61 polypeptide on addition of xylan pretreated at 60.degree. C. to 120.degree. C. Hydrolysis was improved by 0.057, from 0.349.+-.0.00510 to 0.406.+-.0.00394 with 160.degree. C. liquor, by 0.089 from 0.287.+-.0.00233 to 0.376.+-.0.00118 with 180.degree. C. liquor, and by 0.0740, from 0.216.+-.0.00225 to 0.293.+-.0.00843 with 180.degree. C. HCl derived liquor. The effect of increasing pretreatment severity on T. aurantiacus GH61A polypeptide cellulolytic enhancing activity with xylan liquors was similar to those of corn stover pretreatment severity (Example 26), and again suggested a maximal overall hydrolysis was obtained by high GH61 polypeptide concentrations in the presence of liquors of intermediate severity. In this case, 50% (w/w) T. aurantiacus GH61A polypeptide in the presence of 160.degree. C. acid-pretreated xylan produced the highest cellulose conversion. Additionally, as previously observed for corn stover liquors, there appeared to be an optimum balance between GH61 polypeptide cellulolytic enhancing activity and the production of soluble inhibitors. The addition of a specific concentration and tailoring of pretreatment severity can be accomplished for a given GH61 polypeptide concentration within a cellulase composition.
[0517] FIG. 20C shows the fractional hydrolysis of AVICEL.RTM. containing various severity acid-treatments of beechwood xylan by the T. reesei cellulase composition and various concentrations of the T. aurantiacus GH61A polypeptide. From FIG. 20C, it was clear that the maximum conversion was achieved in the presence of a high concentration of the T. aurantiacus GH61A polypeptide (50%) and liquor produced at 170.degree. C. In the presence of liquor generated at 190.degree. C., the observed hydrolysis was lower for all concentrations of the T. aurantiacus GH61A polypeptide tested, and in the presence of liquors generated below 170.degree. C.; the conversion levels reached were not as great. These data suggest that treatment severity of some substrates may be optimized to generate liquors for a given GH61 polypeptide concentration within a cellulase composition.
Example 31: Effect of Solid-Phase Extracted Acid Pretreated Corn Stover Liquors in Combination with the Thermoascus aurantiacus GH61A Polypeptide on Cellulose Hydrolysis by the T. reesei Cellulase Composition
[0518] The Thermoascus aurantiacus GH61A polypeptide was assayed for cellulolytic enhancing activity according to Example 3 with the following exceptions. T. aurantiacus GH61 polypeptide was either not added or was added at 50% (w/w) of total protein and 10% (v/v) solid phase extraction resin-eluted liquor was added to each hydrolysis reaction. Both NREL acid-pretreated corn stover liquor prepared according to Example 2 and acid-pretreated xylan prepared according to Example 23, and then pH adjusted to 5.0, were applied to one of two solid phase extraction cartridges, either a BOND ELUT.TM. C-18 column (Varian, Palo Alto, Calif., USA) or a STRATA.TM.-X Polymeric column (Phenomenex, Torrance, Calif., USA) equilibrated in water. The samples were washed 3-times with 1 ml of water and then eluted with two aliquots of 600 .mu.l methanol. The eluted samples were diluted back to their original volumes with water and assayed for GH61 polypeptide cellulolytic enhancing activity.
[0519] FIG. 21 shows the fractional hydrolysis of AVICEL.RTM. by the T. reesei cellulase composition with and without 50% (w/w) T. aurantiacus GH61A polypeptide addition in the presence of solid-phase extracted liquors as indicated. The fractional hydrolysis was equivalent within error for all samples, indicating that no enhancement of cellulolysis from GH61 polypeptide addition was observed in the presence of solid-phase extracted liquors from acid-pretreated corn stover or from acid-pretreated xylan. A cellulolytic enhancement of the T. reesei cellulase composition was previously demonstrated in the presence of these liquors in combination with addition of the GH61 polypeptide, prior to solid-phase extraction. It was therefore concluded that the compounds present in these liquors that were correlated with the observed GH61 polypeptide cellulolytic enhancing activity were eluted during the wash steps.
Example 32: Effect of Electrodialyzed Acid-Pretreated Corn Stover Liquors on Thermoascus aurantiacus GH61A Polypeptide Enhancing the Trichoderma reesei Cellulase Composition
[0520] The Thermoascus aurantiacus GH61A polypeptide was assayed for cellulolytic-enhancing activity according to Example 3 with the following exceptions: 10% (v/v) NREL pretreated corn stover liquor, acid-pretreated xylan (Example 23) or electrodialyzed NREL pretreated corn stover liquor was added to each hydrolysis reaction. Electrodialyzed NREL pretreated corn stover liquor was generated in the following manner. Two kg of NREL pretreated corn stover liquor was filtered and. the pH was adjusted to 5.0 using concentrated NaOH. The pretreatment liquor was subjected to concentrating electrodialysis using a EUR2B pilot scale electrodialysis unit (Ameridia, Somerset, N.J., USA) equipped with a EUR2B-10 stack Ameridia, Somerset, N.J., USA). The solution of pH adjusted liquor had a conductance of 16.5 mS/cm and a pH of 5.0 and was charged to the diluate tank. A solution of potassium nitrate (20 mS/cm, 2.0 kg) was charged to the electrode rinse tank. A dilute solution of NREL pretreatment liquor (0.657 mS/cm) was charged to the concentrate tank. The solutions described above were circulated through the EUR2B-10 stack at a flow rate of approximately 0.9 gallon per minute (gpm) using a DC power supply set to 14 volts. After 55 minutes, the conductivity change in the diluate and concentrate tanks remained stable and the run was ended. The final conductance in the concentrate tank was 14.01 mS/cm.
[0521] FIG. 22 shows that increasing the T. aurantiacus GH61A polypeptide concentration in the presence of NREL pretreated corn stover liquor, electrodialyzed NREL acid-pretreated corn stover liquor, and acid-pretreated xylan increased the fractional hydrolysis of AVICEL.RTM.. At the highest concentrations of GH61 polypeptide, the electrodialyzed NREL liquor showed slightly lower conversion than the undialyzed sample, 0.541.+-.0.0006 compared with 0.524.+-.0.0118, though at intermediate GH61 polypeptide concentrations the conversion was higher for the electrodialyzed liquor, 0.467.+-.0.00671 compared with 0.502.+-.0.00215.
Example 33: Effect of Addition of Acid-Pretreated Corn Stover Liquors and Thermoascus aurantiacus GH61A Polypeptide to Non-Acid Pretreated Biomass Feedstocks
[0522] The Thermoascus aurantiacus GH61A polypeptide was assayed for cellulolytic-enhancing activity according to Example 3 with the following exceptions: 5% (v/v) added NREL acid-pretreated corn stover liquor was added to either 3% or 5% total solids of various milled biomass feedstocks, and aliquots were removed for analysis at 1, 3, and 7 days of saccharification. Control incubations of each biomass containing either no T. reesei cellulase composition and no GH61 polypeptide, or containing GH61 polypeptide in the absence of the T. reesei cellulase composition with and without liquor, and control incubations containing an equivalent concentration of liquor with the T. reesei cellulase composition and GH61 polypeptide without biomass were performed in parallel. The composition of total accessible cellulose in each biomass was determined by measurement of glucose equivalents produced by saccharification by 50 mg of Cellic CTec.TM. (available Novozymes A/S, Bagsvaerd, Denmark) over 7 days of hydrolysis. Liquors were filtered using a 10 kDa MWCO VIVASPIN 20.RTM. centrifuge filter and pH adjusted to 5.0 by addition of NaOH or HCl, prior to use. Biomass feedstocks included raw sugarcane bagasse and corn stovers pretreated in the following manners: organosolv low severity ethanol, organosolv medium severity ethanol, organosolv high severity ethanol, and organosolv glycerol.
[0523] FIGS. 23A, 23B, 23C and 23D show the fractional hydrolysis of various biomass feedstocks by the T. reesei cellulase composition with varying T. aurantiacus GH61A polypeptide concentrations, with and without supplemented NREL acid-pretreated corn stover liquor, as indicated. For each biomass, addition of 5% (v/v) NREL acid-pretreated corn stover liquor in the absence of the T. reesei cellulase composition added a background sugar concentration equivalent to approximately 0.05 increase in apparent hydrolysis, depending on the relative amount of cellulose present in each substrate. This was not substantially changed by addition of the T. reesei cellulase composition or the GH61 polypeptide. Since these liquor-derived sugars are not subtracted from the fractional hydrolysis values, for each biomass the presence of liquor shifts the apparent hydrolysis to a higher value.
[0524] For each biomass, a higher fractional hydrolysis was obtained by addition of higher T. aurantiacus GH61A polypeptide concentrations from 0 to 50% (w/w) total protein to the T. reesei cellulase composition. For some of these biomasses, the presence of the NREL acid-pretreated corn stover liquor increased the total cellulolytic enhancement by the T. aurantiacus GH61A polypeptide. This was particularly apparent at 3 days of hydrolysis (gray bars, FIG. 24), as exemplified by the enhancement of low and medium severity ethanol organosolv pretreatments. In the absence of GH61 polypeptide, fractional hydrolysis of low severity ethanol organosolv corn stover was 0.148.+-.0.00447 without liquor and 0.165.+-.0.00126 with liquor, whereas at 50% GH61, fractional hydrolysis was increased from 0.237.+-.0.00737 to 0.273.+-.0.00113. Similarly, in the absence of GH61 polypeptide, fractional hydrolysis of medium severity ethanol corn stover was 0.179.+-.0.000737 without liquor and 0.190.+-.0.00401 with liquor, whereas at 50% GH61 polypeptide, fractional hydrolysis was increased from 0.281.+-.0.0136 to 0.328.+-.0.00543. Substrates that showed this augmentation of the GH61 polypeptide cellulolytic enhancing effect included organosolv low and medium severity ethanol and sugarcane bagasse. It appeared that cellulolysis of lower severity pretreatment biomass feedstocks were better enhanced by the T. aurantiacus GH61A polypeptide in the presence of supplemented acid-pretreated biomass liquor. The augmentation of the GH61 polypeptide cellulolytic enhancing activity on the low severity pretreatment by supplemented liquor appeared most dramatic at the early stages of saccharification.
Example 34: Effect of Addition of Acid-Pretreated Monosaccharides on Thermoascus Aurantiacus GH61A Enhancement of Trichoderma reesei Cellulase Composition Cellulolysis of Microcrystalline Cellulose
[0525] The Thermoascus aurantiacus GH61A polypeptide was assayed for cellulolytic enhancing activity as described in Example 3 with the following exceptions. Saccharification reactions were performed with 5 mM MnSO.sub.4, and either 5% or 10% (v/v) liquor derived from acid-pretreatment of monosaccharides was added. Liquors were generated in the following manner: 20 mg per ml of each monosaccharide was incubated with 1% H.sub.2SO.sub.4 in an SBL-2 fluidized sand bath reactor with TC-8D Temperature Controller at 190.degree. C. for 5 minutes, and the resulting liquors were then filtered using a 10 kDa MWCO VIVASPIN 20.RTM. centrifuge filter and pH adjusted to 5.0 by addition of NaOH or HCl prior to use. Ozonolysis was performed in the following manner: NREL acid-pretreated corn stover was slurried at 15% total solids, and the liquor extracted by vacuum filtration using a glass fiber filter. This liquor was incubated with a low concentration of ozone for 30 minutes prior to pH adjustment.
[0526] FIG. 24 shows the fractional hydrolysis of AVICEL.RTM. by the T. reesei cellulase composition with either no GH61A polypeptide or 24% (w/w) T. aurantiacus GH61A polypeptide in the presence of 5% or 10% (v/v) of various acid-pretreated monosaccharides, and with ozone-treated NREL pretreated corn stover liquor. FIG. 25 demonstrates that acid-pretreated monosaccharides were strongly inhibitory to cellulolysis by the T. reesei cellulase composition in the absence of GH61 polypeptide. In the presence of acid-pretreated hemicellulose-derived sugars, e.g., C5 sugars, including arabinose and xylose, the fractional hydrolysis was increased by addition of the T. aurantiacus GH61A polypeptide. In the presence of 5% acid-pretreated arabinose, addition of the GH61 polypeptide increased fractional hydrolysis from 0.183.+-.0.00372 to 0.223.+-.0.00548 at 3 days of hydrolysis, and from 0.229.+-.0.00379 to 0.328.+-.0.00272 at 7 days of hydrolysis. Similarly, in the presence of 5% acid-pretreated xylose, addition of the GH61 polypeptide increased fractional hydrolysis from 0.113.+-.0.0148 to 0.140.+-.0.00356 at 3 days of hydrolysis, and from 0.117.+-.0.00252 to 0.177.+-.0.00602 at 7 days of hydrolysis. Conversely, addition of the T. aurantiacus GH61A polypeptide in the presence of acid-pretreated glucose increased fractional hydrolysis to a much smaller extent, even at the higher concentration of 10% (v/v), from 0.172.+-.0.00658 to 0.206.+-.0.00108. Ozone treatment of NREL acid-pretreated corn stover liquor altered the liquor components in such a manner as to disrupt the ability of the liquor in combination with the GH61 polypeptide to enhance cellulolysis of AVICEL.RTM. by the T. reesei cellulase composition. In the presence of liquor, the GH61 polypeptide increased fractional hydrolysis from 0.522.+-.0.00612 to 0.679.+-.0.00807, whereas in the presence of the ozone-treated liquor, the fractional hydrolysis was increased only from 0.512.+-.0.00347 to 0.540.+-.0.00956.
Example 35: Enhancement of AVICEL.RTM. Cellulolysis by T. reesei Cellulases Using NREL PCS Liquor and Various GH61 Polypeptides
[0527] Microcrystalline cellulose saccharification reactions were performed as described (Example 3), using 29.5 mg of microcrystalline cellulose (AVICEL.RTM.) per ml and 4 mg of the T. reesei cellulase composition per g cellulose in 50 mM sodium acetate, 1 mM manganese sulfate at pH 5.0 in the presence of 10% (v/v) NREL PCS liquor prepared as described (Example 2). GH61 polypeptides including Thermoascus aurantiacus GH61A polypeptide, Aspergillus fumigatus GH61B polypeptide and Penicillium pinophilum GH61 polypeptide were added between 0 and 2 mg per g cellulose (0 and 50% (w/w) of the T. reesei cellulase composition concentration). Alternatively, liquor was added at 10% (v/v) to saccharifications containing either no GH61 polypeptide or 2 mg of the various GH61 polypeptides per g cellulose.
[0528] FIG. 25 shows the fractional hydrolysis of AVICEL.RTM. by the T. reesei cellulase composition with various concentrations of the indicated GH61 polypeptides with 10% (v/v) NREL PCS liquor. Fractional hydrolysis is shown for the T. reesei cellulase composition with Thermoascus aurantiacus GH61A (circles), Aspergillus fumigatus GH61B polypeptide (diamonds) and Penicillium pinophilum GH61 polypeptide (squares) at 1 day (open symbols) and 3 days of hydrolysis (closed symbols). Addition of liquor in combination with all the GH61 polypeptides showed an apparent increase in hydrolysis of the cellulose. Thermoascus aurantiacus GH61A polypeptide produced the greatest enhancement of cellulolysis in the presence of liquor, 0.098.+-.at 7 days of hydrolysis. Conversely, addition of the highest concentration of each of the GH61 polypeptides did not enhance cellulolysis in the absence of supplemented liquor. Multiple GH61 polypeptides are thus shown to enhance cellulolysis by T. reesei cellulases in the presence of biomass liquor.
Example 36: Effect of Addition of Kraft (Indulin) Lignin or Oxidized Kraft (Indulin) Lignin on the Thermoascus aurantiacus GH61A Enhancement of Trichoderma reesei Cellulase Composition Cellulolysis of Microcrystalline Cellulose
[0529] Microcrystalline cellulose saccharification were performed as described (Example 3), using 25 mg of microcrystalline cellulose (AVICEL.RTM.) per ml and 4 mg per g cellulose of a composition containing a blend of an Aspergillus aculeatus GH10 xylanase (WO 94/021785) and a Trichoderma reesei cellulase preparation containing Aspergillus fumigatus beta-glucosidase (WO 2005/047499) and Thermoascus aurantiacus GH61A polypeptide (WO 2005/074656) in 50 mM sodium acetate, 1 mM manganese sulfate at pH 5.0 in the presence of zero or 0.1% (w/w) Kraft (Indulin) lignin or oxidized Kraft (Indulin) lignin. Oxidized Kraft lignin was generated by overnight incubation of a 20% total solids slurry of lignin with sodium periodate at 4.degree. C. at a concentration of 5 g of sodium periodate per 100 g of slurry. The oxidized lignin was washed extensively with water and then freeze dried. The T. reesei cellulase composition was either supplemented with an additional 15% (w/w) T. aurantiacus GH61A polypeptide or was not supplemented.
[0530] FIG. 26 shows the concentration of glucose produced by saccharification at various saccharification times as indicated. Addition of lignin or oxidized lignin enhanced the saccharification of AVICEL.RTM. by the T. reesei cellulase composition containing GH61A polypeptide. Addition of 15% (w/w) supplemental GH61A polypeptide to a T. reesei cellulase composition containing T. aurantiacus GH61A polypeptide further increased the extent of saccharification of a hydrolysis reaction containing Kraft lignin. Addition of 15% supplemental GH61A polypeptide to a cellulase composition containing GH61A polypeptide resulted in lower glucose conversion than comparable reactions without supplemental GH61A polypeptide in hydrolyses of AVICEL.RTM. containing no Kraft lignin or containing oxidized Kraft lignin. These data indicate that products of biomass pretreatment derived from specific lignins or the lignins themselves, in combination with GH61A polypeptide, enhance the conversion of cellulose by a T. reesei cellulase composition.
Example 37: Thermoascus aurantiacus GH61A Enhancement of Cellulolysis of High Total Solids Alkaline Pretreated Corn Stover by a Trichoderma reesei Cellulase Composition
[0531] Raw, washed and milled corn stover was pretreated with 8% (w/w of dry weight) sodium hydroxide at 90.degree. C. for 1 hour. The resulting whole slurry was transferred to a vacuum filtration apparatus and washed exhaustively with tap water until the pH of the filtrate was less than or equal to 8.6. The washed solids were then transferred to hydrolysis reactors to give final solids concentrations of 10%. A composition containing a blend of an Aspergillus aculeatus GH10 xylanase (WO 94/021785) and a Trichoderma reesei cellulase preparation containing Aspergillus fumigatus beta-glucosidase (WO 2005/047499) and Thermoascus aurantiacus GH61A polypeptide (WO 2005/074656) was replaced with increasing concentrations of GH61A polypeptide, maintaining a fixed total protein concentration of 4 mg protein per gram cellulose. The washed, milled, alkaline pretreated corn stover was hydrolyzed at 50.degree. C. for 120 hours in 10-20 g Oak Ridge tubes (Nalge Nunc International Corporation, Rochester, N.Y., USA) containing 1/4 inch steel balls for agitation, using a FINEPCR Hybridization Oven (Daigger, Inc. Vernon Hills, Ill., USA), rotating at 12 rpm.
[0532] FIG. 27 shows that glucose concentration increased with increasing replacement of the cellulase composition with T. aurantiacus GH61A polypeptide, indicating that cellulose hydrolysis was enhanced with 5-10% additional GH61A polypeptide. Thus at high total solids, supplementation of a T. reesei cellulase composition with higher relative concentrations of GH61 polypeptide resulted in higher cellulose conversion of alkaline pretreated corn stovers.
Example 38: Thermoascus aurantiacus GH61A Enhancement of Cellulolysis of High Total Solids Pretreated Corn Stover of Various Pretreatment Severities by a Trichoderma reesei Cellulase Composition
[0533] Raw corn stover was milled in a Thomas Model 10 Wiley Mill (Thomas Scientific, Swedesboro, N.J., USA) with a nominal screen size of 2 mm and then thoroughly washed with tap water. The washed corn stover was allowed to dry in a 45.degree. C. convection oven until the dry solids content was above 90%. The dried solids were then sieved through a #40 mesh screen to ensure a uniform size distribution. An Accelerated Solvent Extractor (ASE) 350 instrument (Dionex Corporation, Bannockburn, Ill., USA) was used for all pretreatments. Approximately 15.0 g of the washed, milled and sieved corn stover was packed into a 100 ml stainless steel extraction cell to ensure consistent solids loading during pretreatment. The extraction cell was loaded into the heating chamber and filled with sulfuric acid solution of various concentrations until the back-pressure reached 1500 psi. The heating chamber then heated the filled cell to the desired temperature. At the end of the heating phase, the pretreatment was stopped by immediately releasing the pressure in the extraction cell, purging with nitrogen gas, and collecting the pretreatment liquor in a designated glass vial. The extraction cell was then immediately removed from the ASE 350 instrument and quenched in ice. After cooling to room temperature, the pretreated solids were removed from the cell and re-slurried with the pretreatment liquor. The following pretreatment conditions were varied: temperature from 150-190.degree. C., static residence times between 1 and 15 minutes, and acid concentrations between 0.4%-1.0% (w/w) H.sub.2SO.sub.4. The range for combined severity factor ranged from 0.5 to 2.0.
[0534] Pretreated corn stover at each pretreatment severity was adjusted to pH 5.0 and a final TS of 15%. Hydrolysis was initiated by adding to each PCS batch either 2 mg of a composition containing a blend of an Aspergillus aculeatus GH10 xylanase (WO 94/021785) and a Trichoderma reesei cellulase preparation containing Aspergillus fumigatus beta-glucosidase (WO 2005/047499) and Thermoascus aurantiacus GH61A polypeptide (WO 2005/074656) with 0.4 mg of T. aurantiacus GH61A polypeptide per gram cellulose or 1.6 mg of the composition containing a blend of an Aspergillus aculeatus GH10 xylanase (WO 94/021785) and a Trichoderma reesei cellulase preparation containing Aspergillus fumigatus beta-glucosidase (WO 2005/047499) and Thermoascus aurantiacus GH61A polypeptide (WO 2005/074656) with 0.4 mg of T. aurantiacus GH61A polypeptide per gram cellulose and incubating at 50.degree. C. for up to 216 hrs in 10-20 g Oak Ridge tubes containing 1/4 inch steel balls for agitation, using a FINEPCR Hybridization Oven, rotating at 12 rpm.
[0535] FIG. 28 shows the cellulose conversion of high total solids (15% dry weight) corn stover of various severity acid pretreatments. At each set of pretreatment conditions, except for a single, low severity pretreatment, replacement of the cellulase composition with 20% additional T. aurantiacus GH61A polypeptide resulted in greater cellulose conversion, particularly at 216 hours of hydrolysis. Gray bars: 120 hours of saccharification; black bars: 216 hours of saccharification. Thus at high total solids, supplementation of a T. reesei cellulase composition with higher relative concentrations of GH61 polypeptide resulted in higher cellulose conversion when the pretreatment severity was sufficient to generate suitable biomass liquor.
Example 39: Thermoascus aurantiacus GH61A Enhancement of Cellulolysis of High Total Solids Pretreated Giant Cane (Arundo donax) of Various Pretreatment Severities by a Trichoderma reesei Cellulase Composition
[0536] Raw Arundo donax was milled in a Thomas Model 10 Wiley Mill with a nominal screen size of 2 mm and then thoroughly washed with tap water. The washed A. donax biomass was allowed to dry in a 45.degree. C. convection oven until the dry solids content was above 90%. The dried solids were then sieved through a #40 mesh screen to ensure a uniform size distribution. An Accelerated Solvent Extractor (ASE) 350 instrument was used for all pretreatments. Approximately 20.0 g of the washed, milled and sieved Arundo donax was packed into a 100 ml stainless steel extraction cell to ensure consistent solids loading during pretreatment. The extraction cell was loaded into the heating chamber and filled with sulfuric acid solution of various concentrations until the back-pressure reached 1500 psi. The heating chamber then heated the filled cell to the desired temperature. At the end of the heating phase, the pretreatment was stopped by immediately releasing the pressure in the extraction cell, purging with nitrogen gas and collecting the pretreatment liquor in a designated glass vial. The extraction cell was then immediately removed from the ASE 350 instrument and quenched in ice. After cooling to room temperature, the pretreated solids were removed from the cell and re-slurried with the pretreatment liquor. The following pretreatment conditions were varied: temperature from 170-190.degree. C., static residence times between 1 and 5 minutes, and acid concentrations between 0.5%-1.0% (w/w) H.sub.2SO.sub.4. The range for combined severity factor ranged from 0.35 to 2.13.
[0537] Arundo donax biomass of each pretreatment severity was adjusted to pH 5.0 and a final TS of 15%. Hydrolysis was initiated by adding either 4 mg of a composition containing a blend of an Aspergillus aculeatus GH10 xylanase (WO 94/021785) and a Trichoderma reesei cellulase preparation containing Aspergillus fumigatus beta-glucosidase (WO 2005/047499) and Thermoascus aurantiacus GH61A polypeptide (WO 2005/074656) per gram cellulose or 3.4 mg of the a composition containing a blend of an Aspergillus aculeatus GH10 xylanase (WO 94/021785) and a Trichoderma reesei cellulase preparation containing Aspergillus fumigatus beta-glucosidase (WO 2005/047499) and Thermoascus aurantiacus GH61A polypeptide (WO 2005/074656) and 0.6 mg of T. aurantiacus GH61A per gram cellulose and incubating at 50.degree. C. for up to 120 hours in 10-20 g Oak Ridge tubes containing 1/4 inch steel balls for agitation, using a FINEPCR Hybridization Oven, rotating at 12 rpm.
[0538] FIG. 29 shows the cellulose conversion of high total solids (15% dry weight) Arundo donax of various severity acid pretreatments. At each set of pretreatment conditions, replacement of the cellulase composition with 15% additional T. aurantiacus GH61A polypeptide resulted in greater cellulose conversion, particularly at 216 hours of hydrolysis. Gray bars: 72 hours of saccharification; black bars: 120 hours of saccharification. Thus at high total solids, supplementation of a T. reesei cellulase composition with higher relative concentrations of GH61 polypeptide resulted in higher cellulose conversion of A. donax biomass.
[0539] The present invention is further described by the following numbered paragraphs:
[0540] [1] A method for degrading or converting a cellulosic material, comprising: treating the cellulosic material with an enzyme composition in the presence of a polypeptide having cellulolytic enhancing activity and a liquor, wherein the combination of the polypeptide having cellulolytic enhancing activity and the liquor enhances hydrolysis of the cellulosic material by the enzyme composition.
[0541] [2] The method of paragraph 1, wherein the cellulosic material is pretreated.
[0542] [3] The method of paragraph 1 or 2, further comprising recovering the degraded cellulosic material.
[0543] [4] The method of any of paragraphs 1-3, wherein the enzyme composition comprises one or more (several) enzymes selected from the group consisting of a cellulase, a hemicellulase, an esterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin.
[0544] [5] The method of paragraph 4, wherein the cellulase one or more (several) enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase.
[0545] [6] The method of paragraph 4, wherein the hemicellulase is one or more (several) enzymes selected from the group consisting of a xylanase, an acetyxylan esterase, a feruloyl esterase, an arabinofuranosidase, a xylosidase, and a glucuronidase.
[0546] [7] The method of any of paragraphs 1-6, wherein the degraded cellulosic material is a sugar.
[0547] [8] The method of paragraph 7, wherein the sugar is selected from the group consisting of glucose, xylose, mannose, galactose, and arabinose.
[0548] [9] A method for producing a fermentation product, comprising:
[0549] (a) saccharifying a cellulosic material with an enzyme composition in the presence of a polypeptide having cellulolytic enhancing activity and a liquor, wherein the combination of the polypeptide having cellulolytic enhancing activity and the liquor enhances hydrolysis of the cellulosic material by the enzyme composition;
[0550] (b) fermenting the saccharified cellulosic material with one or more fermenting microorganisms to produce the fermentation product; and
[0551] (c) recovering the fermentation product from the fermentation.
[0552] [10] The method of paragraph 9, wherein the cellulosic material is pretreated.
[0553] [11] The method of paragraph 9 or 10, wherein the enzyme composition comprises one or more (several) enzymes selected from the group consisting of a cellulase, a hemicellulase, an expansin, an esterase, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin.
[0554] [12] The method of paragraph 11, wherein the cellulase is one or more (several) enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase.
[0555] [13] The method of paragraph 11, wherein the hemicellulase is one or more (several) enzymes selected from the group consisting of a xylanase, an acetyxylan esterase, a feruloyl esterase, an arabinofuranosidase, a xylosidase, and a glucuronidase.
[0556] [14] The method of any of paragraphs 9-13, wherein steps (a) and (b) are performed simultaneously in a simultaneous saccharification and fermentation.
[0557] [15] The method of any of paragraphs 9-14, wherein the fermentation product is an alcohol, an alkane, a cycloalkane, an alkene, an amino acid, a gas, isoprene, a ketone, an organic acid, or polyketide.
[0558] [16] A method of fermenting a cellulosic material, comprising: fermenting the cellulosic material with one or more fermenting microorganisms, wherein the cellulosic material is saccharified with an enzyme composition in the presence of a polypeptide having cellulolytic enhancing activity and a liquor, wherein the combination of the polypeptide having cellulolytic enhancing activity and the liquor enhances hydrolysis of the cellulosic material by the enzyme composition.
[0559] [17] The method of paragraph 16, wherein the cellulosic material is pretreated before saccharification.
[0560] [18] The method of paragraph 16 or 17, wherein the enzyme composition comprises one or more (several) enzymes selected from the group consisting of a cellulase, a hemicellulase, an expansin, an esterase, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin.
[0561] [19] The method of paragraph 18, wherein the cellulase is one or more (several) enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase.
[0562] [20] The method of paragraph 18, wherein the hemicellulase is one or more (several) enzymes selected from the group consisting of a xylanase, an acetyxylan esterase, a feruloyl esterase, an arabinofuranosidase, a xylosidase, and a glucuronidase.
[0563] [21] The method of any of paragraphs 16-20, wherein the fermenting of the cellulosic material produces a fermentation product.
[0564] [22] The method of paragraph 21, further comprising recovering the fermentation product from the fermentation.
[0565] [23] The method of any of paragraphs 16-22, wherein the fermentation product is an alcohol, an alkane, a cycloalkane, an alkene, an amino acid, a gas, isoprene, a ketone, an organic acid, or polyketide.
[0566] [24] The method of any of paragraphs 1-23, wherein the liquor is obtained from a material that is the same as the cellulosic material subjected to saccharification by the enzyme composition.
[0567] [25] The method of any of paragraphs 1-23, wherein the liquor is obtained from a material that is different than the cellulosic material subjected to saccharification by the enzyme composition.
[0568] [26] The method of any of paragraphs 1-23, wherein the liquor is obtained from a material that is the same as the cellulosic material subjected to saccharification by the enzyme composition, and the treatment conditions used to produce the liquor are different from the pretreatment conditions of the cellulosic material.
[0569] [27] The method of any of paragraphs 1-23, wherein the liquor is obtained from a material that is the same as the cellulosic material subjected to saccharification by a cellulase composition, and the treatment conditions used to produce the liquor are the same as the pretreatment conditions of the cellulosic material.
[0570] [28] The method of any of paragraphs 1-23, wherein the liquor is obtained from a material that is the same as the cellulosic material subjected to saccharification by the enzyme composition, and the treatment conditions used to produce the liquor are the same as the pretreatment conditions of the cellulosic material, and the liquor is further processed to remove cellulose inhibitors.
[0571] [39] The method of any of paragraphs 1-23, wherein the liquor is obtained from a material that is different than the cellulosic material subjected to saccharification by the enzyme composition, and the treatment conditions used to produce the liquor are different from the pretreatment conditions of the cellulosic material.
[0572] [30] The method of any of paragraphs 1-23, wherein the liquor is obtained from a material that is different than the cellulosic material subjected to saccharification by the enzyme composition, and the treatment conditions used to produce the liquor are the same as the pretreatment conditions of the cellulosic material.
[0573] [31] The method of any of paragraphs 1-23, wherein the liquor is obtained from a material that is different than the cellulosic material subjected to saccharification by the enzyme composition, and the treatment conditions used to produce the liquor are the same as the pretreatment conditions of the cellulosic material, and the liquor is further processed to remove cellulose inhibitors.
[0574] [32] The method of any of paragraphs 1-31, wherein the liquor optimizes the cellulolytic enhancing activity of a GH61 polypeptide with a GH61 effect of preferably at least 1.05, more preferably at least 1.10, more preferably at least 1.15, more preferably at least 1.2, more preferably at least 1.25, more preferably at least 1.3, more preferably at least 1.35, more preferably at least 1.4, more preferably at least 1.45, more preferably at least 1.5, more preferably at least 1.55, more preferably at least 1.6, more preferably at least 1.65, more preferably at least 1.7, more preferably at least 1.75, more preferably at least 1.8, more preferably at least 1.85, most preferably at least 1.9, most preferably at least 1.95, and even most preferably at least 2.
[0575] [33] The method of any of paragraphs 1-31, wherein an effective amount of the liquor to cellulose is about 10.sup.-6 to about 10 g per g of cellulose, e.g., about 10.sup.-6 to about 7.5 g, about 10.sup.-6 to about 5, about 10.sup.-6 to about 2.5 g, about 10.sup.-6 to about 1 g, about 10.sup.-5 to about 1 g, about 10.sup.-5 to about 10.sup.-1 g, about 10.sup.-4 to about 10.sup.-1 g, about 10.sup.-3 to about 10.sup.-1 g, or about 10.sup.-3 to about 10.sup.-2 g per g of cellulose.
[0576] [34] The method of any of paragraphs 1-31, wherein the liquor is present in an amount that minimizes inhibition of a cellulase composition of about 1 to about 20% (v/v), e.g., about 1 to about 15%, about 1 to about 10%, about 2 to about 7%, about 2 to about 5%, or about 3 to about 5%.
[0577] [35] The method of any of paragraphs 1-31, wherein the liquor is obtained from a lignocellulose material, a hemicellulose material, a lignacious material, monosaccharides of the lignocellulose material, monosaccharides of the hemicellulose material, or a combination thereof
[0578] [36] The method of any of paragraphs 1-35, wherein the liquor is further processed to remove inhibitors of a cellulase, a hemicellulase, or a combination thereof.
[0579] [37] An isolated liquor, which in combination with a polypeptide having cellulolytic enhancing activity enhances hydrolysis of a cellulosic material by a cellulolytic enzyme.
[0580] [38] A composition comprising a polypeptide having cellulolytic enhancing activity and a liquor, wherein the combination of the polypeptide having cellulolytic enhancing activity and the liquor enhances hydrolysis of a cellulosic material by a cellulolytic enzyme.
[0581] [39] The composition of paragraph 38, which further comprises one or more (several) enzymes selected from the group consisting of a cellulase, a hemicellulase, an esterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollen in.
[0582] [40] The composition of paragraph 39, wherein the cellulase one or more (several) enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase.
[0583] [41] The composition of paragraph 39, wherein the hemicellulase is one or more (several) enzymes selected from the group consisting of a xylanase, an acetyxylan esterase, a feruloyl esterase, an arabinofuranosidase, a xylosidase, and a glucuronidase.
[0584] [42] The composition of any of paragraphs 38-41, wherein the liquor optimizes the cellulolytic enhancing activity of a GH61 polypeptide with a GH61 effect of preferably at least 1.05, more preferably at least 1.10, more preferably at least 1.15, more preferably at least 1.2, more preferably at least 1.25, more preferably at least 1.3, more preferably at least 1.35, more preferably at least 1.4, more preferably at least 1.45, more preferably at least 1.5, more preferably at least 1.55, more preferably at least 1.6, more preferably at least 1.65, more preferably at least 1.7, more preferably at least 1.75, more preferably at least 1.8, more preferably at least 1.85, most preferably at least 1.9, most preferably at least 1.95, and even most preferably at least 2.
[0585] [43] The composition of any of paragraphs 38-41, wherein an effective amount of the liquor to cellulose is about 10.sup.-6 to about 10 g per g of cellulose, e.g., about 10.sup.-6 to about 7.5 g, about 10.sup.-6 to about 5, about 10.sup.-6 to about 2.5 g, about 10.sup.-6 to about 1 g, about 10.sup.-5 to about 1 g, about 10.sup.-5 to about 10.sup.-1 g, about 10.sup.-4 to about 10.sup.-1 g, about 10.sup.-3 to about 10.sup.-1 g, or about 10.sup.-3 to about 10.sup.-2 g per g of cellulose.
[0586] [44] The composition of any of paragraphs 38-41, wherein the liquor is obtained from a lignocellulose material, a hemicellulose material, a lignacious material, monosaccharides of the lignocellulose material, monosaccharides of the hemicellulose material, or a combination thereof.
[0587] The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.
Sequence CWU
1
1
16611846DNAThielavia terrestris 1aattgaagga gggagtggcg gagtggccac
caagtcaggc ggctgtcaac taaccaagga 60tgggaacagt tcggctcgcc ttgcccgagg
gcagcgttcc ctgatgggga cgaaccatgg 120gactggggtc agctgctgta taaaagttca
aatcgatgat ctctcagatg gcgctgctgg 180ggtgttctgc gcttttccat cctcgcaacc
tggtatccca ctagtccagc gttcggcacc 240atgaagtcgt tcaccattgc cgccttggca
gccctatggg cccaggaggc cgccgcccac 300gcgaccttcc aggacctctg gattgatgga
gtcgactacg gctcgcaatg tgtccgcctc 360ccggcgtcca actcccccgt caccaatgtt
gcgtccgacg atatccgatg caatgtcggc 420acctcgaggc ccaccgtcaa gtgcccggtc
aaggccggct ccacggtcac gatcgagatg 480caccaggttc gcacgcctct ctgcgtaggc
cccccagcta ctatatggca ctaacacgac 540ctccagcaac ctggcgaccg gtcttgcgcc
aacgaggcta tcggcggcga ccactacggc 600cccgtaatgg tgtacatgtc caaggtcgat
gacgcggtga cagccgacgg ttcatcgggc 660tggttcaagg tgttccagga cagctgggcc
aagaacccgt cgggttcgac gggcgacgac 720gactactggg gcaccaagga cctcaactcg
tgctgcggca agatgaacgt caagatcccc 780gaagacatcg agccgggcga ctacctgctc
cgcgccgagg ttatcgcgct gcacgtggcc 840gccagctcgg gcggcgcgca gttctacatg
tcctgctacc agctgaccgt gacgggctcc 900ggcagcgcca ccccctcgac cgtgaatttc
ccgggcgcct actcggccag cgacccgggc 960atcctgatca acatccacgc gcccatgtcg
acctacgtcg tcccgggccc gaccgtgtac 1020gcgggcggct cgaccaagtc ggctggcagc
tcctgctccg gctgcgaggc gacctgcacg 1080gttggttccg gccccagcgc gacactgacg
cagcccacct ccaccgcgac cgcgacctcc 1140gcccctggcg gcggcggctc cggctgcacg
gcggccaagt accagcagtg cggcggcacc 1200ggctacactg ggtgcaccac ctgcgctgta
agttccctcg tgatatgcag cggaacaccg 1260tctggactgt tttgctaact cgcgtcgtag
tccgggtcta cctgcagcgc cgtctcgcct 1320ccgtactact cgcagtgcct ctaagccggg
agcgcttgct cagcgggctg ctgtgaagga 1380gctccatgtc cccatgccgc catggccgga
gtaccgggct gagcgcccaa ttcttgtata 1440tagttgagtt ttcccaatca tgaatacata
tgcatctgca tggactgttg cgtcgtcagt 1500ctacatcctt tgctccactg aactgtgaga
ccccatgtca tccggaccat tcgatcggtg 1560ctcgctctac catctcggtt gatgggtctg
ggcttgagag tcactggcac gtcctcggcg 1620gtaatgaaat gtggaggaaa gtgtgagctg
tctgacgcac tcggcgctga tgagacgttg 1680agcgcggccc acactggtgt tctgtaagcc
agcacacaaa agaatactcc aggatggccc 1740atagcggcaa atatacagta tcagggatgc
aaaaagtgca aaagtaaggg gctcaatcgg 1800ggatcgaacc cgagacctcg cacatgactt
atttcaagtc aggggt 18462326PRTThielavia terrestris 2Met
Lys Ser Phe Thr Ile Ala Ala Leu Ala Ala Leu Trp Ala Gln Glu1
5 10 15Ala Ala Ala His Ala Thr Phe
Gln Asp Leu Trp Ile Asp Gly Val Asp 20 25
30Tyr Gly Ser Gln Cys Val Arg Leu Pro Ala Ser Asn Ser Pro
Val Thr 35 40 45Asn Val Ala Ser
Asp Asp Ile Arg Cys Asn Val Gly Thr Ser Arg Pro 50 55
60Thr Val Lys Cys Pro Val Lys Ala Gly Ser Thr Val Thr
Ile Glu Met65 70 75
80His Gln Gln Pro Gly Asp Arg Ser Cys Ala Asn Glu Ala Ile Gly Gly
85 90 95Asp His Tyr Gly Pro Val
Met Val Tyr Met Ser Lys Val Asp Asp Ala 100
105 110Val Thr Ala Asp Gly Ser Ser Gly Trp Phe Lys Val
Phe Gln Asp Ser 115 120 125Trp Ala
Lys Asn Pro Ser Gly Ser Thr Gly Asp Asp Asp Tyr Trp Gly 130
135 140Thr Lys Asp Leu Asn Ser Cys Cys Gly Lys Met
Asn Val Lys Ile Pro145 150 155
160Glu Asp Ile Glu Pro Gly Asp Tyr Leu Leu Arg Ala Glu Val Ile Ala
165 170 175Leu His Val Ala
Ala Ser Ser Gly Gly Ala Gln Phe Tyr Met Ser Cys 180
185 190Tyr Gln Leu Thr Val Thr Gly Ser Gly Ser Ala
Thr Pro Ser Thr Val 195 200 205Asn
Phe Pro Gly Ala Tyr Ser Ala Ser Asp Pro Gly Ile Leu Ile Asn 210
215 220Ile His Ala Pro Met Ser Thr Tyr Val Val
Pro Gly Pro Thr Val Tyr225 230 235
240Ala Gly Gly Ser Thr Lys Ser Ala Gly Ser Ser Cys Ser Gly Cys
Glu 245 250 255Ala Thr Cys
Thr Val Gly Ser Gly Pro Ser Ala Thr Leu Thr Gln Pro 260
265 270Thr Ser Thr Ala Thr Ala Thr Ser Ala Pro
Gly Gly Gly Gly Ser Gly 275 280
285Cys Thr Ala Ala Lys Tyr Gln Gln Cys Gly Gly Thr Gly Tyr Thr Gly 290
295 300Cys Thr Thr Cys Ala Ser Gly Ser
Thr Cys Ser Ala Val Ser Pro Pro305 310
315 320Tyr Tyr Ser Gln Cys Leu
3253880DNAThielavia terrestris 3accccgggat cactgcccct aggaaccagc
acacctcggt ccaatcatgc ggttcgacgc 60cctctccgcc ctcgctcttg cgccgcttgt
ggctggccac ggcgccgtga ccagctacat 120catcggcggc aaaacctatc ccggctacga
gggcttctcg cctgcctcga gcccgccgac 180gatccagtac cagtggcccg actacaaccc
gaccctgagc gtgaccgacc cgaagatgcg 240ctgcaacggc ggcacctcgg cagagctcag
cgcgcccgtc caggccggcg agaacgtgac 300ggccgtctgg aagcagtgga cccaccagca
aggccccgtc atggtctgga tgttcaagtg 360ccccggcgac ttctcgtcgt gccacggcga
cggcaagggc tggttcaaga tcgaccagct 420gggcctgtgg ggcaacaacc tcaactcgaa
caactggggc accgcgatcg tctacaagac 480cctccagtgg agcaacccga tccccaagaa
cctcgcgccg ggcaactacc tcatccgcca 540cgagctgctc gccctgcacc aggccaacac
gccgcagttc tacgccgagt gcgcccagct 600ggtcgtctcc ggcagcggct ccgccctgcc
cccgtccgac tacctctaca gcatccccgt 660ctacgcgccc cagaacgacc ccggcatcac
cgtgagtggg cttccgttcc gcggcgagct 720ctgtggaaat cttgctgacg atgggctagg
ttgacatcta caacggcggg cttacctcct 780acaccccgcc cggcggcccc gtctggtctg
gcttcgagtt ttaggcgcat tgagtcgggg 840gctacgaggg gaaggcatct gttcgcatga
gcgtgggtac 8804239PRTThielavia terrestris 4Met
Arg Phe Asp Ala Leu Ser Ala Leu Ala Leu Ala Pro Leu Val Ala1
5 10 15Gly His Gly Ala Val Thr Ser
Tyr Ile Ile Gly Gly Lys Thr Tyr Pro 20 25
30Gly Tyr Glu Gly Phe Ser Pro Ala Ser Ser Pro Pro Thr Ile
Gln Tyr 35 40 45Gln Trp Pro Asp
Tyr Asn Pro Thr Leu Ser Val Thr Asp Pro Lys Met 50 55
60Arg Cys Asn Gly Gly Thr Ser Ala Glu Leu Ser Ala Pro
Val Gln Ala65 70 75
80Gly Glu Asn Val Thr Ala Val Trp Lys Gln Trp Thr His Gln Gln Gly
85 90 95Pro Val Met Val Trp Met
Phe Lys Cys Pro Gly Asp Phe Ser Ser Ser 100
105 110His Gly Asp Gly Lys Gly Trp Phe Lys Ile Asp Gln
Leu Gly Leu Trp 115 120 125Gly Asn
Asn Leu Asn Ser Asn Asn Trp Gly Thr Ala Ile Val Tyr Lys 130
135 140Thr Leu Gln Trp Ser Asn Pro Ile Pro Lys Asn
Leu Ala Pro Gly Asn145 150 155
160Tyr Leu Ile Arg His Glu Leu Leu Ala Leu His Gln Ala Asn Thr Pro
165 170 175Gln Phe Tyr Ala
Glu Cys Ala Gln Leu Val Val Ser Gly Ser Gly Ser 180
185 190Ala Leu Pro Pro Ser Asp Tyr Leu Tyr Ser Ile
Pro Val Tyr Ala Pro 195 200 205Gln
Asn Asp Pro Gly Ile Thr Val Asp Ile Tyr Asn Gly Gly Leu Thr 210
215 220Ser Tyr Thr Pro Pro Gly Gly Pro Val Trp
Ser Gly Phe Glu Phe225 230
23551000DNAThielavia terrestris 5ctcctgttcc tgggccaccg cttgttgcct
gcactattgg tagagttggt ctattgctag 60agttggccat gcttctcaca tcagtcctcg
gctcggctgc cctgcttgct agcggcgctg 120cggcacacgg cgccgtgacc agctacatca
tcgccggcaa gaattacccg gggtgggtag 180ctgattattg agggcgcatt caaggttcat
accggtgtgc atggctgaca accggctggc 240agataccaag gcttttctcc tgcgaactcg
ccgaacgtca tccaatggca atggcatgac 300tacaaccccg tcttgtcgtg cagcgactcg
aagcttcgct gcaacggcgg cacgtcggcc 360accctgaacg ccacggccgc accgggcgac
accatcaccg ccatctgggc gcagtggacg 420cacagccagg gccccatcct ggtgtggatg
tacaagtgcc cgggctcctt cagctcctgt 480gacggctccg gcgctggctg gttcaagatc
gacgaggccg gcttccacgg cgacggcgtc 540aaggtcttcc tcgacaccga gaacccgtcc
ggctgggaca tcgccaagct cgtcggcggc 600aacaagcagt ggagcagcaa ggtccccgag
ggcctcgccc ccggcaacta cctcgtccgc 660cacgagttga tcgccctgca ccaggccaac
aacccgcagt tctacccgga gtgcgcccag 720gtcgtcatca ccggctccgg caccgcgcag
ccggatgcct catacaaggc ggctatcccc 780ggctactgca accagaatga cccgaacatc
aaggtgagat ccaggcgtaa tgcagtctac 840tgctggaaag aaagtggtcc aagctaaacc
gcgctccagg tgcccatcaa cgaccactcc 900atccctcaga cctacaagat tcccggccct
cccgtcttca agggcaccgc cagcaagaag 960gcccgggact tcaccgcctg aagttgttga
atcgatggag 10006258PRTThielavia terrestris 6Met
Leu Leu Thr Ser Val Leu Gly Ser Ala Ala Leu Leu Ala Ser Gly1
5 10 15Ala Ala Ala His Gly Ala Val
Thr Ser Tyr Ile Ile Ala Gly Lys Asn 20 25
30Tyr Pro Gly Tyr Gln Gly Phe Ser Pro Ala Asn Ser Pro Asn
Val Ile 35 40 45Gln Trp Gln Trp
His Asp Tyr Asn Pro Val Leu Ser Cys Ser Asp Ser 50 55
60Lys Leu Arg Cys Asn Gly Gly Thr Ser Ala Thr Leu Asn
Ala Thr Ala65 70 75
80Ala Pro Gly Asp Thr Ile Thr Ala Ile Trp Ala Gln Trp Thr His Ser
85 90 95Gln Gly Pro Ile Leu Val
Trp Met Tyr Lys Cys Pro Gly Ser Phe Ser 100
105 110Ser Cys Asp Gly Ser Gly Ala Gly Trp Phe Lys Ile
Asp Glu Ala Gly 115 120 125Phe His
Gly Asp Gly Val Lys Val Phe Leu Asp Thr Glu Asn Pro Ser 130
135 140Gly Trp Asp Ile Ala Lys Leu Val Gly Gly Asn
Lys Gln Trp Ser Ser145 150 155
160Lys Val Pro Glu Gly Leu Ala Pro Gly Asn Tyr Leu Val Arg His Glu
165 170 175Leu Ile Ala Leu
His Gln Ala Asn Asn Pro Gln Phe Tyr Pro Glu Cys 180
185 190Ala Gln Val Val Ile Thr Gly Ser Gly Thr Ala
Gln Pro Asp Ala Ser 195 200 205Tyr
Lys Ala Ala Ile Pro Gly Tyr Cys Asn Gln Asn Asp Pro Asn Ile 210
215 220Lys Val Pro Ile Asn Asp His Ser Ile Pro
Gln Thr Tyr Lys Ile Pro225 230 235
240Gly Pro Pro Val Phe Lys Gly Thr Ala Ser Lys Lys Ala Arg Asp
Phe 245 250 255Thr
Ala7681DNAThielavia terrestris 7atgctcgcaa acggtgccat cgtcttcctg
gccgccgccc tcggcgtcag tggccactac 60acctggccac gggttaacga cggcgccgac
tggcaacagg tccgtaaggc ggacaactgg 120caggacaacg gctacgtcgg ggatgtcacg
tcgccacaga tccgctgttt ccaggcgacc 180ccgtccccgg ccccatccgt cctcaacacc
acggccggct cgaccgtgac ctactgggcc 240aaccccgacg tctaccaccc cgggcctgtg
cagttttaca tggcccgcgt gcccgatggc 300gaggacatca actcgtggaa cggcgacggc
gccgtgtggt tcaaggtgta cgaggaccat 360cctacctttg gcgctcagct cacatggccc
agcacgggca agagctcgtt cgcggttccc 420atccccccgt gcatcaagtc cggctactac
ctcctccggg cggagcaaat cggcctgcac 480gtcgcccaga gcgtaggcgg agcgcagttc
tacatctcat gcgcccagct cagcgtcacc 540ggcggcggca gcaccgagcc gccgaacaag
gtggccttcc ccggcgctta cagtgcgacg 600gacccgggca ttctgatcaa catctactac
cctgttccca cgtcctacca gaaccccggc 660ccggccgtct tcagctgctg a
6818226PRTThielavia terrestris 8Met Leu
Ala Asn Gly Ala Ile Val Phe Leu Ala Ala Ala Leu Gly Val1 5
10 15Ser Gly His Tyr Thr Trp Pro Arg
Val Asn Asp Gly Ala Asp Trp Gln 20 25
30Gln Val Arg Lys Ala Asp Asn Trp Gln Asp Asn Gly Tyr Val Gly
Asp 35 40 45Val Thr Ser Pro Gln
Ile Arg Cys Phe Gln Ala Thr Pro Ser Pro Ala 50 55
60Pro Ser Val Leu Asn Thr Thr Ala Gly Ser Thr Val Thr Tyr
Trp Ala65 70 75 80Asn
Pro Asp Val Tyr His Pro Gly Pro Val Gln Phe Tyr Met Ala Arg
85 90 95Val Pro Asp Gly Glu Asp Ile
Asn Ser Trp Asn Gly Asp Gly Ala Val 100 105
110Trp Phe Lys Val Tyr Glu Asp His Pro Thr Phe Gly Ala Gln
Leu Thr 115 120 125Trp Pro Ser Thr
Gly Lys Ser Ser Phe Ala Val Pro Ile Pro Pro Cys 130
135 140Ile Lys Ser Gly Tyr Tyr Leu Leu Arg Ala Glu Gln
Ile Gly Leu His145 150 155
160Val Ala Gln Ser Val Gly Gly Ala Gln Phe Tyr Ile Ser Cys Ala Gln
165 170 175Leu Ser Val Thr Gly
Gly Gly Ser Thr Glu Pro Pro Asn Lys Val Ala 180
185 190Phe Pro Gly Ala Tyr Ser Ala Thr Asp Pro Gly Ile
Leu Ile Asn Ile 195 200 205Tyr Tyr
Pro Val Pro Thr Ser Tyr Gln Asn Pro Gly Pro Ala Val Phe 210
215 220Ser Cys2259960DNAThielavia terrestris
9atgaagggac ttttcagtgc cgccgccctc tccctggccg tcggccaggc ttcggcccat
60tacatcttcc agcaactctc catcaacggg aaccagtttc cggtgtacca atatattcgc
120aagaacacca attataacag tcccgttacc gatctcacgt ccgacgatct tcggtgcaat
180gtcggcgccc agggtgctgg gacagacacc gtcacggtga aggccggcga ccagttcacc
240ttcacccttg acacccctgt ttaccaccag gggcccatct ccatctacat gtccaaggcc
300ccgggcgcgg cgtcagacta cgatggcagc ggcggctggt tcaagatcaa ggactggggc
360ccgactttca acgccgacgg cacggccacc tgggacatgg ccggctcata cacctacaac
420atcccgacct gcattcccga cggcgactat ctgctccgca tccagtcgct ggccatccac
480aacccctggc cggcgggcat cccgcagttc tacatctcct gcgcccagat caccgtgacc
540ggcggcggca acggcaaccc tggcccgacg gccctcatcc ccggcgcctt caaggacacc
600gacccgggct acacggtgaa catctacacg aacttccaca actacacggt tcccggcccg
660gaggtcttca gctgcaacgg cggcggctcg aacccgcccc cgccggtgag tagcagcacg
720cccgcgacca cgacgctggt cacgtcgacg cgcaccacgt cctccacgtc ctccgcctcg
780acgccggcct cgaccggcgg ctgcaccgtc gccaagtggg gccagtgcgg cggcaacggg
840tacaccggct gcacgacctg cgcggccggg tccacctgca gcaagcagaa cgactactac
900tcgcagtgct tgtaagggag gccgcaaagc atgaggtgtt tgaagaggag gagaggggtc
96010304PRTThielavia terrestris 10Met Lys Gly Leu Phe Ser Ala Ala Ala Leu
Ser Leu Ala Val Gly Gln1 5 10
15Ala Ser Ala His Tyr Ile Phe Gln Gln Leu Ser Ile Asn Gly Asn Gln
20 25 30Phe Pro Val Tyr Gln Tyr
Ile Arg Lys Asn Thr Asn Tyr Asn Ser Pro 35 40
45Val Thr Asp Leu Thr Ser Asp Asp Leu Arg Cys Asn Val Gly
Ala Gln 50 55 60Gly Ala Gly Thr Asp
Thr Val Thr Val Lys Ala Gly Asp Gln Phe Thr65 70
75 80Phe Thr Leu Asp Thr Pro Val Tyr His Gln
Gly Pro Ile Ser Ile Tyr 85 90
95Met Ser Lys Ala Pro Gly Ala Ala Ser Asp Tyr Asp Gly Ser Gly Gly
100 105 110Trp Phe Lys Ile Lys
Asp Trp Gly Pro Thr Phe Asn Ala Asp Gly Thr 115
120 125Ala Thr Trp Asp Met Ala Gly Ser Tyr Thr Tyr Asn
Ile Pro Thr Cys 130 135 140Ile Pro Asp
Gly Asp Tyr Leu Leu Arg Ile Gln Ser Leu Ala Ile His145
150 155 160Asn Pro Trp Pro Ala Gly Ile
Pro Gln Phe Tyr Ile Ser Cys Ala Gln 165
170 175Ile Thr Val Thr Gly Gly Gly Asn Gly Asn Pro Gly
Pro Thr Ala Leu 180 185 190Ile
Pro Gly Ala Phe Lys Asp Thr Asp Pro Gly Tyr Thr Val Asn Ile 195
200 205Tyr Thr Asn Phe His Asn Tyr Thr Val
Pro Gly Pro Glu Val Phe Ser 210 215
220Cys Asn Gly Gly Gly Ser Asn Pro Pro Pro Pro Val Ser Ser Ser Thr225
230 235 240Pro Ala Thr Thr
Thr Leu Val Thr Ser Thr Arg Thr Thr Ser Ser Thr 245
250 255Ser Ser Ala Ser Thr Pro Ala Ser Thr Gly
Gly Cys Thr Val Ala Lys 260 265
270Trp Gly Gln Cys Gly Gly Asn Gly Tyr Thr Gly Cys Thr Thr Cys Ala
275 280 285Ala Gly Ser Thr Cys Ser Lys
Gln Asn Asp Tyr Tyr Ser Gln Cys Leu 290 295
30011954DNAThielavia terrestris 11atgaagggcc tcagcctcct cgccgctgcg
tcggcagcga ctgctcatac catcttcgtg 60cagctcgagt cagggggaac gacctatccg
gtatcctacg gcatccggga ccctagctac 120gacggtccca tcaccgacgt cacctccgac
tcactggctt gcaatggtcc cccgaacccc 180acgacgccgt ccccgtacat catcaacgtc
accgccggca ccacggtcgc ggcgatctgg 240aggcacaccc tcacatccgg ccccgacgat
gtcatggacg ccagccacaa ggggccgacc 300ctggcctacc tcaagaaggt cgatgatgcc
ttgaccgaca cgggtatcgg cggcggctgg 360ttcaagatcc aggaggccgg ttacgacaat
ggcaattggg ctaccagcac ggtgatcacc 420aacggtggct tccaatatat tgacatcccc
gcctgcattc ccaacggcca gtatctgctc 480cgcgccgaga tgatcgcgct ccacgccgcc
agcacgcagg gtggtgccca gctctacatg 540gagtgcgcgc agatcaacgt ggtgggcggc
tccggcagcg ccagcccgca gacgtacagc 600atcccgggca tctaccaggc aaccgacccg
ggcctgctga tcaacatcta ctccatgacg 660ccgtccagcc agtacaccat tccgggtccg
cccctgttca cctgcagcgg cagcggcaac 720aacggcggcg gcagcaaccc gtcgggcggg
cagaccacga cggcgaagcc cacgacgacg 780acggcggcga cgaccacctc ctccgccgct
cctaccagca gccagggggg cagcagcggt 840tgcaccgttc cccagtggca gcagtgcggt
ggcatctcgt tcaccggctg caccacctgc 900gcggcgggct acacctgcaa gtatctgaac
gactattact cgcaatgcca gtaa 95412317PRTThielavia terrestris 12Met
Lys Gly Leu Ser Leu Leu Ala Ala Ala Ser Ala Ala Thr Ala His1
5 10 15Thr Ile Phe Val Gln Leu Glu
Ser Gly Gly Thr Thr Tyr Pro Val Ser 20 25
30Tyr Gly Ile Arg Asp Pro Ser Tyr Asp Gly Pro Ile Thr Asp
Val Thr 35 40 45Ser Asp Ser Leu
Ala Cys Asn Gly Pro Pro Asn Pro Thr Thr Pro Ser 50 55
60Pro Tyr Ile Ile Asn Val Thr Ala Gly Thr Thr Val Ala
Ala Ile Trp65 70 75
80Arg His Thr Leu Thr Ser Gly Pro Asp Asp Val Met Asp Ala Ser His
85 90 95Lys Gly Pro Thr Leu Ala
Tyr Leu Lys Lys Val Asp Asp Ala Leu Thr 100
105 110Asp Thr Gly Ile Gly Gly Gly Trp Phe Lys Ile Gln
Glu Ala Gly Tyr 115 120 125Asp Asn
Gly Asn Trp Ala Thr Ser Thr Val Ile Thr Asn Gly Gly Phe 130
135 140Gln Tyr Ile Asp Ile Pro Ala Cys Ile Pro Asn
Gly Gln Tyr Leu Leu145 150 155
160Arg Ala Glu Met Ile Ala Leu His Ala Ala Ser Thr Gln Gly Gly Ala
165 170 175Gln Leu Tyr Met
Glu Cys Ala Gln Ile Asn Val Val Gly Gly Ser Gly 180
185 190Ser Ala Ser Pro Gln Thr Tyr Ser Ile Pro Gly
Ile Tyr Gln Ala Thr 195 200 205Asp
Pro Gly Leu Leu Ile Asn Ile Tyr Ser Met Thr Pro Ser Ser Gln 210
215 220Tyr Thr Ile Pro Gly Pro Pro Leu Phe Thr
Cys Ser Gly Ser Gly Asn225 230 235
240Asn Gly Gly Gly Ser Asn Pro Ser Gly Gly Gln Thr Thr Thr Ala
Lys 245 250 255Pro Thr Thr
Thr Thr Ala Ala Thr Thr Thr Ser Ser Ala Ala Pro Thr 260
265 270Ser Ser Gln Gly Gly Ser Ser Gly Cys Thr
Val Pro Gln Trp Gln Gln 275 280
285Cys Gly Gly Ile Ser Phe Thr Gly Cys Thr Thr Cys Ala Ala Gly Tyr 290
295 300Thr Cys Lys Tyr Leu Asn Asp Tyr
Tyr Ser Gln Cys Gln305 310
31513799DNAThermoascus aurantiacus 13atgtcctttt ccaagataat tgctactgcc
ggcgttcttg cctctgcttc tctagtggct 60ggccatggct tcgttcagaa catcgtgatt
gatggtaaaa agtatgtcat tgcaagacgc 120acataagcgg caacagctga caatcgacag
ttatggcggg tatctagtga accagtatcc 180atacatgtcc aatcctccag aggtcatcgc
ctggtctact acggcaactg atcttggatt 240tgtggacggt actggatacc aaaccccaga
tatcatctgc cataggggcg ccaagcctgg 300agccctgact gctccagtct ctccaggagg
aactgttgag cttcaatgga ctccatggcc 360tgattctcac catggcccag ttatcaacta
ccttgctccg tgcaatggtg attgttccac 420tgtggataag acccaattag aattcttcaa
aattgccgag agcggtctca tcaatgatga 480caatcctcct gggatctggg cttcagacaa
tctgatagca gccaacaaca gctggactgt 540caccattcca accacaattg cacctggaaa
ctatgttctg aggcatgaga ttattgctct 600tcactcagct cagaaccagg atggtgccca
gaactatccc cagtgcatca atctgcaggt 660cactggaggt ggttctgata accctgctgg
aactcttgga acggcactct accacgatac 720cgatcctgga attctgatca acatctatca
gaaactttcc agctatatca tccctggtcc 780tcctctgtat actggttaa
79914249PRTThermoascus aurantiacus
14Met Ser Phe Ser Lys Ile Ile Ala Thr Ala Gly Val Leu Ala Ser Ala1
5 10 15Ser Leu Val Ala Gly His
Gly Phe Val Gln Asn Ile Val Ile Asp Gly 20 25
30Lys Tyr Tyr Gly Gly Tyr Leu Val Asn Gln Tyr Pro Tyr
Met Ser Asn 35 40 45Pro Pro Glu
Val Ile Ala Trp Ser Thr Thr Ala Thr Asp Leu Gly Phe 50
55 60Val Asp Gly Thr Gly Tyr Gln Thr Pro Asp Ile Ile
Cys His Arg Gly65 70 75
80Ala Lys Pro Gly Ala Leu Thr Ala Pro Val Ser Pro Gly Gly Thr Val
85 90 95Glu Leu Gln Trp Thr Pro
Trp Pro Asp Ser His His Gly Pro Val Ile 100
105 110Asn Tyr Leu Ala Pro Cys Asn Gly Asp Cys Ser Thr
Val Asp Lys Thr 115 120 125Gln Leu
Glu Phe Phe Lys Ile Ala Glu Ser Gly Leu Ile Asn Asp Asp 130
135 140Asn Pro Pro Gly Ile Trp Ala Ser Asp Asn Leu
Ile Ala Ala Asn Asn145 150 155
160Ser Trp Thr Val Thr Ile Pro Thr Thr Ile Ala Pro Gly Asn Tyr Val
165 170 175Leu Arg His Glu
Ile Ile Ala Leu His Ser Ala Gln Asn Gln Asp Gly 180
185 190Ala Gln Asn Tyr Pro Gln Cys Ile Asn Leu Gln
Val Thr Gly Gly Gly 195 200 205Ser
Asp Asn Pro Ala Gly Thr Leu Gly Thr Ala Leu Tyr His Asp Thr 210
215 220Asp Pro Gly Ile Leu Ile Asn Ile Tyr Gln
Lys Leu Ser Ser Tyr Ile225 230 235
240Ile Pro Gly Pro Pro Leu Tyr Thr Gly
245151172DNATrichoderma reesei 15ggatctaagc cccatcgata tgaagtcctg
cgccattctt gcagcccttg gctgtcttgc 60cgggagcgtt ctcggccatg gacaagtcca
aaacttcacg atcaatggac aatacaatca 120gggtttcatt ctcgattact actatcagaa
gcagaatact ggtcacttcc ccaacgttgc 180tggctggtac gccgaggacc tagacctggg
cttcatctcc cctgaccaat acaccacgcc 240cgacattgtc tgtcacaaga acgcggcccc
aggtgccatt tctgccactg cagcggccgg 300cagcaacatc gtcttccaat ggggccctgg
cgtctggcct cacccctacg gtcccatcgt 360tacctacgtg gctgagtgca gcggatcgtg
cacgaccgtg aacaagaaca acctgcgctg 420ggtcaagatt caggaggccg gcatcaacta
taacacccaa gtctgggcgc agcaggatct 480gatcaaccag ggcaacaagt ggactgtgaa
gatcccgtcg agcctcaggc ccggaaacta 540tgtcttccgc catgaacttc ttgctgccca
tggtgcctct agtgcgaacg gcatgcagaa 600ctatcctcag tgcgtgaaca tcgccgtcac
aggctcgggc acgaaagcgc tccctgccgg 660aactcctgca actcagctct acaagcccac
tgaccctggc atcttgttca acccttacac 720aacaatcacg agctacacca tccctggccc
agccctgtgg caaggctaga tccaggggta 780cggtgttggc gttcgtgaag tcggagctgt
tgacaaggat atctgatgat gaacggagag 840gactgatggg cgtgactgag tgtatatatt
tttgatgacc aaattgtata cgaaatccga 900acgcatggtg atcattgttt atccctgtag
tatattgtct ccaggctgct aagagcccac 960cgggtgtatt acggcaacaa agtcaggaat
ttgggtggca atgaacgcag gtctccatga 1020atgtatatgt gaagaggcat cggctggcat
gggcattacc agatataggc cctgtgaaac 1080atatagtact tgaacgtgct actggaacgg
atcataagca agtcatcaac atgtgaaaaa 1140acactacatg taaaaaaaaa aaaaaaaaaa
aa 117216249PRTTrichoderma reesei 16Met
Lys Ser Cys Ala Ile Leu Ala Ala Leu Gly Cys Leu Ala Gly Ser1
5 10 15Val Leu Gly His Gly Gln Val
Gln Asn Phe Thr Ile Asn Gly Gln Tyr 20 25
30Asn Gln Gly Phe Ile Leu Asp Tyr Tyr Tyr Gln Lys Gln Asn
Thr Gly 35 40 45His Phe Pro Asn
Val Ala Gly Trp Tyr Ala Glu Asp Leu Asp Leu Gly 50 55
60Phe Ile Ser Pro Asp Gln Tyr Thr Thr Pro Asp Ile Val
Cys His Lys65 70 75
80Asn Ala Ala Pro Gly Ala Ile Ser Ala Thr Ala Ala Ala Gly Ser Asn
85 90 95Ile Val Phe Gln Trp Gly
Pro Gly Val Trp Pro His Pro Tyr Gly Pro 100
105 110Ile Val Thr Tyr Val Val Glu Cys Ser Gly Ser Cys
Thr Thr Val Asn 115 120 125Lys Asn
Asn Leu Arg Trp Val Lys Ile Gln Glu Ala Gly Ile Asn Tyr 130
135 140Asn Thr Gln Val Trp Ala Gln Gln Asp Leu Ile
Asn Gln Gly Asn Lys145 150 155
160Trp Thr Val Lys Ile Pro Ser Ser Leu Arg Pro Gly Asn Tyr Val Phe
165 170 175Arg His Glu Leu
Leu Ala Ala His Gly Ala Ser Ser Ala Asn Gly Met 180
185 190Gln Asn Tyr Pro Gln Cys Val Asn Ile Ala Val
Thr Gly Ser Gly Thr 195 200 205Lys
Ala Leu Pro Ala Gly Thr Pro Ala Thr Gln Leu Tyr Lys Pro Thr 210
215 220Asp Pro Gly Ile Leu Phe Asn Pro Tyr Thr
Thr Ile Thr Ser Tyr Thr225 230 235
240Ile Pro Gly Pro Ala Leu Trp Gln Gly
24517924DNAMyceliophthora thermophila 17atgaagttca cctcgtccct cgctgtcctg
gccgctgccg gcgcccaggc tcactgttag 60tcgaccctcg aacccaacac ccccctcccc
ccttttctcc tccatctcct cggcctcact 120tagtagccgc tgacaacgac tagatacctt
ccctagggcc ggcactggtg gctcgctctc 180tggcgagtgg gaggtggtcc gcatgaccga
gaaccattac tcgcacggcc cggtcaccga 240tgtcaccagc cccgagatga cctgctatca
gtccggcgtg cagggtgcgc cccagaccgt 300ccaggtcaag gcgggctccc aattcacctt
cagcgtggat ccctcgatcg gccaccccgg 360ccctctccag ttctacatgg ctaaggtgcc
gtcgggccag acggccgcca cctttgacgg 420cacgggagcc gtgtggttca agatctacca
agacggcccg aacggcctcg gcaccgacag 480cattacctgg cccagcgccg gttcgtgact
tcctccccac tcgctttttt ttttttattt 540tttatttttt tttctttcgg aactcaagaa
tctttctctc tctctcccgt ctttggcctt 600gaacaacact aaaactcttc cttactgtat
taattaggca aaaccgaggt ctcggtcacc 660atccccagct gcatcgatga tggcgagtac
ctgctccggg tcgagcacat cgcgctccac 720agcgccagca gcgtgggcgg cgctcagttc
tacattgcct gcgcccagct ctccgtcacc 780ggcggctccg gcaccctcaa cacgggctcg
ctcgtctccc tgcccggcgc ctacaaggcc 840accgacccgg gcatcctctt ccagctctac
tggcccatcc cgaccgagta catcaacccc 900ggcccggccc ccgtctcttg ctaa
92418232PRTMyceliophthora thermophila
18Met Lys Phe Thr Ser Ser Leu Ala Val Leu Ala Ala Ala Gly Ala Gln1
5 10 15Ala His Tyr Thr Phe Pro
Arg Ala Gly Thr Gly Gly Ser Leu Ser Gly 20 25
30Glu Trp Glu Val Val Arg Met Thr Glu Asn His Tyr Ser
His Gly Pro 35 40 45Val Thr Asp
Val Thr Ser Pro Glu Met Thr Cys Tyr Gln Ser Gly Val 50
55 60Gln Gly Ala Pro Gln Thr Val Gln Val Lys Ala Gly
Ser Gln Phe Thr65 70 75
80Phe Ser Val Asp Pro Ser Ile Gly His Pro Gly Pro Leu Gln Phe Tyr
85 90 95Met Ala Lys Val Pro Ser
Gly Gln Thr Ala Ala Thr Phe Asp Gly Thr 100
105 110Gly Ala Val Trp Phe Lys Ile Tyr Gln Asp Gly Pro
Asn Gly Leu Gly 115 120 125Thr Asp
Ser Ile Thr Trp Pro Ser Ala Gly Lys Thr Glu Val Ser Val 130
135 140Thr Ile Pro Ser Cys Ile Asp Asp Gly Glu Tyr
Leu Leu Arg Val Glu145 150 155
160His Ile Ala Leu His Ser Ala Ser Ser Val Gly Gly Ala Gln Phe Tyr
165 170 175Ile Ala Cys Ala
Gln Leu Ser Val Thr Gly Gly Ser Gly Thr Leu Asn 180
185 190Thr Gly Ser Leu Val Ser Leu Pro Gly Ala Tyr
Lys Ala Thr Asp Pro 195 200 205Gly
Ile Leu Phe Gln Leu Tyr Trp Pro Ile Pro Thr Glu Tyr Ile Asn 210
215 220Pro Gly Pro Ala Pro Val Ser Cys225
23019854DNAMyceliophthora thermophila 19atgaaggccc tctctctcct
tgcggctgcc tcggcagtct ctgcgcatac catcttcgtc 60cagctcgaag cagacggcac
gaggtacccg gtctcgtacg ggatccggga cccaagctac 120gacggcccca tcaccgacgt
cacatccaac gacgttgctt gcaacggcgg gccgaacccg 180acgaccccct ccagcgacgt
catcaccgtc accgcgggca ccacggtcaa ggccatctgg 240aggcacaccc tccaatccgg
cccggacgat gtcatggacg ccagccacaa gggcccgacc 300ctggcctacc tcaagaaggt
cggcgatgcc accaaggact cgggcgtcgg cggtggctgg 360ttcaagattc aggaggacgg
ctacaacaac ggccagtggg gcaccagcac cgttatctcc 420aacggcggcg agcactacat
gtgagccatt cctccgagag aagaccaaga ctcttgacga 480tctcgctgac ccgtgcaaca
agtgacatcc cggcctgcat ccccgagggt cagtacctcc 540tccgcgccga gatgatcgcc
ctccacgcgg ccgggtcccc cggcggtgcc cagctctacg 600taagcctctg cccttccccc
cttcctcttg atcgaatcgg actgcccacc ccccttttcg 660actccgacta acaccgttgc
cagatggaat gtgcccagat caacatcgtc ggcggctccg 720gctcggtgcc cagctcgacc
gtcagcttcc ccggcgcgta cagccccaac gacccgggtc 780tcctcatcaa catctattcc
atgtcgccct cgagctcgta caccatcccg ggcccgcccg 840tcttcaagtg ctag
85420235PRTMyceliophthora
thermophila 20Met Lys Ala Leu Ser Leu Leu Ala Ala Ala Ser Ala Val Ser Ala
His1 5 10 15Thr Ile Phe
Val Gln Leu Glu Ala Asp Gly Thr Arg Tyr Pro Val Ser 20
25 30Tyr Gly Ile Arg Asp Pro Ser Tyr Asp Gly
Pro Ile Thr Asp Val Thr 35 40
45Ser Asn Asp Val Ala Cys Asn Gly Gly Pro Asn Pro Thr Thr Pro Ser 50
55 60Ser Asp Val Ile Thr Val Thr Ala Gly
Thr Thr Val Lys Ala Ile Trp65 70 75
80Arg His Thr Leu Gln Ser Gly Pro Asp Asp Val Met Asp Ala
Ser His 85 90 95Lys Gly
Pro Thr Leu Ala Tyr Leu Lys Lys Val Gly Asp Ala Thr Lys 100
105 110Asp Ser Gly Val Gly Gly Gly Trp Phe
Lys Ile Gln Glu Asp Gly Tyr 115 120
125Asn Asn Gly Gln Trp Gly Thr Ser Thr Val Ile Ser Asn Gly Gly Glu
130 135 140His Tyr Ile Asp Ile Pro Ala
Cys Ile Pro Glu Gly Gln Tyr Leu Leu145 150
155 160Arg Ala Glu Met Ile Ala Leu His Ala Ala Gly Ser
Pro Gly Gly Ala 165 170
175Gln Leu Tyr Met Glu Cys Ala Gln Ile Asn Ile Val Gly Gly Ser Gly
180 185 190Ser Val Pro Ser Ser Thr
Val Ser Phe Pro Gly Ala Tyr Ser Pro Asn 195 200
205Asp Pro Gly Leu Leu Ile Asn Ile Tyr Ser Met Ser Pro Ser
Ser Ser 210 215 220Tyr Thr Ile Pro Gly
Pro Pro Val Phe Lys Cys225 230
235211242DNAMyceliophthora thermophila 21atgaagtcct tcgccctcac cactctggcc
gccctggccg gcaacgccgc cgctcacgcg 60accttccagg ccctctgggt cgacggcgtc
gactacggcg cgcagtgtgc ccgtctgccc 120gcgtccaact ccccggtcac cgacgtgacc
tccaacgcga tccgctgcaa cgccaacccg 180tcgcccgctc ggggcaagtg cccggtcaag
gccggctcga ccgttacggt cgagatgcat 240caggtacgtt ggatgaatga aaggggaaag
gaagcagagg cagaagggga aggcgaaggg 300aaagaaaaag aaaaagaaat ggaaaagaaa
aagaaatgga aaagaaaaag aaaaatgaaa 360aagaaagtgg aaaccgtcag actaactggg
gctcctcccc cccacccctc ctttgatatc 420agcaacccgg tgaccggtcg tgcagcagcg
aggcgatcgg cggggcgcac tacggccccg 480tcatggtgta catgtccaag gtgtcggacg
cggcgtcggc ggacgggtcg tcgggctggt 540tcaaggtgtt cgaggacggc tgggccaaga
acccgtccgg cgggtcgggc gacgacgact 600actggggcac caaggacctg aactcgtgct
gcgggaagat gaacgtcaag atccccgccg 660acctgccctc gggcgactac ctgctccggg
ccgaggccct cgcgctgcac acggcgggca 720gcgccggcgg cgcccagttc tacatgacgt
gctaccagct caccgtgacg ggctccggca 780gcgccagccc gcccaccgtc tccttcccgg
gcgcctacaa ggccaccgac ccgggcatcc 840tcgtcaacat ccacgccccg ctgtccggct
acaccgtgcc cggcccggcc gtctactccg 900gcggctccac caagaaggcc ggcagcgcct
gcaccggctg cgagtccacc tgcgccgtcg 960gctccggccc caccgccacc gtctcccagt
cgcccggttc caccgccacc tccgcccccg 1020gcggcggcgg cggctgcacc gtccagaagt
accagcagtg cggcggcgag ggctacaccg 1080gctgcaccaa ctgcgcggta cgtttttcaa
ccccgttttt ttttttcctt ccctacctta 1140tttggttacc taattaatta ctttccggct
gctgactttt tgctttagtc cggctctacc 1200tgcagcgccg tctcgccgcc ctactactcg
cagtgcgtct aa 124222323PRTMyceliophthora thermophila
22Met Lys Ser Phe Ala Leu Thr Thr Leu Ala Ala Leu Ala Gly Asn Ala1
5 10 15Ala Ala His Ala Thr Phe
Gln Ala Leu Trp Val Asp Gly Val Asp Tyr 20 25
30Gly Ala Gln Cys Ala Arg Leu Pro Ala Ser Asn Ser Pro
Val Thr Asp 35 40 45Val Thr Ser
Asn Ala Ile Arg Cys Asn Ala Asn Pro Ser Pro Ala Arg 50
55 60Gly Lys Cys Pro Val Lys Ala Gly Ser Thr Val Thr
Val Glu Met His65 70 75
80Gln Gln Pro Gly Asp Arg Ser Cys Ser Ser Glu Ala Ile Gly Gly Ala
85 90 95His Tyr Gly Pro Val Met
Val Tyr Met Ser Lys Val Ser Asp Ala Ala 100
105 110Ser Ala Asp Gly Ser Ser Gly Trp Phe Lys Val Phe
Glu Asp Gly Trp 115 120 125Ala Lys
Asn Pro Ser Gly Gly Ser Gly Asp Asp Asp Tyr Trp Gly Thr 130
135 140Lys Asp Leu Asn Ser Cys Cys Gly Lys Met Asn
Val Lys Ile Pro Ala145 150 155
160Asp Leu Pro Ser Gly Asp Tyr Leu Leu Arg Ala Glu Ala Leu Ala Leu
165 170 175His Thr Ala Gly
Ser Ala Gly Gly Ala Gln Phe Tyr Met Thr Cys Tyr 180
185 190Gln Leu Thr Val Thr Gly Ser Gly Ser Ala Ser
Pro Pro Thr Val Ser 195 200 205Phe
Pro Gly Ala Tyr Lys Ala Thr Asp Pro Gly Ile Leu Val Asn Ile 210
215 220His Ala Pro Leu Ser Gly Tyr Thr Val Pro
Gly Pro Ala Val Tyr Ser225 230 235
240Gly Gly Ser Thr Lys Lys Ala Gly Ser Ala Cys Thr Gly Cys Glu
Ser 245 250 255Thr Cys Ala
Val Gly Ser Gly Pro Thr Ala Thr Val Ser Gln Ser Pro 260
265 270Gly Ser Thr Ala Thr Ser Ala Pro Gly Gly
Gly Gly Gly Cys Thr Val 275 280
285Gln Lys Tyr Gln Gln Cys Gly Gly Glu Gly Tyr Thr Gly Cys Thr Asn 290
295 300Cys Ala Ser Gly Ser Thr Cys Ser
Ala Val Ser Pro Pro Tyr Tyr Ser305 310
315 320Gln Cys Val231253DNAMyceliophthora thermophila
23atgaagcctt ttagcctcgt cgccctggcg accgccgtga gcggccatgc catcttccag
60cgggtgtcgg tcaacgggca ggaccagggc cagctcaagg gggtgcgggc gccgtcgagc
120aactccccga tccagaacgt caacgatgcc aacatggcct gcaacgccaa cattgtgtac
180cacgacagca ccatcatcaa ggtgcccgcg ggagcccgcg tcggcgcgtg gtggcagcac
240gtcatcggcg ggccgcaggg cgccaacgac ccggacaacc cgatcgcggc ctcccacaag
300ggtatgatga tcgatgatgc ctctctcttc ccccgttctt gatggacagg cgatggctcc
360caggaacacg cgtgactgac caccgaatcc aggccccatc caggtctacc tggccaaggt
420ggacaacgcg gcgacggcgt cgccgtcggg cctcaggtgg ttcaaggtgg ccgagcgcgg
480cctgaacaac ggcgtgtggg ccgtcgatga gctcatcgcc aacaacggct ggcactactt
540cgacctgccg tcgtgcgtgg cccccggcca gtacctgatg cgcgtcgagc tgctcgccct
600gcacagcgcc tcaagccccg gcggcgccca gttctacatg ggctgcgcac agatcgaagg
660tgcgtcgatc tttgttctcc ttccgtgtcc tctctgatcc tttctctctt ctttttcttt
720cttttactcc ctttccttcc atcttcggag aagcaacgaa gggggaaagg gatagaagag
780aggaatgaga gacgacgaaa gagaggattg gggaaagaca agacagggaa aaaaagacaa
840gaaaaaaaaa aaaaaaaaaa aacagagtga gctaacaaga acaatcagtc actggctccg
900gcaccaactc gggctccgac tttgtctcgt tccccggcgc ctactcggcc aacgatccgg
960gcatcttgct aagcatctac gacagctcgg gcaagcccac caacggcggg cgctcgtacc
1020cgatccccgg cccgcgcccc atctcctgct ccggcagcgg cgacggcggc aacaacggcg
1080gcggcggcga cgacaacaac aataacaacg gtggtggcaa caacggcggc ggcggcggcg
1140gcagcgtccc cctgtacggg cagtgcggcg gcatcggcta cacgggcccg accacctgtg
1200cccagggaac ttgcaaggtg tcgaacgaat actacagcca gtgcctcccc tag
125324310PRTMyceliophthora thermophila 24Met Lys Pro Phe Ser Leu Val Ala
Leu Ala Thr Ala Val Ser Gly His1 5 10
15Ala Ile Phe Gln Arg Val Ser Val Asn Gly Gln Asp Gln Gly
Gln Leu 20 25 30Lys Gly Val
Arg Ala Pro Ser Ser Asn Ser Pro Ile Gln Asn Val Asn 35
40 45Asp Ala Asn Met Ala Cys Asn Ala Asn Ile Val
Tyr His Asp Ser Thr 50 55 60Ile Ile
Lys Val Pro Ala Gly Ala Arg Val Gly Ala Trp Trp Gln His65
70 75 80Val Ile Gly Gly Pro Gln Gly
Ala Asn Asp Pro Asp Asn Pro Ile Ala 85 90
95Ala Ser His Lys Gly Pro Ile Gln Val Tyr Leu Ala Lys
Val Asp Asn 100 105 110Ala Ala
Thr Ala Ser Pro Ser Gly Leu Arg Trp Phe Lys Val Ala Glu 115
120 125Arg Gly Leu Asn Asn Gly Val Trp Ala Val
Asp Glu Leu Ile Ala Asn 130 135 140Asn
Gly Trp His Tyr Phe Asp Leu Pro Ser Cys Val Ala Pro Gly Gln145
150 155 160Tyr Leu Met Arg Val Glu
Leu Leu Ala Leu His Ser Ala Ser Ser Pro 165
170 175Gly Gly Ala Gln Phe Tyr Met Gly Cys Ala Gln Ile
Glu Val Thr Gly 180 185 190Ser
Gly Thr Asn Ser Gly Ser Asp Phe Val Ser Phe Pro Gly Ala Tyr 195
200 205Ser Ala Asn Asp Pro Gly Ile Leu Leu
Ser Ile Tyr Asp Ser Ser Gly 210 215
220Lys Pro Thr Asn Gly Gly Arg Ser Tyr Pro Ile Pro Gly Pro Arg Pro225
230 235 240Ile Ser Cys Ser
Gly Ser Gly Asp Gly Gly Asn Asn Gly Gly Gly Gly 245
250 255Asp Asp Asn Asn Asn Asn Asn Gly Gly Gly
Asn Asn Gly Gly Gly Gly 260 265
270Gly Gly Ser Val Pro Leu Tyr Gly Gln Cys Gly Gly Ile Gly Tyr Thr
275 280 285Gly Pro Thr Thr Cys Ala Gln
Gly Thr Cys Lys Val Ser Asn Glu Tyr 290 295
300Tyr Ser Gln Cys Leu Pro305
31025814DNAMyceliophthora thermophila 25atgaagctct ccctcttctc cgtcctggcc
actgccctca ccgtcgaggg gcatgccatc 60ttccagaagg tctccgtcaa cggagcggac
cagggctccc tcaccggcct ccgcgctccc 120aacaacaaca accccgtgca ggatgtcaac
agccaggaca tgatctgcgg ccagtcggga 180tcgacgtcga acactatcat cgaggtcaag
gccggcgata ggatcggtgc ctggtatcag 240catgtcatcg gcggtgccca gttccccaac
gacccagaca acccgattgc caagtcgcac 300aagggccccg tcatggccta cctcgccaag
gttgacaatg ccgcaaccgc cagcaagacg 360ggcctgaagt ggtatgtatt cccgcggccc
gagggacatc gggttgggca agtcgagact 420gacggagctc gcttctccgt ataggttcaa
gatttgggag gataccttta atcccagcac 480caagacctgg ggtgtcgaca acctcatcaa
taacaacggc tgggtgtact tcaacctccc 540gcagtgcatc gccgacggca actacctcct
ccgcgtcgag gtcctcgctc tgcactcggc 600ctactctcag ggccaggctc agttctacca
gtcctgcgcc cagatcaacg tatccggcgg 660cggctccttc acaccgccgt cgactgtcag
cttcccgggt gcctacagcg ccagcgaccc 720cggtatcctg atcaacatct acggcgccac
cggccagccc gacaacaacg gccagccgta 780cactgcccct gggcccgcgc ccatctcctg
ctga 81426246PRTMyceliophthora thermophila
26Met Lys Leu Ser Leu Phe Ser Val Leu Ala Thr Ala Leu Thr Val Glu1
5 10 15Gly His Ala Ile Phe Gln
Lys Val Ser Val Asn Gly Ala Asp Gln Gly 20 25
30Ser Leu Thr Gly Leu Arg Ala Pro Asn Asn Asn Asn Pro
Val Gln Asp 35 40 45Val Asn Ser
Gln Asp Met Ile Cys Gly Gln Ser Gly Ser Thr Ser Asn 50
55 60Thr Ile Ile Glu Val Lys Ala Gly Asp Arg Ile Gly
Ala Trp Tyr Gln65 70 75
80His Val Ile Gly Gly Ala Gln Phe Pro Asn Asp Pro Asp Asn Pro Ile
85 90 95Ala Lys Ser His Lys Gly
Pro Val Met Ala Tyr Leu Ala Lys Val Asp 100
105 110Asn Ala Ala Thr Ala Ser Lys Thr Gly Leu Lys Trp
Phe Lys Ile Trp 115 120 125Glu Asp
Thr Phe Asn Pro Ser Thr Lys Thr Trp Gly Val Asp Asn Leu 130
135 140Ile Asn Asn Asn Gly Trp Val Tyr Phe Asn Leu
Pro Gln Cys Ile Ala145 150 155
160Asp Gly Asn Tyr Leu Leu Arg Val Glu Val Leu Ala Leu His Ser Ala
165 170 175Tyr Ser Gln Gly
Gln Ala Gln Phe Tyr Gln Ser Cys Ala Gln Ile Asn 180
185 190Val Ser Gly Gly Gly Ser Phe Thr Pro Pro Ser
Thr Val Ser Phe Pro 195 200 205Gly
Ala Tyr Ser Ala Ser Asp Pro Gly Ile Leu Ile Asn Ile Tyr Gly 210
215 220Ala Thr Gly Gln Pro Asp Asn Asn Gly Gln
Pro Tyr Thr Ala Pro Gly225 230 235
240Pro Ala Pro Ile Ser Cys
245271115DNAThermoascus aurantiacus 27atgtcgttct cgaagattgc tgcgatcacc
ggggccatta cctatgcgtc tctggccgcc 60gctcacggtt atgttacagg aatcgtagcc
gatggcacct agtatgtaac gctcatgcca 120agatccgcat tgctgtacta acaattagca
gctacggggg ctatatcgtg acccaatacc 180cctacatgtc gacaccgccg gatgtcatcg
cctggtctac caaagcaact gatcttggtt 240tcgtggatcc cagtagctat gcttcgtctg
atattatctg ccacaagggt gctgagcctg 300gtgccctgag cgccaaggtg gctgctggag
ggaccgtcga gctgcagtgg acggattggc 360ctgagagtca caagggcccg gtcattgact
acctcgccgc ctgtaacggg gactgctcga 420ctgtcgacaa gaccaaacta gagttcttca
agattgatga gagtggccta attgacggca 480gcagcgcccc aggcacatgg gcctctgaca
acttgattgc caataacaac agctggaccg 540tcaccatccc gagcacgatt gctcccggca
actatgtcct gagacatgaa atcattgccc 600tccactccgc cggaaataca aatggtgctc
agaactaccc ccagtgtatc aaccttgagg 660tcacaggcag tggcaccgac acccctgccg
gcaccctcgg aacggagctt tataaggcaa 720cggaccctgg cattctggtc aacatctacc
agaccctgac cagctacgat attcccggcc 780ctgctctgta caccggtggt agctctggta
gctctggttc ctccaacacc gccaaggcca 840ccacttcgac ggcttctagc tctatcgtga
ccccgacgcc tgttaacaac ccaaccgtta 900ctcagactgc cgttgttgat gtcacccaga
ctgtttccca gaatgctgcc gtcgccacca 960cgactccggc ctccactgca gttgctacag
ctgtcccaac gggaaccacc tttagctttg 1020attcgatgac ctcggatgaa ttcgtcagcc
tgatgcgtgc gaccgtgaat tggctgcttt 1080ctaacaagaa gcatgcccgg gatctttctt
actaa 111528354PRTThermoascus aurantiacus
28Met Ser Phe Ser Lys Ile Ala Ala Ile Thr Gly Ala Ile Thr Tyr Ala1
5 10 15Ser Leu Ala Ala Ala His
Gly Tyr Val Thr Gly Ile Val Ala Asp Gly 20 25
30Thr Tyr Tyr Gly Gly Tyr Ile Val Thr Gln Tyr Pro Tyr
Met Ser Thr 35 40 45Pro Pro Asp
Val Ile Ala Trp Ser Thr Lys Ala Thr Asp Leu Gly Phe 50
55 60Val Asp Pro Ser Ser Tyr Ala Ser Ser Asp Ile Ile
Cys His Lys Gly65 70 75
80Ala Glu Pro Gly Ala Leu Ser Ala Lys Val Ala Ala Gly Gly Thr Val
85 90 95Glu Leu Gln Trp Thr Asp
Trp Pro Glu Ser His Lys Gly Pro Val Ile 100
105 110Asp Tyr Leu Ala Ala Cys Asn Gly Asp Cys Ser Thr
Val Asp Lys Thr 115 120 125Lys Leu
Glu Phe Phe Lys Ile Asp Glu Ser Gly Leu Ile Asp Gly Ser 130
135 140Ser Ala Pro Gly Thr Trp Ala Ser Asp Asn Leu
Ile Ala Asn Asn Asn145 150 155
160Ser Trp Thr Val Thr Ile Pro Ser Thr Ile Ala Pro Gly Asn Tyr Val
165 170 175Leu Arg His Glu
Ile Ile Ala Leu His Ser Ala Gly Asn Thr Asn Gly 180
185 190Ala Gln Asn Tyr Pro Gln Cys Ile Asn Leu Glu
Val Thr Gly Ser Gly 195 200 205Thr
Asp Thr Pro Ala Gly Thr Leu Gly Thr Glu Leu Tyr Lys Ala Thr 210
215 220Asp Pro Gly Ile Leu Val Asn Ile Tyr Gln
Thr Leu Thr Ser Tyr Asp225 230 235
240Ile Pro Gly Pro Ala Leu Tyr Thr Gly Gly Ser Ser Gly Ser Ser
Gly 245 250 255Ser Ser Asn
Thr Ala Lys Ala Thr Thr Ser Thr Ala Ser Ser Ser Ile 260
265 270Val Thr Pro Thr Pro Val Asn Asn Pro Thr
Val Thr Gln Thr Ala Val 275 280
285Val Asp Val Thr Gln Thr Val Ser Gln Asn Ala Ala Val Ala Thr Thr 290
295 300Thr Pro Ala Ser Thr Ala Val Ala
Thr Ala Val Pro Thr Gly Thr Thr305 310
315 320Phe Ser Phe Asp Ser Met Thr Ser Asp Glu Phe Val
Ser Leu Met Arg 325 330
335Ala Thr Val Asn Trp Leu Leu Ser Asn Lys Lys His Ala Arg Asp Leu
340 345 350Ser Tyr29862DNAAspergillus
fumigatus 29atgactttgt ccaagatcac ttccattgct ggccttctgg cctcagcgtc
tctcgtggct 60ggccacggct ttgtttctgg cattgttgct gatgggaaat agtatgtgct
tgaaccacac 120aaatgacagc tgcaacagct aacttctatt ccagttacgg agggtacctt
gttaaccaat 180acccctacat gagcaaccct cccgacacca ttgcctggtc caccaccgcc
accgacctcg 240gctttgtgga cggcaccggc taccagtctc cggatattat ctgccacaga
gacgcaaaga 300atggcaagtt gaccgcaacc gttgcagccg gttcacagat cgaattccag
tggacgacgt 360ggccagagtc tcaccatgga ccggtacgac gccgaagaga agagaacata
ttgtgaccag 420ataggctaac atagcatagt tgattactta cctcgctcca tgcaacggcg
actgtgccac 480cgtggacaag accaccctga agtttgtcaa gatcgccgct caaggcttga
tcgacggctc 540caacccacct ggtgtttggg ctgatgatga aatgatcgcc aacaacaaca
cggccacagt 600gaccattcct gcctcctatg cccccggaaa ctacgtcctt cgccacgaga
tcatcgccct 660tcactctgcg ggtaacctga acggcgcgca gaactacccc cagtgtttca
acatccaaat 720caccggtggc ggcagtgctc agggatctgg caccgctggc acgtccctgt
acaagaatac 780tgatcctggc atcaagtttg acatctactc ggatctgagc ggtggatacc
ctattcctgg 840tcctgcactg ttcaacgctt aa
86230250PRTAspergillus fumigatus 30Met Thr Leu Ser Lys Ile
Thr Ser Ile Ala Gly Leu Leu Ala Ser Ala1 5
10 15Ser Leu Val Ala Gly His Gly Phe Val Ser Gly Ile
Val Ala Asp Gly 20 25 30Lys
Tyr Tyr Gly Gly Tyr Leu Val Asn Gln Tyr Pro Tyr Met Ser Asn 35
40 45Pro Pro Asp Thr Ile Ala Trp Ser Thr
Thr Ala Thr Asp Leu Gly Phe 50 55
60Val Asp Gly Thr Gly Tyr Gln Ser Pro Asp Ile Ile Cys His Arg Asp65
70 75 80Ala Lys Asn Gly Lys
Leu Thr Ala Thr Val Ala Ala Gly Ser Gln Ile 85
90 95Glu Phe Gln Trp Thr Thr Trp Pro Glu Ser His
His Gly Pro Leu Ile 100 105
110Thr Tyr Leu Ala Pro Cys Asn Gly Asp Cys Ala Thr Val Asp Lys Thr
115 120 125Thr Leu Lys Phe Val Lys Ile
Ala Ala Gln Gly Leu Ile Asp Gly Ser 130 135
140Asn Pro Pro Gly Val Trp Ala Asp Asp Glu Met Ile Ala Asn Asn
Asn145 150 155 160Thr Ala
Thr Val Thr Ile Pro Ala Ser Tyr Ala Pro Gly Asn Tyr Val
165 170 175Leu Arg His Glu Ile Ile Ala
Leu His Ser Ala Gly Asn Leu Asn Gly 180 185
190Ala Gln Asn Tyr Pro Gln Cys Phe Asn Ile Gln Ile Thr Gly
Gly Gly 195 200 205Ser Ala Gln Gly
Ser Gly Thr Ala Gly Thr Ser Leu Tyr Lys Asn Thr 210
215 220Asp Pro Gly Ile Lys Phe Asp Ile Tyr Ser Asp Leu
Ser Gly Gly Tyr225 230 235
240Pro Ile Pro Gly Pro Ala Leu Phe Asn Ala 245
250311021DNAPenicillium pinophilum 31atgccttcta ctaaagtcgc
tgccctttct gctgttctag ctttggcctc cacggttgct 60ggccatggtt ttgtgcaaaa
catcgttatc gacggtaaat cgtaagcagt gatgcatcca 120ttattaaact agacatgctt
acaaaaaaat cagttactct ggataccttg tgaatcagtt 180cccctacgag tccaacccac
cagctgttat tgggtgggca acaactgcaa ccgacctggg 240attcgtcgct cccagtgagt
acaccaatgc agacattatc tgccacaaga acgccacacc 300tggcgcgctt tctgctccag
ttgctgcagg gggcactgtc gagctccagt ggactacatg 360gcccgatagt catcacggtc
ctgtcatcag ctacctcgcc aactgcaatg gcaattgttc 420taccgtggat aagactaagc
tagactttgt caagattgac caaggtggtt tgatcgacga 480tactaccccc ccgggtacat
gggcttccga caaacttatc gctgccaaca acagctggac 540tgtaactatc ccctccacca
tcgcgcctgg aaactacgtt ttgcgccacg aaatcattgc 600tcttcactcc gctggaaacg
cagacggtgc ccaaaactac cctcaatgca tcaacttgga 660gatcaccggc agcggaaccg
ccgctccctc tggtaccgct ggcgaaaagc tctacacctc 720tactgacccc ggtatcttgg
tcaatatcta ccaatccttg tcgacctacg ttattcccgg 780accaactctg tggagcggtg
ctgccaatgg cgctgttgcc actggttctg ctactgcggt 840tgctacgact gccactgctt
ctgcgaccgc tactcctacc acacttgtta cctctgtcgc 900tccagcttca tctacctttg
ccactgctgt tgtgaccact gtcgctcctg cagtaactga 960tgtcgtgact gtcaccgatg
tagttaccgt gaccaccgtc atcaccacta ctgtcctttg 1020a
102132322PRTPenicillium
pinophilum 32Met Pro Ser Thr Lys Val Ala Ala Leu Ser Ala Val Leu Ala Leu
Ala1 5 10 15Ser Thr Val
Ala Gly His Gly Phe Val Gln Asn Ile Val Ile Asp Gly 20
25 30Lys Ser Tyr Ser Gly Tyr Leu Val Asn Gln
Phe Pro Tyr Glu Ser Asn 35 40
45Pro Pro Ala Val Ile Gly Trp Ala Thr Thr Ala Thr Asp Leu Gly Phe 50
55 60Val Ala Pro Ser Glu Tyr Thr Asn Ala
Asp Ile Ile Cys His Lys Asn65 70 75
80Ala Thr Pro Gly Ala Leu Ser Ala Pro Val Ala Ala Gly Gly
Thr Val 85 90 95Glu Leu
Gln Trp Thr Thr Trp Pro Asp Ser His His Gly Pro Val Ile 100
105 110Ser Tyr Leu Ala Asn Cys Asn Gly Asn
Cys Ser Thr Val Asp Lys Thr 115 120
125Lys Leu Asp Phe Val Lys Ile Asp Gln Gly Gly Leu Ile Asp Asp Thr
130 135 140Thr Pro Pro Gly Thr Trp Ala
Ser Asp Lys Leu Ile Ala Ala Asn Asn145 150
155 160Ser Trp Thr Val Thr Ile Pro Ser Thr Ile Ala Pro
Gly Asn Tyr Val 165 170
175Leu Arg His Glu Ile Ile Ala Leu His Ser Ala Gly Asn Ala Asp Gly
180 185 190Ala Gln Asn Tyr Pro Gln
Cys Ile Asn Leu Glu Ile Thr Gly Ser Gly 195 200
205Thr Ala Ala Pro Ser Gly Thr Ala Gly Glu Lys Leu Tyr Thr
Ser Thr 210 215 220Asp Pro Gly Ile Leu
Val Asn Ile Tyr Gln Ser Leu Ser Thr Tyr Val225 230
235 240Ile Pro Gly Pro Thr Leu Trp Ser Gly Ala
Ala Asn Gly Ala Val Ala 245 250
255Thr Gly Ser Ala Thr Ala Val Ala Thr Thr Ala Thr Ala Ser Ala Thr
260 265 270Ala Thr Pro Thr Thr
Leu Val Thr Ser Val Ala Pro Ala Ser Ser Thr 275
280 285Phe Ala Thr Ala Val Val Thr Thr Val Ala Pro Ala
Val Thr Asp Val 290 295 300Val Thr Val
Thr Asp Val Val Thr Val Thr Thr Val Ile Thr Thr Thr305
310 315 320Val Leu331486DNAThermoascus
sp. 33atgttgtcgt tcgcttctgc caagtcagct gtgctgacga cccttctact tcttggatcc
60gctcaggctc acactttgat gaccaccctg tttgtggatg gcgtcaatca gggagatggt
120gtctgtattc gcatgaacaa caacggtagt actgccaaca cctatatcca gcctgtcacg
180agcaaggata ttgcctgcgg taagtacagt accggtccag atatcatact ctatttcaat
240ccgacaacag tcagagctgg agagcaatgc taaacatccc caggcattca aggcgaaatt
300ggcgccgctc gagtctgtcc agccaaggct tcatccaccc tcacgttcca attccgagag
360cagccatcca acccgaattc cgctcctctc gatccctcgc acaaaggccc cgctgcggtg
420tacctgaaaa aggtagactc cgccatcgcg agcaacaacg ccgctggaga cggctggttc
480aagatctggg agtccgtcta cgacgagtcc acgggcaaat ggggtacgac caagatgatc
540gagaacaacg ggcacatctc tgtcaaggtc cccgacgata tcgagggtgg gtattatctc
600gcgcgtacgg agcttctggc gctgcacgcg gcgaacgaag gggatccgca gttctacgtt
660ggctgcgcgc agctgttcat cgattcagcg gggacagcga aaccgcctac tgtctctatt
720ggagagggga cctacgatct gagcatgcct gccatgacgt acaatatcta ccagactccg
780ttggctctac catacccgat gtatgggcct cctgtctaca cacctggctc tggctcgggt
840tctggctctg gttccgggtc agcttctgca acgagatctt ctgctattcc tactgccacc
900gctgttacgg actgttcttc cgaagaggac agggaagact cagtcatggc aaccggtgtt
960cccgttgcaa gaagcacact cagaacctgg gttgacagac tgtcatggca tggtaaggcc
1020cgtgagaacg tgaaaccagc cgccaggaga agcgcccttg tccagaccga gggtctgaag
1080ccggaaggct gcatcttcgt caacggcaac tggtgcggtt tcgaggtccc cgattacaac
1140gatgcggaaa gctgctgggc tgtacgttcc cgtctaatta cttaaaacga aataaaagct
1200aacagtactt ttctttttct aatcccaggc ctccgacaac tgctggaaac agtccgactc
1260gtgctggaac cagacccagc ccaccggcta caacaactgc cagatctggc aagaccagaa
1320atgcaagccc atccaggact cgtgtagcca atccaacccg actggaccgc cgaacaaggg
1380caaggatata actccaacgt ggccgcccct ggagggctcg atgaagacct tcaccaagcg
1440cactgtcagt taccgtgatt ggattatgaa aaggaaagga gcataa
148634444PRTThermoascus sp. 34Met Leu Ser Phe Ala Ser Ala Lys Ser Ala Val
Leu Thr Thr Leu Leu1 5 10
15Leu Leu Gly Ser Ala Gln Ala His Thr Leu Met Thr Thr Leu Phe Val
20 25 30Asp Gly Val Asn Gln Gly Asp
Gly Val Cys Ile Arg Met Asn Asn Asn 35 40
45Gly Ser Thr Ala Asn Thr Tyr Ile Gln Pro Val Thr Ser Lys Asp
Ile 50 55 60Ala Cys Gly Ile Gln Gly
Glu Ile Gly Ala Ala Arg Val Cys Pro Ala65 70
75 80Lys Ala Ser Ser Thr Leu Thr Phe Gln Phe Arg
Glu Gln Pro Ser Asn 85 90
95Pro Asn Ser Ala Pro Leu Asp Pro Ser His Lys Gly Pro Ala Ala Val
100 105 110Tyr Leu Lys Lys Val Asp
Ser Ala Ile Ala Ser Asn Asn Ala Ala Gly 115 120
125Asp Gly Trp Phe Lys Ile Trp Glu Ser Val Tyr Asp Glu Ser
Thr Gly 130 135 140Lys Trp Gly Thr Thr
Lys Met Ile Glu Asn Asn Gly His Ile Ser Val145 150
155 160Lys Val Pro Asp Asp Ile Glu Gly Gly Tyr
Tyr Leu Ala Arg Thr Glu 165 170
175Leu Leu Ala Leu His Ala Ala Asn Glu Gly Asp Pro Gln Phe Tyr Val
180 185 190Gly Cys Ala Gln Leu
Phe Ile Asp Ser Ala Gly Thr Ala Lys Pro Pro 195
200 205Thr Val Ser Ile Gly Glu Gly Thr Tyr Asp Leu Ser
Met Pro Ala Met 210 215 220Thr Tyr Asn
Ile Tyr Gln Thr Pro Leu Ala Leu Pro Tyr Pro Met Tyr225
230 235 240Gly Pro Pro Val Tyr Thr Pro
Gly Ser Gly Ser Gly Ser Gly Ser Gly 245
250 255Ser Gly Ser Ala Ser Ala Thr Arg Ser Ser Ala Ile
Pro Thr Ala Thr 260 265 270Ala
Val Thr Asp Cys Ser Ser Glu Glu Asp Arg Glu Asp Ser Val Met 275
280 285Ala Thr Gly Val Pro Val Ala Arg Ser
Thr Leu Arg Thr Trp Val Asp 290 295
300Arg Leu Ser Trp His Gly Lys Ala Arg Glu Asn Val Lys Pro Ala Ala305
310 315 320Arg Arg Ser Ala
Leu Val Gln Thr Glu Gly Leu Lys Pro Glu Gly Cys 325
330 335Ile Phe Val Asn Gly Asn Trp Cys Gly Phe
Glu Val Pro Asp Tyr Asn 340 345
350Asp Ala Glu Ser Cys Trp Ala Ala Ser Asp Asn Cys Trp Lys Gln Ser
355 360 365Asp Ser Cys Trp Asn Gln Thr
Gln Pro Thr Gly Tyr Asn Asn Cys Gln 370 375
380Ile Trp Gln Asp Gln Lys Cys Lys Pro Ile Gln Asp Ser Cys Ser
Gln385 390 395 400Ser Asn
Pro Thr Gly Pro Pro Asn Lys Gly Lys Asp Ile Thr Pro Thr
405 410 415Trp Pro Pro Leu Glu Gly Ser
Met Lys Thr Phe Thr Lys Arg Thr Val 420 425
430Ser Tyr Arg Asp Trp Ile Met Lys Arg Lys Gly Ala
435 44035835DNAPenicillium sp. 35atgctgtctt cgacgactcg
caccctcgcc tttacaggcc ttgcgggcct tctgtccgct 60cccctggtca aggcccatgg
ctttgtccag ggcattgtca tcggtgacca attgtaagtc 120cctctcttgc agttctgtcg
attaactgct ggactgcttg cttgactccc tgctgactcc 180caacagctac agcgggtaca
tcgtcaactc gttcccctac gaatccaacc caccccccgt 240catcggctgg gccacgaccg
ccaccgacct gggcttcgtc gacggcacag gataccaagg 300cccggacatc atctgccacc
ggaatgcgac gcccgcgccg ctgacagccc ccgtggccgc 360cggcggcacc gtcgagctgc
agtggacgcc gtggccggac agccaccacg gacccgtcat 420cacctacctg gcgccgtgca
acggcaactg ctcgaccgtc gacaagacga cgctggagtt 480cttcaagatc gaccagcagg
gcctgatcga cgacacgagc ccgccgggca cctgggcgtc 540ggacaacctc atcgccaaca
acaatagctg gaccgtcacc attcccaaca gcgtcgcccc 600cggcaactac gtcctgcgcc
acgagatcat cgccctgcac tcggccaaca acaaggacgg 660cgcccagaac tacccccagt
gcatcaacat cgaggtcacg ggcggcggct ccgacgcgcc 720tgagggtact ctgggcgagg
atctctacca tgacaccgac ccgggcattc tggtcgacat 780ttacgagccc attgcgacgt
ataccattcc ggggccgcct gagccgacgt tctag 83536253PRTPenicillium sp.
36Met Leu Ser Ser Thr Thr Arg Thr Leu Ala Phe Thr Gly Leu Ala Gly1
5 10 15Leu Leu Ser Ala Pro Leu
Val Lys Ala His Gly Phe Val Gln Gly Ile 20 25
30Val Ile Gly Asp Gln Phe Tyr Ser Gly Tyr Ile Val Asn
Ser Phe Pro 35 40 45Tyr Glu Ser
Asn Pro Pro Pro Val Ile Gly Trp Ala Thr Thr Ala Thr 50
55 60Asp Leu Gly Phe Val Asp Gly Thr Gly Tyr Gln Gly
Pro Asp Ile Ile65 70 75
80Cys His Arg Asn Ala Thr Pro Ala Pro Leu Thr Ala Pro Val Ala Ala
85 90 95Gly Gly Thr Val Glu Leu
Gln Trp Thr Pro Trp Pro Asp Ser His His 100
105 110Gly Pro Val Ile Thr Tyr Leu Ala Pro Cys Asn Gly
Asn Cys Ser Thr 115 120 125Val Asp
Lys Thr Thr Leu Glu Phe Phe Lys Ile Asp Gln Gln Gly Leu 130
135 140Ile Asp Asp Thr Ser Pro Pro Gly Thr Trp Ala
Ser Asp Asn Leu Ile145 150 155
160Ala Asn Asn Asn Ser Trp Thr Val Thr Ile Pro Asn Ser Val Ala Pro
165 170 175Gly Asn Tyr Val
Leu Arg His Glu Ile Ile Ala Leu His Ser Ala Asn 180
185 190Asn Lys Asp Gly Ala Gln Asn Tyr Pro Gln Cys
Ile Asn Ile Glu Val 195 200 205Thr
Gly Gly Gly Ser Asp Ala Pro Glu Gly Thr Leu Gly Glu Asp Leu 210
215 220Tyr His Asp Thr Asp Pro Gly Ile Leu Val
Asp Ile Tyr Glu Pro Ile225 230 235
240Ala Thr Tyr Thr Ile Pro Gly Pro Pro Glu Pro Thr Phe
245 25037977DNAThielavia terrestris 37atgaagctgt
catcccagct cgccgccctc acgctggccg cggcctccgt gtcaggccac 60tacatcttcg
agcagattgc ccatggcggc accaagttcc caccttacga gtacatccga 120agaaacacga
actataacag ccctgtcacc agtctctcgt cgaacgacct gcgatgcaac 180gtaggcggcg
agacggctgg caacacgacc gtcctcgacg tgaaggcggg cgactccttc 240accttctact
cggacgtggc cgtgtaccac caggggccca tctcactgtg cgtgccccgg 300gccaactttg
atcagtccca agcggactgt ccgctcgcct ggataaccac aattgactga 360cagcccgcac
agctacatgt ccaaggctcc cggctccgtc gtggactacg acggctccgg 420cgactggttc
aagatccacg actggggccc gaccttcagc aacggccagg cctcgtggcc 480gctgcggggt
gcgtcccttc cctttccctc ccccttcctc ccccttcctc cccccctttc 540cccccttttc
tgtctggtcg cacgccctgc tgacgtcccc gtagacaact accagtacaa 600catcccgacg
tgcatcccga acggcgagta cctgctgcgc atccagtcgc tggcgatcca 660caacccgggc
gccacgccgc agttctacat cagctgcgcg caggtccggg tctcgggcgg 720cggcagcgcc
tccccctccc caacggccaa gatccccggc gcgttcaagg cgaccgatcc 780cgggtatacc
gcgaatgtga gtgccctatg ttccttgcgc tccttgttcc ttgctccttg 840ctcggcgtgc
ttgaacgcta cgggctgtgg agggagggat ggatggatga ataggatgct 900gactgatggt
gggacaccag atttacaata acttccactc gtatacggtg ccgggtccgg 960cggtctttca
gtgctag
97738223PRTThielavia terrestris 38Met Lys Leu Ser Ser Gln Leu Ala Ala Leu
Thr Leu Ala Ala Ala Ser1 5 10
15Val Ser Gly His Tyr Ile Phe Glu Gln Ile Ala His Gly Gly Thr Lys
20 25 30Phe Pro Pro Tyr Glu Tyr
Ile Arg Arg Asn Thr Asn Tyr Asn Ser Pro 35 40
45Val Thr Ser Leu Ser Ser Asn Asp Leu Arg Cys Asn Val Gly
Gly Glu 50 55 60Thr Ala Gly Asn Thr
Thr Val Leu Asp Val Lys Ala Gly Asp Ser Phe65 70
75 80Thr Phe Tyr Ser Asp Val Ala Val Tyr His
Gln Gly Pro Ile Ser Leu 85 90
95Tyr Met Ser Lys Ala Pro Gly Ser Val Val Asp Tyr Asp Gly Ser Gly
100 105 110Asp Trp Phe Lys Ile
His Asp Trp Gly Pro Thr Phe Ser Asn Gly Gln 115
120 125Ala Ser Trp Pro Leu Arg Asp Asn Tyr Gln Tyr Asn
Ile Pro Thr Cys 130 135 140Ile Pro Asn
Gly Glu Tyr Leu Leu Arg Ile Gln Ser Leu Ala Ile His145
150 155 160Asn Pro Gly Ala Thr Pro Gln
Phe Tyr Ile Ser Cys Ala Gln Val Arg 165
170 175Val Ser Gly Gly Gly Ser Ala Ser Pro Ser Pro Thr
Ala Lys Ile Pro 180 185 190Gly
Ala Phe Lys Ala Thr Asp Pro Gly Tyr Thr Ala Asn Ile Tyr Asn 195
200 205Asn Phe His Ser Tyr Thr Val Pro Gly
Pro Ala Val Phe Gln Cys 210 215
22039878DNAThielavia terrestris 39atgaagttct cactggtgtc tctgctggct
tacggcctct cggtcgaggc gcactccatc 60ttccaggttc gtctcgcaca tcacgctcaa
ctcggctcgt ggcgtaaggg caaggattaa 120cacggccggc agagagtctc ggtcaacggc
caagaccaag gcctgctcac cggcctccgc 180gctccaagca acaacaaccc agtgcaagat
gtcaacagcc agaacatgat ttgcggccag 240tcgggctcca agtcgcagac cgttatcaac
gtcaaggccg gcgacaggat cggctcgctc 300tggcagcatg tcatcggcgg cgcccagttt
tcgggtgacc cggacaaccc gatcgcccac 360tcgcacaagg gccccgtgat ggcgtacctt
gctaaggtcg acaatgccgc gtccgcgagc 420caaacgggtc tgaagtggta agtagcgggc
gacgctcagg ggacggggat cgggggcctg 480ctccatccga gactaacacc gtggacaggt
tcaagatctg gcaggacggg ttcgatacca 540gcagcaagac atggggcgtc gacaacctga
tcaagaacaa cggctgggtg tacttccacc 600tgccgcagtg cctcgctccg ggccagtatc
tcctgcgcgt cgaggttctg gcgctgcact 660cggcgtacca gcagggccag gcccagttct
accagtcctg cgcccagatc aacgtctccg 720gctccgggtc cttcagcccg tcccagacgg
tcagcatccc gggcgtctac agcgccaccg 780acccgagcat cctcatcaac atctacggca
gcacggggca gcccgacaac ggcggcaagg 840cttacaaccc ccctggaccc gccccgatct
cctgctga 87840246PRTThielavia terrestris 40Met
Lys Phe Ser Leu Val Ser Leu Leu Ala Tyr Gly Leu Ser Val Glu1
5 10 15Ala His Ser Ile Phe Gln Arg
Val Ser Val Asn Gly Gln Asp Gln Gly 20 25
30Leu Leu Thr Gly Leu Arg Ala Pro Ser Asn Asn Asn Pro Val
Gln Asp 35 40 45Val Asn Ser Gln
Asn Met Ile Cys Gly Gln Ser Gly Ser Lys Ser Gln 50 55
60Thr Val Ile Asn Val Lys Ala Gly Asp Arg Ile Gly Ser
Leu Trp Gln65 70 75
80His Val Ile Gly Gly Ala Gln Phe Ser Gly Asp Pro Asp Asn Pro Ile
85 90 95Ala His Ser His Lys Gly
Pro Val Met Ala Tyr Leu Ala Lys Val Asp 100
105 110Asn Ala Ala Ser Ala Ser Gln Thr Gly Leu Lys Trp
Phe Lys Ile Trp 115 120 125Gln Asp
Gly Phe Asp Thr Ser Ser Lys Thr Trp Gly Val Asp Asn Leu 130
135 140Ile Lys Asn Asn Gly Trp Val Tyr Phe His Leu
Pro Gln Cys Leu Ala145 150 155
160Pro Gly Gln Tyr Leu Leu Arg Val Glu Val Leu Ala Leu His Ser Ala
165 170 175Tyr Gln Gln Gly
Gln Ala Gln Phe Tyr Gln Ser Cys Ala Gln Ile Asn 180
185 190Val Ser Gly Ser Gly Ser Phe Ser Pro Ser Gln
Thr Val Ser Ile Pro 195 200 205Gly
Val Tyr Ser Ala Thr Asp Pro Ser Ile Leu Ile Asn Ile Tyr Gly 210
215 220Ser Thr Gly Gln Pro Asp Asn Gly Gly Lys
Ala Tyr Asn Pro Pro Gly225 230 235
240Pro Ala Pro Ile Ser Cys 245411253DNAThielavia
terrestris 41atgaggacga cattcgccgc cgcgttggca gccttcgctg cgcaggaagt
ggcaggccat 60gccatcttcc aacagctctg ggtggacggc accgactata tacgtgctcc
ccttttcctt 120ttgtgtttgc ccatcctcga ttgataaccc gaggccatcc aatgctgact
cttacagcac 180ggctcctcct gcgtccgcat gccgctgtcg aactcgcccg tcacgaacgt
cggcagcagg 240gacatgatct gcaacgccgg cacgcgcccc gtcagcggga agtgccccgt
caaggccggc 300ggcaccgtga cggttgagat gcaccaggtg ggctgatttc ctgagcgtcc
tattcctccc 360ggaagcccct ttcccatcct ttgccctggc taacccctcc gcccctccca
gcaacccggg 420gatcggtcgt gtaacaacga agccatcggc ggcgcccact ggggaccggt
gcaggtgtac 480ctcagcaagg tggaggacgc gagcacggcg gacgggtcga cgggctggtt
caagatcttc 540gcggacacgt ggtccaagaa ggcgggcagc tcggtggggg acgacgacaa
ctggggcacg 600cgcgacctca acgcgtgctg cggcaagatg caggtcaaga tcccggcgga
catcccgtcg 660ggcgactacc tgctgcgggc ggaggcgctg gcgctgcaca cggcgggcca
ggtgggcggc 720gcgcagttct acatgagctg ctaccagatc accgtgtcgg gcggcggcag
cgccagcccg 780gccaccgtca agttccccgg cgcctacagc gccaacgacc cgggcatcca
catcaacatc 840cacgcggccg tgtccaacta cgtcgcgccc ggcccggccg tctattccgg
cggcacgacc 900aaggtggccg ggtccgggtg ccaaggctgc gagaacacgt gcaaggtcgg
ctcgtcgccc 960acggcgacgg cgccgtcggg caagagcggc gcgggttccg acggcggcgc
tgggaccgac 1020ggcgggtctt cgtcttcgag ccccgacacg ggcagcgcgt gcagcgtgca
ggcctacggg 1080cagtgcggcg ggaacgggta ctcgggttgc acccagtgcg cggtaagttc
ggggtcgtct 1140gtcttttgta ggaacatccg agaggcttgg ctgacgaggc gttgttgtag
cccggctata 1200cttgcaaggc ggtctctccg ccgtactatt cgcagtgcgc cccttcttct
tag 125342334PRTThielavia terrestris 42Met Arg Thr Thr Phe Ala
Ala Ala Leu Ala Ala Phe Ala Ala Gln Glu1 5
10 15Val Ala Gly His Ala Ile Phe Gln Gln Leu Trp His
Gly Ser Ser Cys 20 25 30Val
Arg Met Pro Leu Ser Asn Ser Pro Val Thr Asn Val Gly Ser Arg 35
40 45Asp Met Ile Cys Asn Ala Gly Thr Arg
Pro Val Ser Gly Lys Cys Pro 50 55
60Val Lys Ala Gly Gly Thr Val Thr Val Glu Met His Gln Gln Pro Gly65
70 75 80Asp Arg Ser Cys Asn
Asn Glu Ala Ile Gly Gly Ala His Trp Gly Pro 85
90 95Val Gln Val Tyr Leu Ser Lys Val Glu Asp Ala
Ser Thr Ala Asp Gly 100 105
110Ser Thr Gly Trp Phe Lys Ile Phe Ala Asp Thr Trp Ser Lys Lys Ala
115 120 125Gly Ser Ser Val Gly Asp Asp
Asp Asn Trp Gly Thr Arg Asp Leu Asn 130 135
140Ala Cys Cys Gly Lys Met Gln Val Lys Ile Pro Ala Asp Ile Pro
Ser145 150 155 160Gly Asp
Tyr Leu Leu Arg Ala Glu Ala Leu Ala Leu His Thr Ala Gly
165 170 175Gln Val Gly Gly Ala Gln Phe
Tyr Met Ser Cys Tyr Gln Ile Thr Val 180 185
190Ser Gly Gly Gly Ser Ala Ser Pro Ala Thr Val Lys Phe Pro
Gly Ala 195 200 205Tyr Ser Ala Asn
Asp Pro Gly Ile His Ile Asn Ile His Ala Ala Val 210
215 220Ser Asn Tyr Val Ala Pro Gly Pro Ala Val Tyr Ser
Gly Gly Thr Thr225 230 235
240Lys Val Ala Gly Ser Gly Cys Gln Gly Cys Glu Asn Thr Cys Lys Val
245 250 255Gly Ser Ser Pro Thr
Ala Thr Ala Pro Ser Gly Lys Ser Gly Ala Gly 260
265 270Ser Asp Gly Gly Ala Gly Thr Asp Gly Gly Ser Ser
Ser Ser Ser Pro 275 280 285Asp Thr
Gly Ser Ala Cys Ser Val Gln Ala Tyr Gly Gln Cys Gly Gly 290
295 300Asn Gly Tyr Ser Gly Cys Thr Gln Cys Ala Pro
Gly Tyr Thr Cys Lys305 310 315
320Ala Val Ser Pro Pro Tyr Tyr Ser Gln Cys Ala Pro Ser Ser
325 33043798DNAThielavia terrestris 43atgaagctga
gcgttgccat cgccgtgctg gcgtcggctc ttgccgaggc tcactgtgag 60tgcatcgtct
cactccagct actgcgaagc ttgctgacga tggtccctag acaccttccc 120cagcatcgga
aacaccgctg actggcagta tgtgcggatt acaacgaact accagagcaa 180cgggccggtg
acggacgtca cctcggatca aattcggtgc tacgaacgga acccaggcac 240gggagcgcag
ggcatataca acgtcaccgc cggccagacc atcaactaca acgcgaaggc 300gtccatctcc
cacccggggc ccatgtcctt ctacattgct aaggttcccg ccggccaaac 360cgctgcgacc
tgggacggta agggggctgt gtggaccaag atctaccagg acatgcccaa 420gttcggcagc
agcctgacct ggcccaccat gggtaagaat tctcaccctg gaaatgaacg 480cacatttgca
cagatctaac atggcctaca ggcgccaagt ctgtccccgt caccatccct 540cgttgcctcc
agaacggcga ttaccttctg cgagccgagc acatcgctct acacagcgcg 600agcagcgtcg
gtggcgccca gttctacctc tcgtgcgccc agcttactgt cagcggcggc 660agtggcacct
ggaaccccaa gaaccgggtc tccttccccg gcgcttacaa ggcaacagac 720ccgggcatct
tgatcaacat ctactacccc gtgccgacca gctactcgcc gcccggcccg 780ccggctgaga
cgtgctaa
79844227PRTThielavia terrestris 44Met Lys Leu Ser Val Ala Ile Ala Val Leu
Ala Ser Ala Leu Ala Glu1 5 10
15Ala His Tyr Thr Phe Pro Ser Ile Gly Asn Thr Ala Asp Trp Gln Tyr
20 25 30Val Arg Ile Thr Thr Asn
Tyr Gln Ser Asn Gly Pro Val Thr Asp Val 35 40
45Thr Ser Asp Gln Ile Arg Cys Tyr Glu Arg Asn Pro Gly Thr
Gly Ala 50 55 60Gln Gly Ile Tyr Asn
Val Thr Ala Gly Gln Thr Ile Asn Tyr Asn Ala65 70
75 80Lys Ala Ser Ile Ser His Pro Gly Pro Met
Ser Phe Tyr Ile Ala Lys 85 90
95Val Pro Ala Gly Gln Thr Ala Ala Thr Trp Asp Gly Lys Gly Ala Val
100 105 110Trp Thr Lys Ile Tyr
Gln Asp Met Pro Lys Phe Gly Ser Ser Leu Thr 115
120 125Trp Pro Thr Met Gly Ala Lys Ser Val Pro Val Thr
Ile Pro Arg Cys 130 135 140Leu Gln Asn
Gly Asp Tyr Leu Leu Arg Ala Glu His Ile Ala Leu His145
150 155 160Ser Ala Ser Ser Val Gly Gly
Ala Gln Phe Tyr Leu Ser Cys Ala Gln 165
170 175Leu Thr Val Ser Gly Gly Ser Gly Thr Trp Asn Pro
Lys Asn Arg Val 180 185 190Ser
Phe Pro Gly Ala Tyr Lys Ala Thr Asp Pro Gly Ile Leu Ile Asn 195
200 205Ile Tyr Tyr Pro Val Pro Thr Ser Tyr
Ser Pro Pro Gly Pro Pro Ala 210 215
220Glu Thr Cys225451107DNAThielavia terrestris 45atgccttctt tcgcctccaa
gactctcctt tccaccctgg cgggtgccgc atccgtggcc 60gcccacgggc acgtgtcgaa
catcgtcatc aacggggtct cgtaccaggg ttacgatccg 120acctccttcc cttacatgca
gaacccgccc atcgtggtcg gctggactgc cgccgacacg 180gacaacggct ttgttgcccc
ggatgccttc gccagtggcg atatcatctg ccacaagaac 240gccaccaacg ccaagggcca
cgccgtggtc gccgcgggag acaagatctt catccagtgg 300aacacatggc ccgagtccca
ccacggcccc gtcatcgact acctcgcgag ctgcggcagc 360gcgtcctgcg agaccgtcga
caagaccaag ctcgagttct tcaagatcga cgaggtcggc 420ctggtcgacg gcagctcggc
gcccggtgtg tggggctccg accagctcat cgccaacaac 480aactcgtggc tcgtcgagat
cccgcccacc atcgcgccgg gcaactacgt cctgcgccac 540gagatcatcg cgctgcacag
cgccgaaaac gccgacggcg cccagaacta cccgcagtgc 600ttcaacctgc agatcaccgg
caccggcacc gccaccccct ccggcgtccc cggcacctcg 660ctctacaccc cgaccgaccc
gggcatcctc gtcaacatct acagcgcccc gatcacctac 720accgtcccgg ggccggccct
catctccggc gccgtcagca tcgcccagtc ctcctccgcc 780atcaccgcct ccggcaccgc
cctgaccggc tctgccaccg cacccgccgc cgccgctgct 840accacaactt ccaccaccaa
cgccgcggct gctgctacct ctgctgctgc tgctgctggt 900acttccacaa ccaccaccag
cgccgcggcc gtggtccaga cctcctcctc ctcctcctcc 960gccccgtcct ctgccgccgc
cgccgccacc accaccgcgg ctgccagcgc ccgcccgacc 1020ggctgctcct ctggccgctc
caggaagcag ccgcgccgcc acgcgcggga tatggtggtt 1080gcgcgagggg ctgaggaggc
aaactga 110746368PRTThielavia
terrestris 46Met Pro Ser Phe Ala Ser Lys Thr Leu Leu Ser Thr Leu Ala Gly
Ala1 5 10 15Ala Ser Val
Ala Ala His Gly His Val Ser Asn Ile Val Ile Asn Gly 20
25 30Val Ser Tyr Gln Gly Tyr Asp Pro Thr Ser
Phe Pro Tyr Met Gln Asn 35 40
45Pro Pro Ile Val Val Gly Trp Thr Ala Ala Asp Thr Asp Asn Gly Phe 50
55 60Val Ala Pro Asp Ala Phe Ala Ser Gly
Asp Ile Ile Cys His Lys Asn65 70 75
80Ala Thr Asn Ala Lys Gly His Ala Val Val Ala Ala Gly Asp
Lys Ile 85 90 95Phe Ile
Gln Trp Asn Thr Trp Pro Glu Ser His His Gly Pro Val Ile 100
105 110Asp Tyr Leu Ala Ser Cys Gly Ser Ala
Ser Cys Glu Thr Val Asp Lys 115 120
125Thr Lys Leu Glu Phe Phe Lys Ile Asp Glu Val Gly Leu Val Asp Gly
130 135 140Ser Ser Ala Pro Gly Val Trp
Gly Ser Asp Gln Leu Ile Ala Asn Asn145 150
155 160Asn Ser Trp Leu Val Glu Ile Pro Pro Thr Ile Ala
Pro Gly Asn Tyr 165 170
175Val Leu Arg His Glu Ile Ile Ala Leu His Ser Ala Glu Asn Ala Asp
180 185 190Gly Ala Gln Asn Tyr Pro
Gln Cys Phe Asn Leu Gln Ile Thr Gly Thr 195 200
205Gly Thr Ala Thr Pro Ser Gly Val Pro Gly Thr Ser Leu Tyr
Thr Pro 210 215 220Thr Asp Pro Gly Ile
Leu Val Asn Ile Tyr Ser Ala Pro Ile Thr Tyr225 230
235 240Thr Val Pro Gly Pro Ala Leu Ile Ser Gly
Ala Val Ser Ile Ala Gln 245 250
255Ser Ser Ser Ala Ile Thr Ala Ser Gly Thr Ala Leu Thr Gly Ser Ala
260 265 270Thr Ala Pro Ala Ala
Ala Ala Ala Thr Thr Thr Ser Thr Thr Asn Ala 275
280 285Ala Ala Ala Ala Thr Ser Ala Ala Ala Ala Ala Gly
Thr Ser Thr Thr 290 295 300Thr Thr Ser
Ala Ala Ala Val Val Gln Thr Ser Ser Ser Ser Ser Ser305
310 315 320Ala Pro Ser Ser Ala Ala Ala
Ala Ala Thr Thr Thr Ala Ala Ala Ser 325
330 335Ala Arg Pro Thr Gly Cys Ser Ser Gly Arg Ser Arg
Lys Gln Pro Arg 340 345 350Arg
His Ala Arg Asp Met Val Val Ala Arg Gly Ala Glu Glu Ala Asn 355
360 36547993DNAThielavia terrestris
47atgccgcccg cactccctca actcctaacc acggtcctga ccgccctcac cctcggttcc
60accgccctcg cccactcaca cctcgcgtac attatcgtta acggcaagct ctaccagggc
120ttcgacccgc gcccgcacca ggccaactac ccttcccggg tcgggtggtc caccggcgcc
180gtcgacgacg gcttcgtcac gccggccaac tactccaccc cggacatcat ttgccacatc
240gccggcacca gcccggccgg ccacgcgccc gtgcgcccgg gcgaccgcat ccacgtccag
300tggaacggct ggccggtcgg ccacatcggt cccgtgctgt cgtacctcgc ccgctgcgag
360tcggacacgg gctgcacggg ccagaacaag accgcgctgc ggtggaccaa gatcgacgac
420tccagcccga ccatgcagaa cgtcgccggc gcgggcaccc agggcgaggg cacccccggc
480aagcgctggg ccaccgacgt gctgatcgcc gccaacaaca gctggcaggt cgccgtgccg
540gcggggctgc cgaccggcgc gtacgtgctg cgcaacgaga tcatcgcgct gcactacgcg
600gcgaggaaga acggggcgca gaactatccg ctctgcatga acctgtgggt ggacgccagt
660ggtgataata gtagtgtggc tgcaacgacg gcggcggtga cggcgggggg tctgcagatg
720gatgcgtatg acgcgcgcgg gttctacaag gagaacgatc cgggcgtgct ggtcaatgtc
780acggccgcgc tgtcgtcgta tgtcgtgccc gggccgacgg tggcggcggg cgccacgccg
840gtgccgtacg cgcagcagag cccgagcgtg tcgacggcgg cgggcacgcc cgtcgtcgtt
900acaaggacta gcgagacggc gccgtacacg ggcgccatga cgccgacggt tgcggcgagg
960atgaagggga gggggtatga tcggcggggt tag
99348330PRTThielavia terrestris 48Met Pro Pro Ala Leu Pro Gln Leu Leu Thr
Thr Val Leu Thr Ala Leu1 5 10
15Thr Leu Gly Ser Thr Ala Leu Ala His Ser His Leu Ala Tyr Ile Ile
20 25 30Val Asn Gly Lys Leu Tyr
Gln Gly Phe Asp Pro Arg Pro His Gln Ala 35 40
45Asn Tyr Pro Ser Arg Val Gly Trp Ser Thr Gly Ala Val Asp
Asp Gly 50 55 60Phe Val Thr Pro Ala
Asn Tyr Ser Thr Pro Asp Ile Ile Cys His Ile65 70
75 80Ala Gly Thr Ser Pro Ala Gly His Ala Pro
Val Arg Pro Gly Asp Arg 85 90
95Ile His Val Gln Trp Asn Gly Trp Pro Val Gly His Ile Gly Pro Val
100 105 110Leu Ser Tyr Leu Ala
Arg Cys Glu Ser Asp Thr Gly Cys Thr Gly Gln 115
120 125Asn Lys Thr Ala Leu Arg Trp Thr Lys Ile Asp Asp
Ser Ser Pro Thr 130 135 140Met Gln Asn
Val Ala Gly Ala Gly Thr Gln Gly Glu Gly Thr Pro Gly145
150 155 160Lys Arg Trp Ala Thr Asp Val
Leu Ile Ala Ala Asn Asn Ser Trp Gln 165
170 175Val Ala Val Pro Ala Gly Leu Pro Thr Gly Ala Tyr
Val Leu Arg Asn 180 185 190Glu
Ile Ile Ala Leu His Tyr Ala Ala Arg Lys Asn Gly Ala Gln Asn 195
200 205Tyr Pro Leu Cys Met Asn Leu Trp Val
Asp Ala Ser Gly Asp Asn Ser 210 215
220Ser Val Ala Ala Thr Thr Ala Ala Val Thr Ala Gly Gly Leu Gln Met225
230 235 240Asp Ala Tyr Asp
Ala Arg Gly Phe Tyr Lys Glu Asn Asp Pro Gly Val 245
250 255Leu Val Asn Val Thr Ala Ala Leu Ser Ser
Tyr Val Val Pro Gly Pro 260 265
270Thr Val Ala Ala Gly Ala Thr Pro Val Pro Tyr Ala Gln Gln Ser Pro
275 280 285Ser Val Ser Thr Ala Ala Gly
Thr Pro Val Val Val Thr Arg Thr Ser 290 295
300Glu Thr Ala Pro Tyr Thr Gly Ala Met Thr Pro Thr Val Ala Ala
Arg305 310 315 320Met Lys
Gly Arg Gly Tyr Asp Arg Arg Gly 325
330491221DNAThielavia terrestris 49atgaagacat tcaccgccct cctggccgca
gccggcctcg tcgccggcca tggatatgtc 60gacaacgcca ccattggcgg ccagttttat
caggtactct accgcttcac ccaaggtccg 120ctggccacaa ctctataggt gtcataaatt
aacaagccac cgtcccgcag ttctatcagg 180tgtgctcgct accgaccatg tggtcccgtc
tcagcaagcc actcacacgc ccatgatccc 240ctagccttac gtcgacccgt atttagcaac
cttggcacgt agtatttatt gtcccaaata 300ttgagctgaa ctgcacctcc ctagaatccc
gcggtgctaa cattctttca gcccgacagg 360gtctctcgat ccatcccggg caacggcccg
gtcacggacg tcactctcat cgacctgcag 420tgcaacgcca attccacccc ggccaagctc
cacgccactg ccgctgccgg ctcggacgtg 480attctccgct ggacgctctg gcctgagtcg
cacgttggcc ccgtcatcac ctacatggcc 540cgctgccccg acacgggctg ccaggactgg
atgccgggca cttcgtagga gcccatcttg 600caccatatcc atttcaaccg gccacacgca
ctgacccata tgtctgtcta cccctgcagt 660gcggtctggt tcaagatcaa ggagggcggc
cgcgacggca cttccaacac ctgggccgac 720gtacgtgtac cccgtcccag agagccaaag
cccccccttc aacaaagcaa acatctcaat 780agcccgagcc tacgcactaa cccctctcct
tccccctcga aaacacagac cccgctgatg 840acggcgccca cctcgtacac gtacacgatc
ccctcctgcc tgaagaaggg ctactacctg 900gtccgccacg agatcatcgc gctgcacgcc
gcctacacct accccggcgc gcagttctac 960ccgggctgcc accagctcaa cgtcacgggc
ggcgggtcca ccgtaccgtc gagcggcctg 1020gtggcctttc ccggggcgta caagggcagt
gaccccggga ttacgtacga tgcgtataaa 1080ggtgggttgg ctggttggcc caggtcttgg
tgatggggga atgtggtgat gaggtttatt 1140atttgggatc ccgtggctaa cgtaaccctg
ggtgtagcgc aaacgtacca gattcctggg 1200ccggcggtct ttacttgctg a
122150236PRTThielavia terrestris 50Met
Lys Thr Phe Thr Ala Leu Leu Ala Ala Ala Gly Leu Val Ala Gly1
5 10 15His Gly Tyr Val Asp Asn Ala
Thr Ile Gly Gly Gln Phe Tyr Gln Asn 20 25
30Pro Ala Val Leu Thr Phe Phe Gln Pro Asp Arg Val Ser Arg
Ser Ile 35 40 45Pro Gly Asn Gly
Pro Val Thr Asp Val Thr Leu Ile Asp Leu Gln Cys 50 55
60Asn Ala Asn Ser Thr Pro Ala Lys Leu His Ala Thr Ala
Ala Ala Gly65 70 75
80Ser Asp Val Ile Leu Arg Trp Thr Leu Trp Pro Glu Ser His Val Gly
85 90 95Pro Val Ile Thr Tyr Met
Ala Arg Cys Pro Asp Thr Gly Cys Gln Asp 100
105 110Trp Met Pro Gly Thr Ser Ala Val Trp Phe Lys Ile
Lys Glu Gly Gly 115 120 125Arg Asp
Gly Thr Ser Asn Thr Trp Ala Asp Thr Pro Leu Met Thr Ala 130
135 140Pro Thr Ser Tyr Thr Tyr Thr Ile Pro Ser Cys
Leu Lys Lys Gly Tyr145 150 155
160Tyr Leu Val Arg His Glu Ile Ile Ala Leu His Ala Ala Tyr Thr Tyr
165 170 175Pro Gly Ala Gln
Phe Tyr Pro Gly Cys His Gln Leu Asn Val Thr Gly 180
185 190Gly Gly Ser Thr Val Pro Ser Ser Gly Leu Val
Ala Phe Pro Gly Ala 195 200 205Tyr
Lys Gly Ser Asp Pro Gly Ile Thr Tyr Asp Ala Tyr Lys Ala Gln 210
215 220Thr Tyr Gln Ile Pro Gly Pro Ala Val Phe
Thr Cys225 230 23551933DNAThielavia
terrestris 51atggccttgc tgctcttggc aggcttggcc attctggccg ggccggctca
tgcccacggc 60ggcctcgcca actacacagt gggcaacacc tggtataggg ggtgcgtaag
gggggcaccg 120acaacgcctg cttagtaact ccaccatttc gagcgggcta acaccgggcg
cagctacgac 180cccttcacgc cggcggccga ccagatcggc cagccgtgga tgatccaacg
cgcgtgggac 240tcgatcgacc cgatcttcag cgtcaacgac aaggcgctcg cctgcaacac
cccggccacg 300gcgccgacct cttacattcc catccgcgcg ggcgagaaca tcacggccgt
gtactggtac 360tggctgcacc cggtgggccc catgacggcg tggctggcgc ggtgcgacgg
cgactgccgc 420gacgccgacg tcaacgaggc gcgctggttc aagatctggg aggccggcct
gctcagcggg 480ccgaacctgg ccgagggcat gtggtaccag aaggcgttcc agaactggga
cggcagcccg 540gacctgtggc ccgtcacgat cccggccggg ctgaagagcg gcctgtacat
gatccggcac 600gagatcttgt cgatccacgt cgaggataaa ccgcagtttt atcccgagtg
tgcgcatctg 660aatgtgaccg ggggtgggga cctgctgccg cctgatgagt ttttggtgaa
gttcccgggc 720gcttacaaag aagatagtga gtgaaacgcg aagcttcggt agccattggg
ttgcgctgat 780ggaggttaga cccgtcgatc aagatcaata tctactcgga ccagtacgcc
aatacaacgg 840tgagtgtaac aggtcgagca aaaccaaaca gatgccgatg actgatgatc
tcagaattac 900acaattcccg gagggccgat atgggatggg tga
93352250PRTThielavia terrestris 52Met Ala Leu Leu Leu Leu Ala
Gly Leu Ala Ile Leu Ala Gly Pro Ala1 5 10
15His Ala His Gly Gly Leu Ala Asn Tyr Thr Val Gly Asn
Thr Trp Tyr 20 25 30Arg Gly
Tyr Asp Pro Phe Thr Pro Ala Ala Asp Gln Ile Gly Gln Pro 35
40 45Trp Met Ile Gln Arg Ala Trp Asp Ser Ile
Asp Pro Ile Phe Ser Val 50 55 60Asn
Asp Lys Ala Leu Ala Cys Asn Thr Pro Ala Thr Ala Pro Thr Ser65
70 75 80Tyr Ile Pro Ile Arg Ala
Gly Glu Asn Ile Thr Ala Val Tyr Trp Tyr 85
90 95Trp Leu His Pro Val Gly Pro Met Thr Ala Trp Leu
Ala Arg Cys Asp 100 105 110Gly
Asp Cys Arg Asp Ala Asp Val Asn Glu Ala Arg Trp Phe Lys Ile 115
120 125Trp Glu Ala Gly Leu Leu Ser Gly Pro
Asn Leu Ala Glu Gly Met Trp 130 135
140Tyr Gln Lys Ala Phe Gln Asn Trp Asp Gly Ser Pro Asp Leu Trp Pro145
150 155 160Val Thr Ile Pro
Ala Gly Leu Lys Ser Gly Leu Tyr Met Ile Arg His 165
170 175Glu Ile Leu Ser Ile His Val Glu Asp Lys
Pro Gln Phe Tyr Pro Glu 180 185
190Cys Ala His Leu Asn Val Thr Gly Gly Gly Asp Leu Leu Pro Pro Asp
195 200 205Glu Phe Leu Val Lys Phe Pro
Gly Ala Tyr Lys Glu Asp Asn Pro Ser 210 215
220Ile Lys Ile Asn Ile Tyr Ser Asp Gln Tyr Ala Asn Thr Thr Asn
Tyr225 230 235 240Thr Ile
Pro Gly Gly Pro Ile Trp Asp Gly 245
250531584DNAThielavia terrestris 53atgatgccgt cccttgttcg cttctcaatg
ggtctggcga ccgccttcgc ctcgctgtcc 60acagcacata ccgtcttcac cacgcttttc
atcaacggcg tcgaccaagg ggacgggacc 120tgcatccgca tggccaagaa gggcagcgtt
tgcacccatc ccattgctgg tggcctcgac 180agcccagaca tggcttgtgg tatgccctct
gcgtttcccc tgcgagagct ttcctcgagc 240taacccaatg ccgcgttgcc caggccgaga
cggacaacaa gccgtggcat tcacctgccc 300agccccggcg ggctccaagt tgagcttcga
gttccgcatg tgggccgacg cctctcagcc 360cggctctatc gacccatccc acctcggctc
gacggcaatc tacctcaaac aagtctccaa 420catcagctcc gactcggctg ccggccctgg
ctggttcaag atctacgccg agggctacga 480cacagccgcc aagaagtggg ccacagagaa
gctcatcgac aacggcggcc tgctgagcat 540cgagcttccg cccactctgc cggcgggata
ctacctcgcc cgcagcgaga tcgtcaccat 600ccagaacgtc accaacgacc acgtcgaccc
gcagttctac gttggctgcg cacagctctt 660cgtccagggg cctccgacca cccccaccgt
cccgccagac agactcgtct ccatcccggg 720ccacgtccat gcctccgacc cggggctgac
cttcaacatc tggcgcgacg acccctccaa 780gacggcctac accgtcgtcg gcccggcccc
cttctccccc accgccgccc ccacccccac 840ctccaccaac accaacgggc agcaacaaca
acaacagcaa caggcgataa agcagacgga 900cggcgtgatc cccgccgact gccagctcaa
gaacgccaac tggtgcggcg ccgaggtgcc 960cgcgtacgcc gacgaggccg gctgctgggc
gtcgtcggcc gactgcttcg cccagctgga 1020cgcctgctac acgtcggcgc cgcccacggg
cagccgcggc tgccggctgt gggaggactg 1080gtgcaccggc attcagcagg gctgccgcgc
ggggcggtgg cgggggccgc cgccctttca 1140tggggagggg gcagcagcgg aggtgtgaac
ggttcgggga cgggtggcgg tggtggtggt 1200ggtggtggtg gcactggctc ttcttcggct
tctgccccga cggagacggc ctctgctggc 1260cgggggggcg caagaatagc tgccgtggcc
ggctgcggag gcgggacagg agacatggtt 1320gaagaggttt tcctctttta ttgggacgct
tgcagcggct ggcgacggag ccgtggtggt 1380ggttcgattc ttgcgaggct tatccttcat
gtccttcttc cacttttgag accgaggcga 1440gcccctcgag tccatttact tctcttccac
ctgtacctca acttctgtta tccaggaacc 1500agtggtttct ataatcgcct gagcattaaa
ctaggcatat ggccaagcaa aatgtcgcct 1560gatgtagcgc attacgtgaa ataa
158454478PRTThielavia terrestris 54Met
Met Pro Ser Leu Val Arg Phe Ser Met Gly Leu Ala Thr Ala Phe1
5 10 15Ala Ser Leu Ser Thr Ala His
Thr Val Phe Thr Thr Leu Phe Ile Asn 20 25
30Gly Val Asp Gln Gly Asp Gly Thr Cys Ile Arg Met Ala Lys
Lys Gly 35 40 45Ser Val Cys Thr
His Pro Ile Ala Gly Gly Leu Asp Ser Pro Asp Met 50 55
60Ala Cys Gly Arg Asp Gly Gln Gln Ala Val Ala Phe Thr
Cys Pro Ala65 70 75
80Pro Ala Gly Ser Lys Leu Ser Phe Glu Phe Arg Met Trp Ala Asp Ala
85 90 95Ser Gln Pro Gly Ser Ile
Asp Pro Ser His Leu Gly Ser Thr Ala Ile 100
105 110Tyr Leu Lys Gln Val Ser Asn Ile Ser Ser Asp Ser
Ala Ala Gly Pro 115 120 125Gly Trp
Phe Lys Ile Tyr Ala Glu Gly Tyr Asp Thr Ala Ala Lys Lys 130
135 140Trp Ala Thr Glu Lys Leu Ile Asp Asn Gly Gly
Leu Leu Ser Ile Glu145 150 155
160Leu Pro Pro Thr Leu Pro Ala Gly Tyr Tyr Leu Ala Arg Ser Glu Ile
165 170 175Val Thr Ile Gln
Asn Val Thr Asn Asp His Val Asp Pro Gln Phe Tyr 180
185 190Val Gly Cys Ala Gln Leu Phe Val Gln Gly Pro
Pro Thr Thr Pro Thr 195 200 205Val
Pro Pro Asp Arg Leu Val Ser Ile Pro Gly His Val His Ala Ser 210
215 220Asp Pro Gly Leu Thr Phe Asn Ile Trp Arg
Asp Asp Pro Ser Lys Thr225 230 235
240Ala Tyr Thr Val Val Gly Pro Ala Pro Phe Ser Pro Thr Ala Ala
Pro 245 250 255Thr Pro Thr
Ser Thr Asn Thr Asn Gly Gln Gln Gln Gln Gln Gln Gln 260
265 270Gln Ala Ile Lys Gln Thr Asp Gly Val Ile
Pro Ala Asp Cys Gln Leu 275 280
285Lys Asn Ala Asn Trp Cys Gly Ala Glu Val Pro Ala Tyr Ala Asp Glu 290
295 300Ala Gly Cys Trp Ala Ser Ser Ala
Asp Cys Phe Ala Gln Leu Asp Ala305 310
315 320Cys Tyr Thr Ser Ala Pro Pro Thr Gly Ser Arg Gly
Cys Arg Leu Trp 325 330
335Glu Asp Trp Cys Thr Gly Ile Gln Gln Gly Cys Arg Ala Gly Arg Trp
340 345 350Arg Gly Pro Pro Pro Phe
His Gly Glu Gly Ala Ala Ala Glu Thr Ala 355 360
365Ser Ala Gly Arg Gly Gly Ala Arg Ile Ala Ala Val Ala Gly
Cys Gly 370 375 380Gly Gly Thr Gly Asp
Met Val Glu Glu Val Phe Leu Phe Tyr Trp Asp385 390
395 400Ala Cys Ser Gly Trp Arg Arg Ser Arg Gly
Gly Gly Ser Ile Leu Ala 405 410
415Arg Leu Ile Leu His Val Leu Leu Pro Leu Leu Arg Pro Arg Arg Ala
420 425 430Pro Arg Val His Leu
Leu Leu Phe His Leu Tyr Leu Asn Phe Cys Tyr 435
440 445Pro Gly Thr Ser Gly Phe Tyr Asn Arg Leu Ser Ile
Lys Leu Gly Ile 450 455 460Trp Pro Ser
Lys Met Ser Pro Asp Val Ala His Tyr Val Lys465 470
47555868DNAThielavia terrestris 55atgcagctcc tcgtgggctt
gctgcttgca gccgtggctg ctcgagcaca ttgtatttct 60acccctttcc gcgtgcctcc
cagcctcaag gcaagaagac gcacgcagca gctaacggac 120cctatcagac acatttccca
gactcgtggt aaatgggcag cccgaggaca aggactggtc 180ggttacgcgc atgaccaaga
acgcgcagag caagcaggga gtccaggacc cgaccagtcc 240cgacattcgc tgctacacgt
cgcagacggc gcctaacgtg gctacggtcc ctgccggagc 300caccgtccat tacatatcga
ctcagcagat caaccacccg ggcccgacgc agtactacct 360cgccaaggta ccggcggggt
cgtcggccaa gacgtgggac gggtcagggg ccgtctggtt 420caagatctcg accaccatgc
cttacttgga caacaacaag cagcttgtct ggccgaatca 480gagtaggaac aattcccgct
ccaatcttcg atttggcctt gagctacggc cgattgcatg 540ggagagaccg ttgactgacg
gggcaaccca accttcatca gacacgtaca cgacggtcaa 600cacgaccatc cccgccgata
cgcccagtgg ggaatacctc ctccgggtcg agcagatcgc 660gctgcacctg gcctcgcagc
ccaacggggc tcagttctac ctggcctgct cgcagatcca 720gattacgggc ggcggcaacg
gcacgcccgg cccgctagtc gcgttgccgg gggcgtacaa 780gagcaacgac ccgggcattt
tggtcaacat ctactctatg cagcccggcg attacaagcc 840gcccgggccg ccggtgtgga
gtggctga 86856230PRTThielavia
terrestris 56Met Gln Leu Leu Val Gly Leu Leu Leu Ala Ala Val Ala Ala Arg
Ala1 5 10 15His Tyr Thr
Phe Pro Arg Leu Val Val Asn Gly Gln Pro Glu Asp Lys 20
25 30Asp Trp Ser Val Thr Arg Met Thr Lys Asn
Ala Gln Ser Lys Gln Gly 35 40
45Val Gln Asp Pro Thr Ser Pro Asp Ile Arg Cys Tyr Thr Ser Gln Thr 50
55 60Ala Pro Asn Val Ala Thr Val Pro Ala
Gly Ala Thr Val His Tyr Ile65 70 75
80Ser Thr Gln Gln Ile Asn His Pro Gly Pro Thr Gln Tyr Tyr
Leu Ala 85 90 95Lys Val
Pro Ala Gly Ser Ser Ala Lys Thr Trp Asp Gly Ser Gly Ala 100
105 110Val Trp Phe Lys Ile Ser Thr Thr Met
Pro Tyr Leu Asp Asn Asn Lys 115 120
125Gln Leu Val Trp Pro Asn Gln Asn Thr Tyr Thr Thr Val Asn Thr Thr
130 135 140Ile Pro Ala Asp Thr Pro Ser
Gly Glu Tyr Leu Leu Arg Val Glu Gln145 150
155 160Ile Ala Leu His Leu Ala Ser Gln Pro Asn Gly Ala
Gln Phe Tyr Leu 165 170
175Ala Cys Ser Gln Ile Gln Ile Thr Gly Gly Gly Asn Gly Thr Pro Gly
180 185 190Pro Leu Val Ala Leu Pro
Gly Ala Tyr Lys Ser Asn Asp Pro Gly Ile 195 200
205Leu Val Asn Ile Tyr Ser Met Gln Pro Gly Asp Tyr Lys Pro
Pro Gly 210 215 220Pro Pro Val Trp Ser
Gly225 230571068DNAThielavia terrestris 57atgaagctgt
acctggcggc ctttctaggc gccgtcgcca ccccgggagc gttcgctcat 60cgtaggttcc
ccgtctatct ccctaggggt agcaccacga ctaatttctc gtcgtccccc 120tgtagaaatc
cacgggattc tacttgtcaa cggcaccgaa acgccggaat ggaaatacgt 180ccggtaatat
ctaccttgct ctccttcttc cacaaccagc ctaacacatc atcagtgacg 240tggcctggga
gggcgcctac gaaccggaaa aataccccaa caccgagttc tttaagacgc 300ccccgcagac
ggacatcaac aacccgaaca tcacctgcgg caggaacgcg ttcgactcgg 360ccagcaagac
tgagacggcc gacatactgg ccggctcaga ggtcggcttc cgcgtctcgt 420gggacggcaa
cggcaagtac ggcgtgttct ggcatcccgg gccggggcag atctacctct 480ctcgtgctcc
gaacgacgac ctggaggact accgcggcga cggagactgg ttcaagatcg 540caaccggcgc
cgccgtctcc aataccgagt ggctgctgtg gaacaagcat gacgtgagcc 600ccaacattcc
tcgcccaatc gatccccaac ctggtcacca tggcggcgtc cgggatgcaa 660agagactaac
tccagaggaa cctacctagt tcaacttcac catccccaag acgacgccgc 720cgggcaagta
cctgatgcgc atcgagcagt tcatgccctc cacggtcgaa tacagccagt 780ggtacgtcaa
ctgcgcccac gtcaacatca tcggccccgg cggaggcacg ccgacgggct 840ttgccaggtt
tcccggcacc tacactgttg acgatcccgg taagccggac ctaccggaca 900cagaggcctc
gggatagctt gctaaccttg tttgctctct ctctttttct ctcccgacta 960ggcatcaagg
tgccgttgaa ccagatcgtc aacagcggag agttgccgca ggaccaactg 1020aggctgctcg
agtacaagcc cccgggccca gcgctgtgga ctggttga
106858257PRTThielavia terrestris 58Met Lys Leu Tyr Leu Ala Ala Phe Leu
Gly Ala Val Ala Thr Pro Gly1 5 10
15Ala Phe Ala His Gln Ile His Gly Ile Leu Leu Val Asn Gly Thr
Glu 20 25 30Thr Pro Glu Trp
Lys Tyr Val Arg Asp Val Ala Trp Glu Gly Ala Tyr 35
40 45Glu Pro Glu Lys Tyr Pro Asn Thr Glu Phe Phe Lys
Thr Pro Pro Gln 50 55 60Thr Asp Ile
Asn Asn Pro Asn Ile Thr Cys Gly Arg Asn Ala Phe Asp65 70
75 80Ser Ala Ser Lys Thr Glu Thr Ala
Asp Ile Leu Ala Gly Ser Glu Val 85 90
95Gly Phe Arg Val Ser Trp Asp Gly Asn Gly Lys Tyr Gly Val
Phe Trp 100 105 110His Pro Gly
Pro Gly Gln Ile Tyr Leu Ser Arg Ala Pro Asn Asp Asp 115
120 125Leu Glu Asp Tyr Arg Gly Asp Gly Asp Trp Phe
Lys Ile Ala Thr Gly 130 135 140Ala Ala
Val Ser Asn Thr Glu Trp Leu Leu Trp Asn Lys His Asp Phe145
150 155 160Asn Phe Thr Ile Pro Lys Thr
Thr Pro Pro Gly Lys Tyr Leu Met Arg 165
170 175Ile Glu Gln Phe Met Pro Ser Thr Val Glu Tyr Ser
Gln Trp Tyr Val 180 185 190Asn
Cys Ala His Val Asn Ile Ile Gly Pro Gly Gly Gly Thr Pro Thr 195
200 205Gly Phe Ala Arg Phe Pro Gly Thr Tyr
Thr Val Asp Asp Pro Gly Ile 210 215
220Lys Val Pro Leu Asn Gln Ile Val Asn Ser Gly Glu Leu Pro Gln Asp225
230 235 240Gln Leu Arg Leu
Leu Glu Tyr Lys Pro Pro Gly Pro Ala Leu Trp Thr 245
250 255Gly59871DNAThermoascus crustaceus
59atggcctttt cccagataat ggctattacc ggcgtttttc ttgcctctgc ttccctggtg
60gctggccatg gctttgttca gaatatcgtg attgatggta aaaggtacct aactacctac
120cttactatct gatgtcattt acaagaaagg gcacagacac aagcggcaaa aaaaagaaag
180aaagaaagaa agaaagaaag ctgacaaaaa ttcaacaagt tatggcgggt acatcgtgaa
240ccaatatcca tacatgtcag atcctccgga ggtcgtcggc tggtctacca ccgcaaccga
300cctcggattc gtggacggta ccggatacca aggacctgat atcatctgcc acaggggcgc
360caagcctgca gccctgactg cccaagtggc cgccggagga accgtcaagc tggaatggac
420tccatggcct gattctcacc acggcccggt gatcaactac cttgctcctt gcaacggtga
480ctgttccacc gtggacaaga cccaattgaa attcttcaag atcgcccagg ccggtctcat
540cgatgacaac agtcctcctg gtatctgggc ctcagacaat ctgatagcgg ccaacaacag
600ctggactgtc accatcccaa ccacaactgc acctggaaac tatgttctaa ggcatgagat
660cattgctctc cactcagctg ggaacaagga tggtgcgcag aactatcccc agtgcatcaa
720cctgaaggtc actggaaatg gttctggcaa tcctcctgct ggtgctcttg gaacggcact
780ctacaaggat acagatccgg gaattctgat caatatctac cagaaacttt ccagctatgt
840tattcctggt cctgctttgt acactggtta g
87160251PRTThermoascus crustaceus 60Met Ala Phe Ser Gln Ile Met Ala Ile
Thr Gly Val Phe Leu Ala Ser1 5 10
15Ala Ser Leu Val Ala Gly His Gly Phe Val Gln Asn Ile Val Ile
Asp 20 25 30Gly Lys Ser Tyr
Gly Gly Tyr Ile Val Asn Gln Tyr Pro Tyr Met Ser 35
40 45Asp Pro Pro Glu Val Val Gly Trp Ser Thr Thr Ala
Thr Asp Leu Gly 50 55 60Phe Val Asp
Gly Thr Gly Tyr Gln Gly Pro Asp Ile Ile Cys His Arg65 70
75 80Gly Ala Lys Pro Ala Ala Leu Thr
Ala Gln Val Ala Ala Gly Gly Thr 85 90
95Val Lys Leu Glu Trp Thr Pro Trp Pro Asp Ser His His Gly
Pro Val 100 105 110Ile Asn Tyr
Leu Ala Pro Cys Asn Gly Asp Cys Ser Thr Val Asp Lys 115
120 125Thr Gln Leu Lys Phe Phe Lys Ile Ala Gln Ala
Gly Leu Ile Asp Asp 130 135 140Asn Ser
Pro Pro Gly Ile Trp Ala Ser Asp Asn Leu Ile Ala Ala Asn145
150 155 160Asn Ser Trp Thr Val Thr Ile
Pro Thr Thr Thr Ala Pro Gly Asn Tyr 165
170 175Val Leu Arg His Glu Ile Ile Ala Leu His Ser Ala
Gly Asn Lys Asp 180 185 190Gly
Ala Gln Asn Tyr Pro Gln Cys Ile Asn Leu Lys Val Thr Gly Asn 195
200 205Gly Ser Gly Asn Pro Pro Ala Gly Ala
Leu Gly Thr Ala Leu Tyr Lys 210 215
220Asp Thr Asp Pro Gly Ile Leu Ile Asn Ile Tyr Gln Lys Leu Ser Ser225
230 235 240Tyr Val Ile Pro
Gly Pro Ala Leu Tyr Thr Gly 245
250611102DNAThermoascus crustaceus 61atgtcattct cgaagatact tgctatcgct
ggggccatta cctacgcatc ttcagctgcc 60gctcatggtt atgtccaggg aattgttgtc
gatggcagct agtatgtcac tctggatgga 120accttcagca cgtactgtac taacaatcag
cagctacggg ggatatatgg tgacccaata 180tccctacacc gctcaacctc cggaactcat
cgcctggtcc actaaagcaa ccgatcttgg 240gtttgtggac ggcagtggct atacttctcc
tgatatcatc tgccataagg gtgctgagcc 300tggtgcccag agcgccaaag tggcagctgg
agggaccgtt gagctgcagt ggacggcatg 360gcccgagtct cacaagggcc cagttattga
ctacctcgcc gcctgcgacg gggactgctc 420atctgttgat aagactgcac taaagttctt
taagattgac gagagtggtc tgattgacgg 480caacggtgct ggaacatggg cctctgatac
gttgatcaaa aataacaaca gctggactgt 540caccatccca agcacaattg cttccggaaa
ctacgtacta agacacgaaa taattgcgct 600ccattctgcc ggaaacaaag atggtgctca
gaactatccc cagtgtatca acctcgaggt 660cactggtagt ggcaccgaaa accctgctgg
cactctcgga acagcgcttt acacagacac 720tgatcctggc cttctggtca acatctacca
gggtctgtcc aactattcaa tccctggtcc 780tgctctgtat agcggcaaca gtgataacgc
tggttccctc aaccctacca ccacgccgtc 840aattcagaat gctgctgctg ctccctccac
ttccacagca tctgttgtca ctgattcttc 900gtcagccacc cagactgcta gtgtcgccgc
cacgactcca gcctccactt cggctgttac 960agcctcacca gctcccgata ctggaagcga
cgtaaccaaa tatctggatt cgatgagctc 1020ggatgaggtc ctcaccctgg tgcgcgggac
cctgtcttgg ctggtttcta acaagaaaca 1080tgcgcgggat ctttctcact ga
110262349PRTThermoascus crustaceus 62Met
Ser Phe Ser Lys Ile Leu Ala Ile Ala Gly Ala Ile Thr Tyr Ala1
5 10 15Ser Ser Ala Ala Ala His Gly
Tyr Val Gln Gly Ile Val Val Asp Gly 20 25
30Ser Tyr Tyr Gly Gly Tyr Met Val Thr Gln Tyr Pro Tyr Thr
Ala Gln 35 40 45Pro Pro Glu Leu
Ile Ala Trp Ser Thr Lys Ala Thr Asp Leu Gly Phe 50 55
60Val Asp Gly Ser Gly Tyr Thr Ser Pro Asp Ile Ile Cys
His Lys Gly65 70 75
80Ala Glu Pro Gly Ala Gln Ser Ala Lys Val Ala Ala Gly Gly Thr Val
85 90 95Glu Leu Gln Trp Thr Ala
Trp Pro Glu Ser His Lys Gly Pro Val Ile 100
105 110Asp Tyr Leu Ala Ala Cys Asp Gly Asp Cys Ser Ser
Val Asp Lys Thr 115 120 125Ala Leu
Lys Phe Phe Lys Ile Asp Glu Ser Gly Leu Ile Asp Gly Asn 130
135 140Gly Ala Gly Thr Trp Ala Ser Asp Thr Leu Ile
Lys Asn Asn Asn Ser145 150 155
160Trp Thr Val Thr Ile Pro Ser Thr Ile Ala Ser Gly Asn Tyr Val Leu
165 170 175Arg His Glu Ile
Ile Ala Leu His Ser Ala Gly Asn Lys Asp Gly Ala 180
185 190Gln Asn Tyr Pro Gln Cys Ile Asn Leu Glu Val
Thr Gly Ser Gly Thr 195 200 205Glu
Asn Pro Ala Gly Thr Leu Gly Thr Ala Leu Tyr Thr Asp Thr Asp 210
215 220Pro Gly Leu Leu Val Asn Ile Tyr Gln Gly
Leu Ser Asn Tyr Ser Ile225 230 235
240Pro Gly Pro Ala Leu Tyr Ser Gly Asn Ser Asp Asn Ala Gly Ser
Leu 245 250 255Asn Pro Thr
Thr Thr Pro Ser Ile Gln Asn Ala Ala Ala Ala Pro Ser 260
265 270Thr Ser Thr Ala Ser Val Val Thr Asp Ser
Ser Ser Ala Thr Gln Thr 275 280
285Ala Ser Val Ala Ala Thr Thr Pro Ala Ser Thr Ser Ala Val Thr Ala 290
295 300Ser Pro Ala Pro Asp Thr Gly Ser
Asp Val Thr Lys Tyr Leu Asp Ser305 310
315 320Met Ser Ser Asp Glu Val Leu Thr Leu Val Arg Gly
Thr Leu Ser Trp 325 330
335Leu Val Ser Asn Lys Lys His Ala Arg Asp Leu Ser His 340
345631493DNAThermoascus crustaceus 63atgttgtcat tcattcccac
caagtcagct gcgctgacga ctcttctact tcttggaaca 60gctcatgctc acactttgat
gaccaccatg tttgtggacg gcgtcaacca gggagatggt 120gtctgcattc gcatgaacaa
tgacggcgga actgccaata cctatatcca gcctatcacg 180agcaaggata tcgcctgcgg
taagtaccca gatgtcatca tactctgcca taacatccgt 240catatctact agaatcggag
caatgttaag tatttccagg catccaaggc gaaatcggcg 300cctcccgagt ctgcccagtc
aaggcatctt ccaccctaac cttccaattc cgcgagcaac 360ccaacaaccc aaactcctcc
cctctcgatc catcgcacaa aggccccgcc gcggtgtacc 420tgaaaaaggt cgactccgcc
atcgcgagca acaacgccgc cggagacagc tggttcaaga 480tctgggagtc cgtctacgac
gagtccacgg gcaaatgggg cacgaccaag atgatcgaga 540acaacgggca catctccgtc
aaggtgcccg atgatatcga gggtggttac tatcttgccc 600ggacggagct gctggcgcta
cattctgcgg atcaggggga tccgcagttc tatgttggct 660gtgcgcagct gtttatcgat
tcggatggga cggcgaaacc gcccactgtt tctattggag 720aggggacgta cgatctgagc
atgcctgcca tgacgtataa tatctgggag acaccgttgg 780ctctgccgta tccgatgtat
gggcctcctg tctatacgcc tggctctggt tctggatcag 840tccgtgcgac gagctcttct
gctgtcccta ctgcaaccga atcctctttt gtagaggaaa 900gagcaaaccc cgtcacggca
aacagtgttt attctgcaag gggcaaattc aaaacctgga 960ttgataaact gtcatggcgc
gggaaggtcc gtgagaacgt cagacaagcc gcgggaagaa 1020gaagcactct cgtccagact
gtgggtctaa agccaaaagg ctgcatcttc gtcaatggaa 1080actggtgcgg cttcgaggtt
cccgactaca acgatgcgga gagctgctgg gctgtatgtt 1140cccctcctta gcctcttaca
tccctaagta ctacatttga aaacaacaaa aagaaatgta 1200tatactaact acgtacgctc
tactctaggc ctccgacaac tgctggaaac agtccgacgc 1260ctgctggaac aagacccaac
ccacgggcta caataactgc cagatctggc aggacaagaa 1320atgcaaggtc atccaggatt
cctgtagcgg acccaacccg catggaccac cgaataaggg 1380caaggatttg actccggagt
ggccgccact gaagggctcg atggatacgt tctccaagcg 1440tactatcggt taccgcgatt
ggattgttag aaggagaggt gcatgagggt gta 149364436PRTThermoascus
crustaceus 64Met Leu Ser Phe Ile Pro Thr Lys Ser Ala Ala Leu Thr Thr Leu
Leu1 5 10 15Leu Leu Gly
Thr Ala His Ala His Thr Leu Met Thr Thr Met Phe Val 20
25 30Asp Gly Val Asn Gln Gly Asp Gly Val Cys
Ile Arg Met Asn Asn Asp 35 40
45Gly Gly Thr Ala Asn Thr Tyr Ile Gln Pro Ile Thr Ser Lys Asp Ile 50
55 60Ala Cys Gly Ile Gln Gly Glu Ile Gly
Ala Ser Arg Val Cys Pro Val65 70 75
80Lys Ala Ser Ser Thr Leu Thr Phe Gln Phe Arg Glu Gln Pro
Asn Asn 85 90 95Pro Asn
Ser Ser Pro Leu Asp Pro Ser His Lys Gly Pro Ala Ala Val 100
105 110Tyr Leu Lys Lys Val Asp Ser Ala Ile
Ala Ser Asn Asn Ala Ala Gly 115 120
125Asp Ser Trp Phe Lys Ile Trp Glu Ser Val Tyr Asp Glu Ser Thr Gly
130 135 140Lys Trp Gly Thr Thr Lys Met
Ile Glu Asn Asn Gly His Ile Ser Val145 150
155 160Lys Val Pro Asp Asp Ile Glu Gly Gly Tyr Tyr Leu
Ala Arg Thr Glu 165 170
175Leu Leu Ala Leu His Ser Ala Asp Gln Gly Asp Pro Gln Phe Tyr Val
180 185 190Gly Cys Ala Gln Leu Phe
Ile Asp Ser Asp Gly Thr Ala Lys Pro Pro 195 200
205Thr Val Ser Ile Gly Glu Gly Thr Tyr Asp Leu Ser Met Pro
Ala Met 210 215 220Thr Tyr Asn Ile Trp
Glu Thr Pro Leu Ala Leu Pro Tyr Pro Met Tyr225 230
235 240Gly Pro Pro Val Tyr Thr Pro Gly Ser Gly
Ser Gly Ser Val Arg Ala 245 250
255Thr Ser Ser Ser Ala Val Pro Thr Ala Thr Glu Ser Ser Phe Val Glu
260 265 270Glu Arg Ala Asn Pro
Val Thr Ala Asn Ser Val Tyr Ser Ala Arg Gly 275
280 285Lys Phe Lys Thr Trp Ile Asp Lys Leu Ser Trp Arg
Gly Lys Val Arg 290 295 300Glu Asn Val
Arg Gln Ala Ala Gly Arg Arg Ser Thr Leu Val Gln Thr305
310 315 320Val Gly Leu Lys Pro Lys Gly
Cys Ile Phe Val Asn Gly Asn Trp Cys 325
330 335Gly Phe Glu Val Pro Asp Tyr Asn Asp Ala Glu Ser
Cys Trp Ala Ala 340 345 350Ser
Asp Asn Cys Trp Lys Gln Ser Asp Ala Cys Trp Asn Lys Thr Gln 355
360 365Pro Thr Gly Tyr Asn Asn Cys Gln Ile
Trp Gln Asp Lys Lys Cys Lys 370 375
380Val Ile Gln Asp Ser Cys Ser Gly Pro Asn Pro His Gly Pro Pro Asn385
390 395 400Lys Gly Lys Asp
Leu Thr Pro Glu Trp Pro Pro Leu Lys Gly Ser Met 405
410 415Asp Thr Phe Ser Lys Arg Thr Ile Gly Tyr
Arg Asp Trp Ile Val Arg 420 425
430Arg Arg Gly Ala 435651377DNATrichoderma reesei 65atggcgccct
cagttacact gccgttgacc acggccatcc tggccattgc ccggctcgtc 60gccgcccagc
aaccgggtac cagcaccccc gaggtccatc ccaagttgac aacctacaag 120tgtacaaagt
ccggggggtg cgtggcccag gacacctcgg tggtccttga ctggaactac 180cgctggatgc
acgacgcaaa ctacaactcg tgcaccgtca acggcggcgt caacaccacg 240ctctgccctg
acgaggcgac ctgtggcaag aactgcttca tcgagggcgt cgactacgcc 300gcctcgggcg
tcacgacctc gggcagcagc ctcaccatga accagtacat gcccagcagc 360tctggcggct
acagcagcgt ctctcctcgg ctgtatctcc tggactctga cggtgagtac 420gtgatgctga
agctcaacgg ccaggagctg agcttcgacg tcgacctctc tgctctgccg 480tgtggagaga
acggctcgct ctacctgtct cagatggacg agaacggggg cgccaaccag 540tataacacgg
ccggtgccaa ctacgggagc ggctactgcg atgctcagtg ccccgtccag 600acatggagga
acggcaccct caacactagc caccagggct tctgctgcaa cgagatggat 660atcctggagg
gcaactcgag ggcgaatgcc ttgacccctc actcttgcac ggccacggcc 720tgcgactctg
ccggttgcgg cttcaacccc tatggcagcg gctacaaaag ctactacggc 780cccggagata
ccgttgacac ctccaagacc ttcaccatca tcacccagtt caacacggac 840aacggctcgc
cctcgggcaa ccttgtgagc atcacccgca agtaccagca aaacggcgtc 900gacatcccca
gcgcccagcc cggcggcgac accatctcgt cctgcccgtc cgcctcagcc 960tacggcggcc
tcgccaccat gggcaaggcc ctgagcagcg gcatggtgct cgtgttcagc 1020atttggaacg
acaacagcca gtacatgaac tggctcgaca gcggcaacgc cggcccctgc 1080agcagcaccg
agggcaaccc atccaacatc ctggccaaca accccaacac gcacgtcgtc 1140ttctccaaca
tccgctgggg agacattggg tctactacga actcgactgc gcccccgccc 1200ccgcctgcgt
ccagcacgac gttttcgact acacggagga gctcgacgac ttcgagcagc 1260ccgagctgca
cgcagactca ctgggggcag tgcggtggca ttgggtacag cgggtgcaag 1320acgtgcacgt
cgggcactac gtgccagtat agcaacgact actactcgca atgcctt
137766459PRTTrichoderma reesei 66Met Ala Pro Ser Val Thr Leu Pro Leu Thr
Thr Ala Ile Leu Ala Ile1 5 10
15Ala Arg Leu Val Ala Ala Gln Gln Pro Gly Thr Ser Thr Pro Glu Val
20 25 30His Pro Lys Leu Thr Thr
Tyr Lys Cys Thr Lys Ser Gly Gly Cys Val 35 40
45Ala Gln Asp Thr Ser Val Val Leu Asp Trp Asn Tyr Arg Trp
Met His 50 55 60Asp Ala Asn Tyr Asn
Ser Cys Thr Val Asn Gly Gly Val Asn Thr Thr65 70
75 80Leu Cys Pro Asp Glu Ala Thr Cys Gly Lys
Asn Cys Phe Ile Glu Gly 85 90
95Val Asp Tyr Ala Ala Ser Gly Val Thr Thr Ser Gly Ser Ser Leu Thr
100 105 110Met Asn Gln Tyr Met
Pro Ser Ser Ser Gly Gly Tyr Ser Ser Val Ser 115
120 125Pro Arg Leu Tyr Leu Leu Asp Ser Asp Gly Glu Tyr
Val Met Leu Lys 130 135 140Leu Asn Gly
Gln Glu Leu Ser Phe Asp Val Asp Leu Ser Ala Leu Pro145
150 155 160Cys Gly Glu Asn Gly Ser Leu
Tyr Leu Ser Gln Met Asp Glu Asn Gly 165
170 175Gly Ala Asn Gln Tyr Asn Thr Ala Gly Ala Asn Tyr
Gly Ser Gly Tyr 180 185 190Cys
Asp Ala Gln Cys Pro Val Gln Thr Trp Arg Asn Gly Thr Leu Asn 195
200 205Thr Ser His Gln Gly Phe Cys Cys Asn
Glu Met Asp Ile Leu Glu Gly 210 215
220Asn Ser Arg Ala Asn Ala Leu Thr Pro His Ser Cys Thr Ala Thr Ala225
230 235 240Cys Asp Ser Ala
Gly Cys Gly Phe Asn Pro Tyr Gly Ser Gly Tyr Lys 245
250 255Ser Tyr Tyr Gly Pro Gly Asp Thr Val Asp
Thr Ser Lys Thr Phe Thr 260 265
270Ile Ile Thr Gln Phe Asn Thr Asp Asn Gly Ser Pro Ser Gly Asn Leu
275 280 285Val Ser Ile Thr Arg Lys Tyr
Gln Gln Asn Gly Val Asp Ile Pro Ser 290 295
300Ala Gln Pro Gly Gly Asp Thr Ile Ser Ser Cys Pro Ser Ala Ser
Ala305 310 315 320Tyr Gly
Gly Leu Ala Thr Met Gly Lys Ala Leu Ser Ser Gly Met Val
325 330 335Leu Val Phe Ser Ile Trp Asn
Asp Asn Ser Gln Tyr Met Asn Trp Leu 340 345
350Asp Ser Gly Asn Ala Gly Pro Cys Ser Ser Thr Glu Gly Asn
Pro Ser 355 360 365Asn Ile Leu Ala
Asn Asn Pro Asn Thr His Val Val Phe Ser Asn Ile 370
375 380Arg Trp Gly Asp Ile Gly Ser Thr Thr Asn Ser Thr
Ala Pro Pro Pro385 390 395
400Pro Pro Ala Ser Ser Thr Thr Phe Ser Thr Thr Arg Arg Ser Ser Thr
405 410 415Thr Ser Ser Ser Pro
Ser Cys Thr Gln Thr His Trp Gly Gln Cys Gly 420
425 430Gly Ile Gly Tyr Ser Gly Cys Lys Thr Cys Thr Ser
Gly Thr Thr Cys 435 440 445Gln Tyr
Ser Asn Asp Tyr Tyr Ser Gln Cys Leu 450
455671254DNATrichoderma reesei 67atgaacaagt ccgtggctcc attgctgctt
gcagcgtcca tactatatgg cggcgccgtc 60gcacagcaga ctgtctgggg ccagtgtgga
ggtattggtt ggagcggacc tacgaattgt 120gctcctggct cagcttgttc gaccctcaat
ccttattatg cgcaatgtat tccgggagcc 180actactatca ccacttcgac ccggccacca
tccggtccaa ccaccaccac cagggctacc 240tcaacaagct catcaactcc acccacgagc
tctggggtcc gatttgccgg cgttaacatc 300gcgggttttg actttggctg taccacagat
ggcacttgcg ttacctcgaa ggtttatcct 360ccgttgaaga acttcaccgg ctcaaacaac
taccccgatg gcatcggcca gatgcagcac 420ttcgtcaacg aggacgggat gactattttc
cgcttacctg tcggatggca gtacctcgtc 480aacaacaatt tgggcggcaa tcttgattcc
acgagcattt ccaagtatga tcagcttgtt 540caggggtgcc tgtctctggg cgcatactgc
atcgtcgaca tccacaatta tgctcgatgg 600aacggtggga tcattggtca gggcggccct
actaatgctc aattcacgag cctttggtcg 660cagttggcat caaagtacgc atctcagtcg
agggtgtggt tcggcatcat gaatgagccc 720cacgacgtga acatcaacac ctgggctgcc
acggtccaag aggttgtaac cgcaatccgc 780aacgctggtg ctacgtcgca attcatctct
ttgcctggaa atgattggca atctgctggg 840gctttcatat ccgatggcag tgcagccgcc
ctgtctcaag tcacgaaccc ggatgggtca 900acaacgaatc tgatttttga cgtgcacaaa
tacttggact cagacaactc cggtactcac 960gccgaatgta ctacaaataa cattgacggc
gccttttctc cgcttgccac ttggctccga 1020cagaacaatc gccaggctat cctgacagaa
accggtggtg gcaacgttca gtcctgcata 1080caagacatgt gccagcaaat ccaatatctc
aaccagaact cagatgtcta tcttggctat 1140gttggttggg gtgccggatc atttgatagc
acgtatgtcc tgacggaaac accgactagc 1200agtggtaact catggacgga cacatccttg
gtcagctcgt gtctcgcaag aaag 125468418PRTTrichoderma reesei 68Met
Asn Lys Ser Val Ala Pro Leu Leu Leu Ala Ala Ser Ile Leu Tyr1
5 10 15Gly Gly Ala Val Ala Gln Gln
Thr Val Trp Gly Gln Cys Gly Gly Ile 20 25
30Gly Trp Ser Gly Pro Thr Asn Cys Ala Pro Gly Ser Ala Cys
Ser Thr 35 40 45Leu Asn Pro Tyr
Tyr Ala Gln Cys Ile Pro Gly Ala Thr Thr Ile Thr 50 55
60Thr Ser Thr Arg Pro Pro Ser Gly Pro Thr Thr Thr Thr
Arg Ala Thr65 70 75
80Ser Thr Ser Ser Ser Thr Pro Pro Thr Ser Ser Gly Val Arg Phe Ala
85 90 95Gly Val Asn Ile Ala Gly
Phe Asp Phe Gly Cys Thr Thr Asp Gly Thr 100
105 110Cys Val Thr Ser Lys Val Tyr Pro Pro Leu Lys Asn
Phe Thr Gly Ser 115 120 125Asn Asn
Tyr Pro Asp Gly Ile Gly Gln Met Gln His Phe Val Asn Glu 130
135 140Asp Gly Met Thr Ile Phe Arg Leu Pro Val Gly
Trp Gln Tyr Leu Val145 150 155
160Asn Asn Asn Leu Gly Gly Asn Leu Asp Ser Thr Ser Ile Ser Lys Tyr
165 170 175Asp Gln Leu Val
Gln Gly Cys Leu Ser Leu Gly Ala Tyr Cys Ile Val 180
185 190Asp Ile His Asn Tyr Ala Arg Trp Asn Gly Gly
Ile Ile Gly Gln Gly 195 200 205Gly
Pro Thr Asn Ala Gln Phe Thr Ser Leu Trp Ser Gln Leu Ala Ser 210
215 220Lys Tyr Ala Ser Gln Ser Arg Val Trp Phe
Gly Ile Met Asn Glu Pro225 230 235
240His Asp Val Asn Ile Asn Thr Trp Ala Ala Thr Val Gln Glu Val
Val 245 250 255Thr Ala Ile
Arg Asn Ala Gly Ala Thr Ser Gln Phe Ile Ser Leu Pro 260
265 270Gly Asn Asp Trp Gln Ser Ala Gly Ala Phe
Ile Ser Asp Gly Ser Ala 275 280
285Ala Ala Leu Ser Gln Val Thr Asn Pro Asp Gly Ser Thr Thr Asn Leu 290
295 300Ile Phe Asp Val His Lys Tyr Leu
Asp Ser Asp Asn Ser Gly Thr His305 310
315 320Ala Glu Cys Thr Thr Asn Asn Ile Asp Gly Ala Phe
Ser Pro Leu Ala 325 330
335Thr Trp Leu Arg Gln Asn Asn Arg Gln Ala Ile Leu Thr Glu Thr Gly
340 345 350Gly Gly Asn Val Gln Ser
Cys Ile Gln Asp Met Cys Gln Gln Ile Gln 355 360
365Tyr Leu Asn Gln Asn Ser Asp Val Tyr Leu Gly Tyr Val Gly
Trp Gly 370 375 380Ala Gly Ser Phe Asp
Ser Thr Tyr Val Leu Thr Glu Thr Pro Thr Ser385 390
395 400Ser Gly Asn Ser Trp Thr Asp Thr Ser Leu
Val Ser Ser Cys Leu Ala 405 410
415Arg Lys69702DNATrichoderma reesei 69atgaagttcc ttcaagtcct
ccctgccctc ataccggccg ccctggccca aaccagctgt 60gaccagtggg caaccttcac
tggcaacggc tacacagtca gcaacaacct ttggggagca 120tcagccggct ctggatttgg
ctgcgtgacg gcggtatcgc tcagcggcgg ggcctcctgg 180cacgcagact ggcagtggtc
cggcggccag aacaacgtca agtcgtacca gaactctcag 240attgccattc cccagaagag
gaccgtcaac agcatcagca gcatgcccac cactgccagc 300tggagctaca gcgggagcaa
catccgcgct aatgttgcgt atgacttgtt caccgcagcc 360aacccgaatc atgtcacgta
ctcgggagac tacgaactca tgatctggct tggcaaatac 420ggcgatattg ggccgattgg
gtcctcacag ggaacagtca acgtcggtgg ccagagctgg 480acgctctact atggctacaa
cggagccatg caagtctatt cctttgtggc ccagaccaac 540actaccaact acagcggaga
tgtcaagaac ttcttcaatt atctccgaga caataaagga 600tacaacgctg caggccaata
tgttcttagc taccaatttg gtaccgagcc cttcacgggc 660agtggaactc tgaacgtcgc
atcctggacc gcatctatca ac 70270234PRTTrichoderma
reesei 70Met Lys Phe Leu Gln Val Leu Pro Ala Leu Ile Pro Ala Ala Leu Ala1
5 10 15Gln Thr Ser Cys
Asp Gln Trp Ala Thr Phe Thr Gly Asn Gly Tyr Thr 20
25 30Val Ser Asn Asn Leu Trp Gly Ala Ser Ala Gly
Ser Gly Phe Gly Cys 35 40 45Val
Thr Ala Val Ser Leu Ser Gly Gly Ala Ser Trp His Ala Asp Trp 50
55 60Gln Trp Ser Gly Gly Gln Asn Asn Val Lys
Ser Tyr Gln Asn Ser Gln65 70 75
80Ile Ala Ile Pro Gln Lys Arg Thr Val Asn Ser Ile Ser Ser Met
Pro 85 90 95Thr Thr Ala
Ser Trp Ser Tyr Ser Gly Ser Asn Ile Arg Ala Asn Val 100
105 110Ala Tyr Asp Leu Phe Thr Ala Ala Asn Pro
Asn His Val Thr Tyr Ser 115 120
125Gly Asp Tyr Glu Leu Met Ile Trp Leu Gly Lys Tyr Gly Asp Ile Gly 130
135 140Pro Ile Gly Ser Ser Gln Gly Thr
Val Asn Val Gly Gly Gln Ser Trp145 150
155 160Thr Leu Tyr Tyr Gly Tyr Asn Gly Ala Met Gln Val
Tyr Ser Phe Val 165 170
175Ala Gln Thr Asn Thr Thr Asn Tyr Ser Gly Asp Val Lys Asn Phe Phe
180 185 190Asn Tyr Leu Arg Asp Asn
Lys Gly Tyr Asn Ala Ala Gly Gln Tyr Val 195 200
205Leu Ser Tyr Gln Phe Gly Thr Glu Pro Phe Thr Gly Ser Gly
Thr Leu 210 215 220Asn Val Ala Ser Trp
Thr Ala Ser Ile Asn225 23071726DNATrichoderma reesei
71atgaaggcaa ctctggttct cggctccctc attgtaggcg ccgtttccgc gtacaaggcc
60accaccacgc gctactacga tgggcaggag ggtgcttgcg gatgcggctc gagctccggc
120gcattcccgt ggcagctcgg catcggcaac ggagtctaca cggctgccgg ctcccaggct
180ctcttcgaca cggccggagc ttcatggtgc ggcgccggct gcggtaaatg ctaccagctc
240acctcgacgg gccaggcgcc ctgctccagc tgcggcacgg gcggtgctgc tggccagagc
300atcatcgtca tggtgaccaa cctgtgcccg aacaatggga acgcgcagtg gtgcccggtg
360gtcggcggca ccaaccaata cggctacagc taccatttcg acatcatggc gcagaacgag
420atctttggag acaatgtcgt cgtcgacttt gagcccattg cttgccccgg gcaggctgcc
480tctgactggg ggacgtgcct ctgcgtggga cagcaagaga cggatcccac gcccgtcctc
540ggcaacgaca cgggctcaac tcctcccggg agctcgccgc cagcgacatc gtcgagtccg
600ccgtctggcg gcggccagca gacgctctat ggccagtgtg gaggtgccgg ctggacggga
660cctacgacgt gccaggcccc agggacctgc aaggttcaga accagtggta ctcccagtgt
720cttcct
72672242PRTTrichoderma reesei 72Met Lys Ala Thr Leu Val Leu Gly Ser Leu
Ile Val Gly Ala Val Ser1 5 10
15Ala Tyr Lys Ala Thr Thr Thr Arg Tyr Tyr Asp Gly Gln Glu Gly Ala
20 25 30Cys Gly Cys Gly Ser Ser
Ser Gly Ala Phe Pro Trp Gln Leu Gly Ile 35 40
45Gly Asn Gly Val Tyr Thr Ala Ala Gly Ser Gln Ala Leu Phe
Asp Thr 50 55 60Ala Gly Ala Ser Trp
Cys Gly Ala Gly Cys Gly Lys Cys Tyr Gln Leu65 70
75 80Thr Ser Thr Gly Gln Ala Pro Cys Ser Ser
Cys Gly Thr Gly Gly Ala 85 90
95Ala Gly Gln Ser Ile Ile Val Met Val Thr Asn Leu Cys Pro Asn Asn
100 105 110Gly Asn Ala Gln Trp
Cys Pro Val Val Gly Gly Thr Asn Gln Tyr Gly 115
120 125Tyr Ser Tyr His Phe Asp Ile Met Ala Gln Asn Glu
Ile Phe Gly Asp 130 135 140Asn Val Val
Val Asp Phe Glu Pro Ile Ala Cys Pro Gly Gln Ala Ala145
150 155 160Ser Asp Trp Gly Thr Cys Leu
Cys Val Gly Gln Gln Glu Thr Asp Pro 165
170 175Thr Pro Val Leu Gly Asn Asp Thr Gly Ser Thr Pro
Pro Gly Ser Ser 180 185 190Pro
Pro Ala Thr Ser Ser Ser Pro Pro Ser Gly Gly Gly Gln Gln Thr 195
200 205Leu Tyr Gly Gln Cys Gly Gly Ala Gly
Trp Thr Gly Pro Thr Thr Cys 210 215
220Gln Ala Pro Gly Thr Cys Lys Val Gln Asn Gln Trp Tyr Ser Gln Cys225
230 235 240Leu
Pro73923DNATrichoderma reesei 73atgcgttcct cccccctcct ccgctccgcc
gttgtggccg ccctgccggt gttggccctt 60gccgctgatg gcaggtccac ccgctactgg
gactgctgca agccttcgtg cggctgggcc 120aagaaggctc ccgtgaacca gcctgtcttt
tcctgcaacg ccaacttcca gcgtatcacg 180gacttcgacg ccaagtccgg ctgcgagccg
ggcggtgtcg cctactcgtg cgccgaccag 240accccatggg ctgtgaacga cgacttcgcg
ctcggttttg ctgccacctc tattgccggc 300agcaatgagg cgggctggtg ctgcgcctgc
tacgagctca ccttcacatc cggtcctgtt 360gctggcaaga agatggtcgt ccagtccacc
agcactggcg gtgatcttgg cagcaaccac 420ttcgatctca acatccccgg cggcggcgtc
ggcatcttcg acggatgcac tccccagttc 480ggcggtctgc ccggccagcg ctacggcggc
atctcgtccc gcaacgagtg cgatcggttc 540cccgacgccc tcaagcccgg ctgctactgg
cgcttcgact ggttcaagaa cgccgacaat 600ccgagcttca gcttccgtca ggtccagtgc
ccagccgagc tcgtcgctcg caccggatgc 660cgccgcaacg acgacggcaa cttccctgcc
gtccagatcc cctccagcag caccagctct 720ccggtcaacc agcctaccag caccagcacc
acgtccacct ccaccacctc gagcccgcca 780gtccagccta cgactcccag cggctgcact
gctgagaggt gggctcagtg cggcggcaat 840ggctggagcg gctgcaccac ctgcgtcgct
ggcagcactt gcacgaagat taatgactgg 900taccatcagt gcctgtagaa ttc
92374305PRTTrichoderma reesei 74Met Arg
Ser Ser Pro Leu Leu Arg Ser Ala Val Val Ala Ala Leu Pro1 5
10 15Val Leu Ala Leu Ala Ala Asp Gly
Arg Ser Thr Arg Tyr Trp Asp Cys 20 25
30Cys Lys Pro Ser Cys Gly Trp Ala Lys Lys Ala Pro Val Asn Gln
Pro 35 40 45Val Phe Ser Cys Asn
Ala Asn Phe Gln Arg Ile Thr Asp Phe Asp Ala 50 55
60Lys Ser Gly Cys Glu Pro Gly Gly Val Ala Tyr Ser Cys Ala
Asp Gln65 70 75 80Thr
Pro Trp Ala Val Asn Asp Asp Phe Ala Leu Gly Phe Ala Ala Thr
85 90 95Ser Ile Ala Gly Ser Asn Glu
Ala Gly Trp Cys Cys Ala Cys Tyr Glu 100 105
110Leu Thr Phe Thr Ser Gly Pro Val Ala Gly Lys Lys Met Val
Val Gln 115 120 125Ser Thr Ser Thr
Gly Gly Asp Leu Gly Ser Asn His Phe Asp Leu Asn 130
135 140Ile Pro Gly Gly Gly Val Gly Ile Phe Asp Gly Cys
Thr Pro Gln Phe145 150 155
160Gly Gly Leu Pro Gly Gln Arg Tyr Gly Gly Ile Ser Ser Arg Asn Glu
165 170 175Cys Asp Arg Phe Pro
Asp Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe 180
185 190Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser
Phe Arg Gln Val 195 200 205Gln Cys
Pro Ala Glu Leu Val Ala Arg Thr Gly Cys Arg Arg Asn Asp 210
215 220Asp Gly Asn Phe Pro Ala Val Gln Ile Pro Ser
Ser Ser Thr Ser Ser225 230 235
240Pro Val Asn Gln Pro Thr Ser Thr Ser Thr Thr Ser Thr Ser Thr Thr
245 250 255Ser Ser Pro Pro
Val Gln Pro Thr Thr Pro Ser Gly Cys Thr Ala Glu 260
265 270Arg Trp Ala Gln Cys Gly Gly Asn Gly Trp Ser
Gly Cys Thr Thr Cys 275 280 285Val
Ala Gly Ser Thr Cys Thr Lys Ile Asn Asp Trp Tyr His Gln Cys 290
295 300Leu305751188DNAMyceliophthora thermophila
75cgacttgaaa cgccccaaat gaagtcctcc atcctcgcca gcgtcttcgc cacgggcgcc
60gtggctcaaa gtggtccgtg gcagcaatgt ggtggcatcg gatggcaagg atcgaccgac
120tgtgtgtcgg gctaccactg cgtctaccag aacgattggt acagccagtg cgtgcctggc
180gcggcgtcga caacgctgca gacatcgacc acgtccaggc ccaccgccac cagcaccgcc
240cctccgtcgt ccaccacctc gcctagcaag ggcaagctga agtggctcgg cagcaacgag
300tcgggcgccg agttcgggga gggcaattac cccggcctct ggggcaagca cttcatcttc
360ccgtcgactt cggcgattca gacgctcatc aatgatggat acaacatctt ccggatcgac
420ttctcgatgg agcgtctggt gcccaaccag ttgacgtcgt ccttcgacca gggttacctc
480cgcaacctga ccgaggtggt caacttcgtg acgaacgcgg gcaagtacgc cgtcctggac
540ccgcacaact acggccggta ctacggcaac atcatcacgg acacgaacgc gttccggacc
600ttctggacca acctggccaa gcagttcgcc tccaactcgc tcgtcatctt cgacaccaac
660aacgagtaca acacgatgga ccagaccctg gtgctcaacc tcaaccaggc cgccatcgac
720ggcatccggg ccgccggcgc gacctcgcag tacatcttcg tcgagggcaa cgcgtggagc
780ggggcctgga gctggaacac gaccaacacc aacatggccg ccctgacgga cccgcagaac
840aagatcgtgt acgagatgca ccagtacctc gactcggaca gctcgggcac ccacgccgag
900tgcgtcagca gcaccatcgg cgcccagcgc gtcgtcggag ccacccagtg gctccgcgcc
960aacggcaagc tcggcgtcct cggcgagttc gccggcggcg ccaacgccgt ctgccagcag
1020gccgtcaccg gcctcctcga ccacctccag gacaacagcg acgtctggct gggtgccctc
1080tggtgggccg ccggtccctg gtggggcgac tacatgtact cgttcgagcc tccttcgggc
1140accggctatg tcaactacaa ctcgatcttg aagaagtact tgccgtaa
118876389PRTMyceliophthora thermophila 76Met Lys Ser Ser Ile Leu Ala Ser
Val Phe Ala Thr Gly Ala Val Ala1 5 10
15Gln Ser Gly Pro Trp Gln Gln Cys Gly Gly Ile Gly Trp Gln
Gly Ser 20 25 30Thr Asp Cys
Val Ser Gly Tyr His Cys Val Tyr Gln Asn Asp Trp Tyr 35
40 45Ser Gln Cys Val Pro Gly Ala Ala Ser Thr Thr
Leu Gln Thr Ser Thr 50 55 60Thr Ser
Arg Pro Thr Ala Thr Ser Thr Ala Pro Pro Ser Ser Thr Thr65
70 75 80Ser Pro Ser Lys Gly Lys Leu
Lys Trp Leu Gly Ser Asn Glu Ser Gly 85 90
95Ala Glu Phe Gly Glu Gly Asn Tyr Pro Gly Leu Trp Gly
Lys His Phe 100 105 110Ile Phe
Pro Ser Thr Ser Ala Ile Gln Thr Leu Ile Asn Asp Gly Tyr 115
120 125Asn Ile Phe Arg Ile Asp Phe Ser Met Glu
Arg Leu Val Pro Asn Gln 130 135 140Leu
Thr Ser Ser Phe Asp Gln Gly Tyr Leu Arg Asn Leu Thr Glu Val145
150 155 160Val Asn Phe Val Thr Asn
Ala Gly Lys Tyr Ala Val Leu Asp Pro His 165
170 175Asn Tyr Gly Arg Tyr Tyr Gly Asn Ile Ile Thr Asp
Thr Asn Ala Phe 180 185 190Arg
Thr Phe Trp Thr Asn Leu Ala Lys Gln Phe Ala Ser Asn Ser Leu 195
200 205Val Ile Phe Asp Thr Asn Asn Glu Tyr
Asn Thr Met Asp Gln Thr Leu 210 215
220Val Leu Asn Leu Asn Gln Ala Ala Ile Asp Gly Ile Arg Ala Ala Gly225
230 235 240Ala Thr Ser Gln
Tyr Ile Phe Val Glu Gly Asn Ala Trp Ser Gly Ala 245
250 255Trp Ser Trp Asn Thr Thr Asn Thr Asn Met
Ala Ala Leu Thr Asp Pro 260 265
270Gln Asn Lys Ile Val Tyr Glu Met His Gln Tyr Leu Asp Ser Asp Ser
275 280 285Ser Gly Thr His Ala Glu Cys
Val Ser Ser Thr Ile Gly Ala Gln Arg 290 295
300Val Val Gly Ala Thr Gln Trp Leu Arg Ala Asn Gly Lys Leu Gly
Val305 310 315 320Leu Gly
Glu Phe Ala Gly Gly Ala Asn Ala Val Cys Gln Gln Ala Val
325 330 335Thr Gly Leu Leu Asp His Leu
Gln Asp Asn Ser Asp Val Trp Leu Gly 340 345
350Ala Leu Trp Trp Ala Ala Gly Pro Trp Trp Gly Asp Tyr Met
Tyr Ser 355 360 365Phe Glu Pro Pro
Ser Gly Thr Gly Tyr Val Asn Tyr Asn Ser Ile Leu 370
375 380Lys Lys Tyr Leu Pro385771232DNABASIDIOMYCETE CBS
495.95 77ggatccactt agtaacggcc gccagtgtgc tggaaagcat gaagtctctc
ttcctgtcac 60ttgtagcgac cgtcgcgctc agctcgccag tattctctgt cgcagtctgg
gggcaatgcg 120gcggcattgg cttcagcgga agcaccgtct gtgatgcagg cgccggctgt
gtgaagctca 180acgactatta ctctcaatgc caacccggcg ctcccactgc tacatccgcg
gcgccaagta 240gcaacgcacc gtccggcact tcgacggcct cggccccctc ctccagcctt
tgctctggca 300gccgcacgcc gttccagttc ttcggtgtca acgaatccgg cgcggagttc
ggcaacctga 360acatccccgg tgttctgggc accgactaca cctggccgtc gccatccagc
attgacttct 420tcatgggcaa gggaatgaat accttccgta ttccgttcct catggagcgt
cttgtccccc 480ctgccactgg catcacagga cctctcgacc agacgtactt gggcggcctg
cagacgattg 540tcaactacat caccggcaaa ggcggctttg ctctcattga cccgcacaac
tttatgatct 600acaatggcca gacgatctcc agtaccagcg acttccagaa gttctggcag
aacctcgcag 660gagtgtttaa atcgaacagt cacgtcatct tcgatgttat gaacgagcct
cacgatattc 720ccgcccagac cgtgttccaa ctgaaccaag ccgctgtcaa tggcatccgt
gcgagcggtg 780cgacgtcgca gctcattctg gtcgagggca caagctggac tggagcctgg
acctggacga 840cctctggcaa cagcgatgca ttcggtgcca ttaaggatcc caacaacaac
gtcgcgatcc 900agatgcatca gtacctggat agcgatggct ctggcacttc gcagacctgc
gtgtctccca 960ccatcggtgc cgagcggttg caggctgcga ctcaatggtt gaagcagaac
aacctcaagg 1020gcttcctggg cgagatcggc gccggctcta actccgcttg catcagcgct
gtgcagggtg 1080cgttgtgttc gatgcagcaa tctggtgtgt ggctcggcgc tctctggtgg
gctgcgggcc 1140cgtggtgggg cgactactac cagtccatcg agccgccctc tggcccggcg
gtgtccgcga 1200tcctcccgca ggccctgctg ccgttcgcgt aa
123278397PRTBASIDIOMYCETE CBS 495.95 78Met Lys Ser Leu Phe Leu
Ser Leu Val Ala Thr Val Ala Leu Ser Ser1 5
10 15Pro Val Phe Ser Val Ala Val Trp Gly Gln Cys Gly
Gly Ile Gly Phe 20 25 30Ser
Gly Ser Thr Val Cys Asp Ala Gly Ala Gly Cys Val Lys Leu Asn 35
40 45Asp Tyr Tyr Ser Gln Cys Gln Pro Gly
Ala Pro Thr Ala Thr Ser Ala 50 55
60Ala Pro Ser Ser Asn Ala Pro Ser Gly Thr Ser Thr Ala Ser Ala Pro65
70 75 80Ser Ser Ser Leu Cys
Ser Gly Ser Arg Thr Pro Phe Gln Phe Phe Gly 85
90 95Val Asn Glu Ser Gly Ala Glu Phe Gly Asn Leu
Asn Ile Pro Gly Val 100 105
110Leu Gly Thr Asp Tyr Thr Trp Pro Ser Pro Ser Ser Ile Asp Phe Phe
115 120 125Met Gly Lys Gly Met Asn Thr
Phe Arg Ile Pro Phe Leu Met Glu Arg 130 135
140Leu Val Pro Pro Ala Thr Gly Ile Thr Gly Pro Leu Asp Gln Thr
Tyr145 150 155 160Leu Gly
Gly Leu Gln Thr Ile Val Asn Tyr Ile Thr Gly Lys Gly Gly
165 170 175Phe Ala Leu Ile Asp Pro His
Asn Phe Met Ile Tyr Asn Gly Gln Thr 180 185
190Ile Ser Ser Thr Ser Asp Phe Gln Lys Phe Trp Gln Asn Leu
Ala Gly 195 200 205Val Phe Lys Ser
Asn Ser His Val Ile Phe Asp Val Met Asn Glu Pro 210
215 220His Asp Ile Pro Ala Gln Thr Val Phe Gln Leu Asn
Gln Ala Ala Val225 230 235
240Asn Gly Ile Arg Ala Ser Gly Ala Thr Ser Gln Leu Ile Leu Val Glu
245 250 255Gly Thr Ser Trp Thr
Gly Ala Trp Thr Trp Thr Thr Ser Gly Asn Ser 260
265 270Asp Ala Phe Gly Ala Ile Lys Asp Pro Asn Asn Asn
Val Ala Ile Gln 275 280 285Met His
Gln Tyr Leu Asp Ser Asp Gly Ser Gly Thr Ser Gln Thr Cys 290
295 300Val Ser Pro Thr Ile Gly Ala Glu Arg Leu Gln
Ala Ala Thr Gln Trp305 310 315
320Leu Lys Gln Asn Asn Leu Lys Gly Phe Leu Gly Glu Ile Gly Ala Gly
325 330 335Ser Asn Ser Ala
Cys Ile Ser Ala Val Gln Gly Ala Leu Cys Ser Met 340
345 350Gln Gln Ser Gly Val Trp Leu Gly Ala Leu Trp
Trp Ala Ala Gly Pro 355 360 365Trp
Trp Gly Asp Tyr Tyr Gln Ser Ile Glu Pro Pro Ser Gly Pro Ala 370
375 380Val Ser Ala Ile Leu Pro Gln Ala Leu Leu
Pro Phe Ala385 390
395791303DNABASIDIOMYCETE CBS 495.95 79ggaaagcgtc agtatggtga aatttgcgct
tgtggcaact gtcggcgcaa tcttgagcgc 60ttctgcggcc aatgcggctt ctatctacca
gcaatgtgga ggcattggat ggtctgggtc 120cactgtttgc gacgccggtc tcgcttgcgt
tatcctcaat gcgtactact ttcagtgctt 180gacgcccgcc gcgggccaga caacgacggg
ctcgggcgca ccggcgtcaa catcaacctc 240tcactcaacg gtcactacgg ggagctcaca
ctcaacaacc gggacgacgg cgacgaaaac 300aactaccact ccgtcgacca ccacgaccct
acccgccatc tctgtgtctg gtcgcgtctg 360ctctggctcc aggacgaagt tcaagttctt
cggtgtgaat gaaagcggcg ccgaattcgg 420gaacactgct tggccagggc agctcgggaa
agactataca tggccttcgc ctagcagcgt 480ggactacttc atgggggctg gattcaatac
attccgtatc accttcttga tggagcgtat 540gagccctccg gctaccggac tcactggccc
attcaaccag acgtacctgt cgggcctcac 600caccattgtc gactacatca cgaacaaagg
aggatacgct cttattgacc cccacaactt 660catgcgttac aacaacggca taatcagcag
cacatctgac ttcgcgactt ggtggagcaa 720tttggccact gtattcaaat ccacgaagaa
cgccatcttc gacatccaga acgagccgta 780cggaatcgat gcgcagaccg tatacgaact
gaatcaagct gccatcaatt cgatccgcgc 840cgctggcgct acgtcacagt tgattctggt
tgaaggaacg tcatacactg gagcttggac 900gtgggtctcg tccggaaacg gagctgcttt
cgcggccgtt acggatcctt acaacaacac 960ggcaattgaa atgcaccaat acctcgacag
cgacggttct gggacaaacg aagactgtgt 1020ctcctccacc attgggtcgc aacgtctcca
agctgccact gcgtggctgc aacaaacagg 1080actcaaggga ttcctcggag agacgggtgc
tgggtcgaat tcccagtgca tcgacgccgt 1140gttcgatgaa ctttgctata tgcaacagca
aggcggctcc tggatcggtg cactctggtg 1200ggctgcgggt ccctggtggg gcacgtacat
ttactcgatt gaacctccga gcggtgccgc 1260tatcccagaa gtccttcctc agggtctcgc
tccattcctc tag 130380429PRTBASIDIOMYCETE CBS 495.95
80Met Val Lys Phe Ala Leu Val Ala Thr Val Gly Ala Ile Leu Ser Ala1
5 10 15Ser Ala Ala Asn Ala Ala
Ser Ile Tyr Gln Gln Cys Gly Gly Ile Gly 20 25
30Trp Ser Gly Ser Thr Val Cys Asp Ala Gly Leu Ala Cys
Val Ile Leu 35 40 45Asn Ala Tyr
Tyr Phe Gln Cys Leu Thr Pro Ala Ala Gly Gln Thr Thr 50
55 60Thr Gly Ser Gly Ala Pro Ala Ser Thr Ser Thr Ser
His Ser Thr Val65 70 75
80Thr Thr Gly Ser Ser His Ser Thr Thr Gly Thr Thr Ala Thr Lys Thr
85 90 95Thr Thr Thr Pro Ser Thr
Thr Thr Thr Leu Pro Ala Ile Ser Val Ser 100
105 110Gly Arg Val Cys Ser Gly Ser Arg Thr Lys Phe Lys
Phe Phe Gly Val 115 120 125Asn Glu
Ser Gly Ala Glu Phe Gly Asn Thr Ala Trp Pro Gly Gln Leu 130
135 140Gly Lys Asp Tyr Thr Trp Pro Ser Pro Ser Ser
Val Asp Tyr Phe Met145 150 155
160Gly Ala Gly Phe Asn Thr Phe Arg Ile Thr Phe Leu Met Glu Arg Met
165 170 175Ser Pro Pro Ala
Thr Gly Leu Thr Gly Pro Phe Asn Gln Thr Tyr Leu 180
185 190Ser Gly Leu Thr Thr Ile Val Asp Tyr Ile Thr
Asn Lys Gly Gly Tyr 195 200 205Ala
Leu Ile Asp Pro His Asn Phe Met Arg Tyr Asn Asn Gly Ile Ile 210
215 220Ser Ser Thr Ser Asp Phe Ala Thr Trp Trp
Ser Asn Leu Ala Thr Val225 230 235
240Phe Lys Ser Thr Lys Asn Ala Ile Phe Asp Ile Gln Asn Glu Pro
Tyr 245 250 255Gly Ile Asp
Ala Gln Thr Val Tyr Glu Leu Asn Gln Ala Ala Ile Asn 260
265 270Ser Ile Arg Ala Ala Gly Ala Thr Ser Gln
Leu Ile Leu Val Glu Gly 275 280
285Thr Ser Tyr Thr Gly Ala Trp Thr Trp Val Ser Ser Gly Asn Gly Ala 290
295 300Ala Phe Ala Ala Val Thr Asp Pro
Tyr Asn Asn Thr Ala Ile Glu Met305 310
315 320His Gln Tyr Leu Asp Ser Asp Gly Ser Gly Thr Asn
Glu Asp Cys Val 325 330
335Ser Ser Thr Ile Gly Ser Gln Arg Leu Gln Ala Ala Thr Ala Trp Leu
340 345 350Gln Gln Thr Gly Leu Lys
Gly Phe Leu Gly Glu Thr Gly Ala Gly Ser 355 360
365Asn Ser Gln Cys Ile Asp Ala Val Phe Asp Glu Leu Cys Tyr
Met Gln 370 375 380Gln Gln Gly Gly Ser
Trp Ile Gly Ala Leu Trp Trp Ala Ala Gly Pro385 390
395 400Trp Trp Gly Thr Tyr Ile Tyr Ser Ile Glu
Pro Pro Ser Gly Ala Ala 405 410
415Ile Pro Glu Val Leu Pro Gln Gly Leu Ala Pro Phe Leu
420 425811580DNAThielavia terrestris 81agccccccgt
tcaggcacac ttggcatcag atcagcttag cagcgcctgc acagcatgaa 60gctctcgcag
tcggccgcgc tggcggcact caccgcgacg gcgctcgccg ccccctcgcc 120cacgacgccg
caggcgccga ggcaggcttc agccggctgc tcgtctgcgg tcacgctcga 180cgccagcacc
aacgtttgga agaagtacac gctgcacccc aacagctact accgcaagga 240ggttgaggcc
gcggtggcgc agatctcgga cccggacctc gccgccaagg ccaagaaggt 300ggccgacgtc
ggcaccttcc tgtggctcga ctcgatcgag aacatcggca agctggagcc 360ggcgatccag
gacgtgccct gcgagaacat cctgggcctg gtcatctacg acctgccggg 420ccgcgactgc
gcggccaagg cgtccaacgg cgagctcaag gtcggcgaga tcgaccgcta 480caagaccgag
tacatcgaca gtgagtgctg ccccccgggt tcgagaagag cgtgggggaa 540agggaaaggg
ttgactgact gacacggcgc actgcagaga tcgtgtcgat cctcaaggca 600caccccaaca
cggcgttcgc gctggtcatc gagccggact cgctgcccaa cctggtgacc 660aacagcaact
tggacacgtg ctcgagcagc gcgtcgggct accgcgaagg cgtggcttac 720gccctcaaga
acctcaacct gcccaacgtg atcatgtacc tcgacgccgg ccacggcggc 780tggctcggct
gggacgccaa cctgcagccc ggcgcgcagg agctagccaa ggcgtacaag 840aacgccggct
cgcccaagca gctccgcggc ttctcgacca acgtggccgg ctggaactcc 900tggtgagctt
ttttccattc catttcttct tcctcttctc tcttcgctcc cactctgcag 960ccccccctcc
cccaagcacc cactggcgtt ccggcttgct gactcggcct ccctttcccc 1020gggcaccagg
gatcaatcgc ccggcgaatt ctcccaggcg tccgacgcca agtacaacaa 1080gtgccagaac
gagaagatct acgtcagcac cttcggctcc gcgctccagt cggccggcat 1140gcccaaccac
gccatcgtcg acacgggccg caacggcgtc accggcctgc gcaaggagtg 1200gggtgactgg
tgcaacgtca acggtgcagg ttcgttgtct tctttttctc ctcttttgtt 1260tgcacgtcgt
ggtccttttc aagcagccgt gtttggttgg gggagatgga ctccggctga 1320tgttctgctt
cctctctagg cttcggcgtg cgcccgacga gcaacacggg cctcgagctg 1380gccgacgcgt
tcgtgtgggt caagcccggc ggcgagtcgg acggcaccag cgacagctcg 1440tcgccgcgct
acgacagctt ctgcggcaag gacgacgcct tcaagccctc gcccgaggcc 1500ggcacctgga
acgaggccta cttcgagatg ctgctcaaga acgccgtgcc gtcgttctaa 1560gacggtccag
catcatccgg
158082396PRTThielavia terrestris 82Met Lys Leu Ser Gln Ser Ala Ala Leu
Ala Ala Leu Thr Ala Thr Ala1 5 10
15Leu Ala Ala Pro Ser Pro Thr Thr Pro Gln Ala Pro Arg Gln Ala
Ser 20 25 30Ala Gly Cys Ser
Ser Ala Val Thr Leu Asp Ala Ser Thr Asn Val Trp 35
40 45Lys Lys Tyr Thr Leu His Pro Asn Ser Tyr Tyr Arg
Lys Glu Val Glu 50 55 60Ala Ala Val
Ala Gln Ile Ser Asp Pro Asp Leu Ala Ala Lys Ala Lys65 70
75 80Lys Val Ala Asp Val Gly Thr Phe
Leu Trp Leu Asp Ser Ile Glu Asn 85 90
95Ile Gly Lys Leu Glu Pro Ala Ile Gln Asp Val Pro Cys Glu
Asn Ile 100 105 110Leu Gly Leu
Val Ile Tyr Asp Leu Pro Gly Arg Asp Cys Ala Ala Lys 115
120 125Ala Ser Asn Gly Glu Leu Lys Val Gly Glu Ile
Asp Arg Tyr Lys Thr 130 135 140Glu Tyr
Ile Asp Lys Ile Val Ser Ile Leu Lys Ala His Pro Asn Thr145
150 155 160Ala Phe Ala Leu Val Ile Glu
Pro Asp Ser Leu Pro Asn Leu Val Thr 165
170 175Asn Ser Asn Leu Asp Thr Cys Ser Ser Ser Ala Ser
Gly Tyr Arg Glu 180 185 190Gly
Val Ala Tyr Ala Leu Lys Asn Leu Asn Leu Pro Asn Val Ile Met 195
200 205Tyr Leu Asp Ala Gly His Gly Gly Trp
Leu Gly Trp Asp Ala Asn Leu 210 215
220Gln Pro Gly Ala Gln Glu Leu Ala Lys Ala Tyr Lys Asn Ala Gly Ser225
230 235 240Pro Lys Gln Leu
Arg Gly Phe Ser Thr Asn Val Ala Gly Trp Asn Ser 245
250 255Trp Asp Gln Ser Pro Gly Glu Phe Ser Gln
Ala Ser Asp Ala Lys Tyr 260 265
270Asn Lys Cys Gln Asn Glu Lys Ile Tyr Val Ser Thr Phe Gly Ser Ala
275 280 285Leu Gln Ser Ala Gly Met Pro
Asn His Ala Ile Val Asp Thr Gly Arg 290 295
300Asn Gly Val Thr Gly Leu Arg Lys Glu Trp Gly Asp Trp Cys Asn
Val305 310 315 320Asn Gly
Ala Gly Phe Gly Val Arg Pro Thr Ser Asn Thr Gly Leu Glu
325 330 335Leu Ala Asp Ala Phe Val Trp
Val Lys Pro Gly Gly Glu Ser Asp Gly 340 345
350Thr Ser Asp Ser Ser Ser Pro Arg Tyr Asp Ser Phe Cys Gly
Lys Asp 355 360 365Asp Ala Phe Lys
Pro Ser Pro Glu Ala Gly Thr Trp Asn Glu Ala Tyr 370
375 380Phe Glu Met Leu Leu Lys Asn Ala Val Pro Ser Phe385
390 395831203DNAThielavia terrestris
83atgaagtacc tcaacctcct cgcagctctc ctcgccgtcg ctcctctctc cctcgctgca
60cccagcatcg aggccagaca gtcgaacgtc aacccataca tcggcaagag cccgctcgtt
120attaggtcgt acgcccaaaa gcttgaggag accgtcagga ccttccagca acgtggcgac
180cagctcaacg ctgcgaggac acggacggtg cagaacgttg cgactttcgc ctggatctcg
240gataccaatg gtattggagc cattcgacct ctcatccaag atgctctcgc ccagcaggct
300cgcactggac agaaggtcat cgtccaaatc gtcgtctaca acctcccaga tcgcgactgc
360tctgccaacg cctcgactgg agagttcacc gtaggaaacg acggtctcaa ccgatacaag
420aactttgtca acaccatcgc ccgcgagctc tcgactgctg acgctgacaa gctccacttt
480gccctcctcc tcgaacccga cgcacttgcc aacctcgtca ccaacgcgaa tgcccccagg
540tgccgaatcg ccgctcccgc ttacaaggag ggtatcgcct acaccctcgc caccttgtcc
600aagcccaacg tcgacgtcta catcgacgcc gccaacggtg gctggctcgg ctggaacgac
660aacctccgcc ccttcgccga actcttcaag gaagtctacg acctcgcccg ccgcatcaac
720cccaacgcca aggtccgcgg cgtccccgtc aacgtctcca actacaacca gtaccgcgct
780gaagtccgcg agcccttcac cgagtggaag gacgcctggg acgagagccg ctacgtcaac
840gtcctcaccc cgcacctcaa cgccgtcggc ttctccgcgc acttcatcgt tgaccaggga
900cgcggtggca agggcggtat caggacggag tggggccagt ggtgcaacgt taggaacgct
960gggttcggta tcaggcctac tgcggatcag ggcgtgctcc agaacccgaa tgtggatgcg
1020attgtgtggg ttaagccggg tggagagtcg gatggcacga gtgatttgaa ctcgaacagg
1080tatgatccta cgtgcaggag tccggtggcg catgttcccg ctcctgaggc tggccagtgg
1140ttcaacgagt atgttgttaa cctcgttttg aacgctaacc cccctcttga gcctacctgg
1200taa
120384400PRTThielavia terrestris 84Met Lys Tyr Leu Asn Leu Leu Ala Ala
Leu Leu Ala Val Ala Pro Leu1 5 10
15Ser Leu Ala Ala Pro Ser Ile Glu Ala Arg Gln Ser Asn Val Asn
Pro 20 25 30Tyr Ile Gly Lys
Ser Pro Leu Val Ile Arg Ser Tyr Ala Gln Lys Leu 35
40 45Glu Glu Thr Val Arg Thr Phe Gln Gln Arg Gly Asp
Gln Leu Asn Ala 50 55 60Ala Arg Thr
Arg Thr Val Gln Asn Val Ala Thr Phe Ala Trp Ile Ser65 70
75 80Asp Thr Asn Gly Ile Gly Ala Ile
Arg Pro Leu Ile Gln Asp Ala Leu 85 90
95Ala Gln Gln Ala Arg Thr Gly Gln Lys Val Ile Val Gln Ile
Val Val 100 105 110Tyr Asn Leu
Pro Asp Arg Asp Cys Ser Ala Asn Ala Ser Thr Gly Glu 115
120 125Phe Thr Val Gly Asn Asp Gly Leu Asn Arg Tyr
Lys Asn Phe Val Asn 130 135 140Thr Ile
Ala Arg Glu Leu Ser Thr Ala Asp Ala Asp Lys Leu His Phe145
150 155 160Ala Leu Leu Leu Glu Pro Asp
Ala Leu Ala Asn Leu Val Thr Asn Ala 165
170 175Asn Ala Pro Arg Cys Arg Ile Ala Ala Pro Ala Tyr
Lys Glu Gly Ile 180 185 190Ala
Tyr Thr Leu Ala Thr Leu Ser Lys Pro Asn Val Asp Val Tyr Ile 195
200 205Asp Ala Ala Asn Gly Gly Trp Leu Gly
Trp Asn Asp Asn Leu Arg Pro 210 215
220Phe Ala Glu Leu Phe Lys Glu Val Tyr Asp Leu Ala Arg Arg Ile Asn225
230 235 240Pro Asn Ala Lys
Val Arg Gly Val Pro Val Asn Val Ser Asn Tyr Asn 245
250 255Gln Tyr Arg Ala Glu Val Arg Glu Pro Phe
Thr Glu Trp Lys Asp Ala 260 265
270Trp Asp Glu Ser Arg Tyr Val Asn Val Leu Thr Pro His Leu Asn Ala
275 280 285Val Gly Phe Ser Ala His Phe
Ile Val Asp Gln Gly Arg Gly Gly Lys 290 295
300Gly Gly Ile Arg Thr Glu Trp Gly Gln Trp Cys Asn Val Arg Asn
Ala305 310 315 320Gly Phe
Gly Ile Arg Pro Thr Ala Asp Gln Gly Val Leu Gln Asn Pro
325 330 335Asn Val Asp Ala Ile Val Trp
Val Lys Pro Gly Gly Glu Ser Asp Gly 340 345
350Thr Ser Asp Leu Asn Ser Asn Arg Tyr Asp Pro Thr Cys Arg
Ser Pro 355 360 365Val Ala His Val
Pro Ala Pro Glu Ala Gly Gln Trp Phe Asn Glu Tyr 370
375 380Val Val Asn Leu Val Leu Asn Ala Asn Pro Pro Leu
Glu Pro Thr Trp385 390 395
400851501DNAThielavia terrestris 85gccgttgtca agatgggcca gaagacgctg
cacggattcg ccgccacggc tttggccgtt 60ctcccctttg tgaaggctca gcagcccggc
aacttcacgc cggaggtgca cccgcaactg 120ccaacgtgga agtgcacgac cgccggcggc
tgcgttcagc aggacacttc ggtggtgctc 180gactggaact accgttggat ccacaatgcc
gacggcaccg cctcgtgcac gacgtccagc 240ggggtcgacc acacgctgtg tccagatgag
gcgacctgcg cgaagaactg cttcgtggaa 300ggcgtcaact acacgagcag cggtgtcacc
acatccggca gttcgctgac gatgaggcag 360tatttcaagg ggagcaacgg gcagaccaac
agcgtttcgc ctcgtctcta cctgctcggc 420tcggatggaa actacgtaat gctcaagctg
ctcggccagg agctgagctt cgatgtcgat 480ctctccacgc tcccctgcgg cgagaacggc
gcgctgtacc tgtccgagat ggacgcgacc 540ggtggcagga accagtacaa caccggcggt
gccaactacg gctcgggcta ctgtgacgcc 600cagtgtcccg tgcagacgtg gatgaacggc
acgctgaaca ccaacgggca gggctactgc 660tgcaacgaga tggacatcct cgaggccaac
tcccgcgcca acgcgatgac acctcacccc 720tgcgccaacg gcagctgcga caagagcggg
tgcggactca acccctacgc cgagggctac 780aagagctact acggaccggg cctcacggtt
gacacgtcga agcccttcac catcattacc 840cgcttcatca ccgacgacgg cacgaccagc
ggcaccctca accagatcca gcggatctat 900gtgcagaatg gcaagacggt cgcgtcggct
gcgtccggag gcgacatcat cacggcatcc 960ggctgcacct cggcccaggc gttcggcggg
ctggccaaca tgggcgcggc gcttggacgg 1020ggcatggtgc tgaccttcag catctggaac
gacgctgggg gctacatgaa ctggctcgac 1080agcggcaaca acggcccgtg cagcagcacc
gagggcaacc cgtccaacat cctggccaac 1140tacccggaca cccacgtggt cttctccaac
atccgctggg gagacatcgg ctcgacggtc 1200caggtctcgg gaggcggcaa cggcggctcg
accaccacca cgtcgaccac cacgctgagg 1260acctcgacca cgaccaccac caccgccccg
acggccactg ccacgcactg gggacaatgc 1320ggcggaatcg gggtacgtca accgcctcct
gcattctgtt gaggaagtta actaacgtgg 1380cctacgcagt ggactggacc gaccgtctgc
gaatcgccgt acgcatgcaa ggagctgaac 1440ccctggtact accagtgcct ctaaagtatt
gcagtgaagc catactccgt gctcggcatg 1500g
150186464PRTThielavia terrestris 86Met
Gly Gln Lys Thr Leu His Gly Phe Ala Ala Thr Ala Leu Ala Val1
5 10 15Leu Pro Phe Val Lys Ala Gln
Gln Pro Gly Asn Phe Thr Pro Glu Val 20 25
30His Pro Gln Leu Pro Thr Trp Lys Cys Thr Thr Ala Gly Gly
Cys Val 35 40 45Gln Gln Asp Thr
Ser Val Val Leu Asp Trp Asn Tyr Arg Trp Ile His 50 55
60Asn Ala Asp Gly Thr Ala Ser Cys Thr Thr Ser Ser Gly
Val Asp His65 70 75
80Thr Leu Cys Pro Asp Glu Ala Thr Cys Ala Lys Asn Cys Phe Val Glu
85 90 95Gly Val Asn Tyr Thr Ser
Ser Gly Val Thr Thr Ser Gly Ser Ser Leu 100
105 110Thr Met Arg Gln Tyr Phe Lys Gly Ser Asn Gly Gln
Thr Asn Ser Val 115 120 125Ser Pro
Arg Leu Tyr Leu Leu Gly Ser Asp Gly Asn Tyr Val Met Leu 130
135 140Lys Leu Leu Gly Gln Glu Leu Ser Phe Asp Val
Asp Leu Ser Thr Leu145 150 155
160Pro Cys Gly Glu Asn Gly Ala Leu Tyr Leu Ser Glu Met Asp Ala Thr
165 170 175Gly Gly Arg Asn
Gln Tyr Asn Thr Gly Gly Ala Asn Tyr Gly Ser Gly 180
185 190Tyr Cys Asp Ala Gln Cys Pro Val Gln Thr Trp
Met Asn Gly Thr Leu 195 200 205Asn
Thr Asn Gly Gln Gly Tyr Cys Cys Asn Glu Met Asp Ile Leu Glu 210
215 220Ala Asn Ser Arg Ala Asn Ala Met Thr Pro
His Pro Cys Ala Asn Gly225 230 235
240Ser Cys Asp Lys Ser Gly Cys Gly Leu Asn Pro Tyr Ala Glu Gly
Tyr 245 250 255Lys Ser Tyr
Tyr Gly Pro Gly Leu Thr Val Asp Thr Ser Lys Pro Phe 260
265 270Thr Ile Ile Thr Arg Phe Ile Thr Asp Asp
Gly Thr Thr Ser Gly Thr 275 280
285Leu Asn Gln Ile Gln Arg Ile Tyr Val Gln Asn Gly Lys Thr Val Ala 290
295 300Ser Ala Ala Ser Gly Gly Asp Ile
Ile Thr Ala Ser Gly Cys Thr Ser305 310
315 320Ala Gln Ala Phe Gly Gly Leu Ala Asn Met Gly Ala
Ala Leu Gly Arg 325 330
335Gly Met Val Leu Thr Phe Ser Ile Trp Asn Asp Ala Gly Gly Tyr Met
340 345 350Asn Trp Leu Asp Ser Gly
Asn Asn Gly Pro Cys Ser Ser Thr Glu Gly 355 360
365Asn Pro Ser Asn Ile Leu Ala Asn Tyr Pro Asp Thr His Val
Val Phe 370 375 380Ser Asn Ile Arg Trp
Gly Asp Ile Gly Ser Thr Val Gln Val Ser Gly385 390
395 400Gly Gly Asn Gly Gly Ser Thr Thr Thr Thr
Ser Thr Thr Thr Leu Arg 405 410
415Thr Ser Thr Thr Thr Thr Thr Thr Ala Pro Thr Ala Thr Ala Thr His
420 425 430Trp Gly Gln Cys Gly
Gly Ile Gly Trp Thr Gly Pro Thr Val Cys Glu 435
440 445Ser Pro Tyr Ala Cys Lys Glu Leu Asn Pro Trp Tyr
Tyr Gln Cys Leu 450 455
460871368DNAThielavia terrestris 87accgatccgc tcgaagatgg cgcccaagtc
tacagttctg gccgcctggc tgctctcctc 60gctggccgcg gcccagcaga tcggcaaagc
cgtgcccgag gtccacccca aactgacaac 120gcagaagtgc actctccgcg gcgggtgcaa
gcctgtccgc acctcggtcg tgctcgactc 180gtccgcgcgc tcgctgcaca aggtcgggga
ccccaacacc agctgcagcg tcggcggcga 240cctgtgctcg gacgcgaagt cgtgcggcaa
gaactgcgcg ctcgagggcg tcgactacgc 300ggcccacggc gtggcgacca agggcgacgc
cctcacgctg caccagtggc tcaagggggc 360cgacggcacc tacaggaccg tctcgccgcg
cgtatacctc ctgggcgagg acgggaagaa 420ctacgaggac ttcaagctgc tcaacgccga
gctcagcttc gacgtcgacg tgtcccagct 480cgtctgcggc atgaacggcg ccctgtactt
ctccgagatg gagatggacg gcggccgcag 540cccgctgaac ccggcgggcg ccacgtacgg
cacgggctac tgcgacgcgc agtgccccaa 600gttggacttt atcaacggcg aggtatttct
tctctcttct gtttttcttt tccatcgctt 660tttctgaccg gaatccgccc tcttagctca
acaccaacca cacgtacggg gcgtgctgca 720acgagatgga catctgggag gccaacgcgc
tggcgcaggc gctcacgccg cacccgtgca 780acgcgacgcg ggtgtacaag tgcgacacgg
cggacgagtg cgggcagccg gtgggcgtgt 840gcgacgaatg ggggtgctcg tacaacccgt
ccaacttcgg ggtcaaggac tactacgggc 900gcaacctgac ggtggacacg aaccgcaagt
tcacggtgac gacgcagttc gtgacgtcca 960acgggcgggc ggacggcgag ctgaccgaga
tccggcggct gtacgtgcag gacggcgtgg 1020tgatccagaa ccacgcggtc acggcgggcg
gggcgacgta cgacagcatc acggacggct 1080tctgcaacgc gacggccacc tggacgcagc
agcggggcgg gctcgcgcgc atgggcgagg 1140ccatcggccg cggcatggtg ctcatcttca
gcctgtgggt tgacaacggc ggcttcatga 1200actggctcga cagcggcaac gccgggccct
gcaacgccac cgagggcgac ccggccctga 1260tcctgcagca gcacccggac gccagcgtca
ccttctccaa catccgatgg ggcgagatcg 1320gcagcacgta caagagcgag tgcagccact
agagtagagc ttgtaatt 136888423PRTThielavia terrestris 88Met
Ala Pro Lys Ser Thr Val Leu Ala Ala Trp Leu Leu Ser Ser Leu1
5 10 15Ala Ala Ala Gln Gln Ile Gly
Lys Ala Val Pro Glu Val His Pro Lys 20 25
30Leu Thr Thr Gln Lys Cys Thr Leu Arg Gly Gly Cys Lys Pro
Val Arg 35 40 45Thr Ser Val Val
Leu Asp Ser Ser Ala Arg Ser Leu His Lys Val Gly 50 55
60Asp Pro Asn Thr Ser Cys Ser Val Gly Gly Asp Leu Cys
Ser Asp Ala65 70 75
80Lys Ser Cys Gly Lys Asn Cys Ala Leu Glu Gly Val Asp Tyr Ala Ala
85 90 95His Gly Val Ala Thr Lys
Gly Asp Ala Leu Thr Leu His Gln Trp Leu 100
105 110Lys Gly Ala Asp Gly Thr Tyr Arg Thr Val Ser Pro
Arg Val Tyr Leu 115 120 125Leu Gly
Glu Asp Gly Lys Asn Tyr Glu Asp Phe Lys Leu Leu Asn Ala 130
135 140Glu Leu Ser Phe Asp Val Asp Val Ser Gln Leu
Val Cys Gly Met Asn145 150 155
160Gly Ala Leu Tyr Phe Ser Glu Met Glu Met Asp Gly Gly Arg Ser Pro
165 170 175Leu Asn Pro Ala
Gly Ala Thr Tyr Gly Thr Gly Tyr Cys Asp Ala Gln 180
185 190Cys Pro Lys Leu Asp Phe Ile Asn Gly Glu Leu
Asn Thr Asn His Thr 195 200 205Tyr
Gly Ala Cys Cys Asn Glu Met Asp Ile Trp Glu Ala Asn Ala Leu 210
215 220Ala Gln Ala Leu Thr Pro His Pro Cys Asn
Ala Thr Arg Val Tyr Lys225 230 235
240Cys Asp Thr Ala Asp Glu Cys Gly Gln Pro Val Gly Val Cys Asp
Glu 245 250 255Trp Gly Cys
Ser Tyr Asn Pro Ser Asn Phe Gly Val Lys Asp Tyr Tyr 260
265 270Gly Arg Asn Leu Thr Val Asp Thr Asn Arg
Lys Phe Thr Val Thr Thr 275 280
285Gln Phe Val Thr Ser Asn Gly Arg Ala Asp Gly Glu Leu Thr Glu Ile 290
295 300Arg Arg Leu Tyr Val Gln Asp Gly
Val Val Ile Gln Asn His Ala Val305 310
315 320Thr Ala Gly Gly Ala Thr Tyr Asp Ser Ile Thr Asp
Gly Phe Cys Asn 325 330
335Ala Thr Ala Thr Trp Thr Gln Gln Arg Gly Gly Leu Ala Arg Met Gly
340 345 350Glu Ala Ile Gly Arg Gly
Met Val Leu Ile Phe Ser Leu Trp Val Asp 355 360
365Asn Gly Gly Phe Met Asn Trp Leu Asp Ser Gly Asn Ala Gly
Pro Cys 370 375 380Asn Ala Thr Glu Gly
Asp Pro Ala Leu Ile Leu Gln Gln His Pro Asp385 390
395 400Ala Ser Val Thr Phe Ser Asn Ile Arg Trp
Gly Glu Ile Gly Ser Thr 405 410
415Tyr Lys Ser Glu Cys Ser His 420891011DNAThielavia
terrestris 89atgaccctac ggctccctgt catcagcctg ctggcctcgc tggcagcagg
cgccgtcgtc 60gtcccacggg cggagtttca cccccctctc ccgacttgga aatgcacgac
ctccgggggc 120tgcgtgcagc agaacaccag cgtcgtcctg gaccgtgact cgaagtacgc
cgcacacagc 180gccggctcgc ggacggaatc ggattacgcg gcaatgggag tgtccacttc
gggcaatgcc 240gtgacgctgt accactacgt caagaccaac ggcaccctcg tccccgcttc
gccgcgcatc 300tacctcctgg gcgcggacgg caagtacgtg cttatggacc tcctcaacca
ggagctgtcg 360gtggacgtcg acttctcggc gctgccgtgc ggcgagaacg gggccttcta
cctgtccgag 420atggcggcgg acgggcgggg cgacgcgggg gcgggcgacg ggtactgcga
cgcgcagtgc 480cagggctact gctgcaacga gatggacatc ctcgaggcca actcgatggc
gacggccatg 540acgccgcacc cgtgcaaggg caacaactgc gaccgcagcg gctgcggcta
caacccgtac 600gccagcggcc agcgcggctt ctacgggccc ggcaagacgg tcgacacgag
caagcccttc 660accgtcgtca cgcagttcgc cgccagcggc ggcaagctga cccagatcac
ccgcaagtac 720atccagaacg gccgggagat cggcggcggc ggcaccatct ccagctgcgg
ctccgagtct 780tcgacgggcg gcctgaccgg catgggcgag gcgctggggc gcggaatggt
gctggccatg 840agcatctgga acgacgcggc ccaggagatg gcatggctcg atgccggcaa
caacggccct 900tgcgccagtg gccagggcag cccgtccgtc attcagtcgc agcatcccga
cacccacgtc 960gtcttctcca acatcaggtg gggcgacatc gggtctacca cgaagaacta g
101190336PRTThielavia terrestris 90Met Thr Leu Arg Leu Pro Val
Ile Ser Leu Leu Ala Ser Leu Ala Ala1 5 10
15Gly Ala Val Val Val Pro Arg Ala Glu Phe His Pro Pro
Leu Pro Thr 20 25 30Trp Lys
Cys Thr Thr Ser Gly Gly Cys Val Gln Gln Asn Thr Ser Val 35
40 45Val Leu Asp Arg Asp Ser Lys Tyr Ala Ala
His Ser Ala Gly Ser Arg 50 55 60Thr
Glu Ser Asp Tyr Ala Ala Met Gly Val Ser Thr Ser Gly Asn Ala65
70 75 80Val Thr Leu Tyr His Tyr
Val Lys Thr Asn Gly Thr Leu Val Pro Ala 85
90 95Ser Pro Arg Ile Tyr Leu Leu Gly Ala Asp Gly Lys
Tyr Val Leu Met 100 105 110Asp
Leu Leu Asn Gln Glu Leu Ser Val Asp Val Asp Phe Ser Ala Leu 115
120 125Pro Cys Gly Glu Asn Gly Ala Phe Tyr
Leu Ser Glu Met Ala Ala Asp 130 135
140Gly Arg Gly Asp Ala Gly Ala Gly Asp Gly Tyr Cys Asp Ala Gln Cys145
150 155 160Gln Gly Tyr Cys
Cys Asn Glu Met Asp Ile Leu Glu Ala Asn Ser Met 165
170 175Ala Thr Ala Met Thr Pro His Pro Cys Lys
Gly Asn Asn Cys Asp Arg 180 185
190Ser Gly Cys Gly Tyr Asn Pro Tyr Ala Ser Gly Gln Arg Gly Phe Tyr
195 200 205Gly Pro Gly Lys Thr Val Asp
Thr Ser Lys Pro Phe Thr Val Val Thr 210 215
220Gln Phe Ala Ala Ser Gly Gly Lys Leu Thr Gln Ile Thr Arg Lys
Tyr225 230 235 240Ile Gln
Asn Gly Arg Glu Ile Gly Gly Gly Gly Thr Ile Ser Ser Cys
245 250 255Gly Ser Glu Ser Ser Thr Gly
Gly Leu Thr Gly Met Gly Glu Ala Leu 260 265
270Gly Arg Gly Met Val Leu Ala Met Ser Ile Trp Asn Asp Ala
Ala Gln 275 280 285Glu Met Ala Trp
Leu Asp Ala Gly Asn Asn Gly Pro Cys Ala Ser Gly 290
295 300Gln Gly Ser Pro Ser Val Ile Gln Ser Gln His Pro
Asp Thr His Val305 310 315
320Val Phe Ser Asn Ile Arg Trp Gly Asp Ile Gly Ser Thr Thr Lys Asn
325 330 335911480DNACladorrhinum
foecundissimum 91gatccgaatt cctcctctcg ttctttagtc acagaccaga catctgccca
cgatggttca 60caagttcgcc ctcctcaccg gcctcgccgc ctccctcgca tctgcccagc
agatcggcac 120cgtcgtcccc gagtctcacc ccaagcttcc caccaagcgc tgcactctcg
ccggtggctg 180ccagaccgtc gacacctcca tcgtcatcga cgccttccag cgtcccctcc
acaagatcgg 240cgacccttcc actccttgcg tcgtcggcgg ccctctctgc cccgacgcca
agtcctgcgc 300tgagaactgc gcgctcgagg gtgtcgacta tgcctcctgg ggcatcaaga
ccgagggcga 360cgccctaact ctcaaccagt ggatgcccga cccggcgaac cctggccagt
acaagacgac 420tactccccgt acttaccttg ttgctgagga cggcaagaac tacgaggatg
tgaagctcct 480ggctaaggag atctcgtttg atgccgatgt cagcaacctt ccctgcggca
tgaacggtgc 540tttctacttg tctgagatgt tgatggatgg tggacgtggc gacctcaacc
ctgctggtgc 600cgagtatggt accggttact gtgatgcgca gtgcttcaag ttggatttca
tcaacggcga 660ggccaacatc gaccaaaagc acggcgcctg ctgcaacgaa atggacattt
tcgaatccaa 720ctcgcgcgcc aagaccttcg tcccccaccc ctgcaacatc acgcaggtct
acaagtgcga 780aggcgaagac gagtgcggcc agcccgtcgg cgtgtgcgac aagtgggggt
gcggcttcaa 840cgagtacaaa tggggcgtcg agtccttcta cggccggggc tcgcagttcg
ccatcgactc 900ctccaagaag ttcaccgtca ccacgcagtt cctgaccgac aacggcaagg
aggacggcgt 960cctcgtcgag atccgccgct tgtggcacca ggatggcaag ctgatcaaga
acaccgctat 1020ccaggttgag gagaactaca gcacggactc ggtgagcacc gagttctgcg
agaagactgc 1080ttctttcacc atgcagcgcg gtggtctcaa ggcgatgggc gaggctatcg
gtcgtggtat 1140ggtgctggtt ttcagcatct gggcggatga ttcgggtttt atgaactggt
tggatgcgga 1200gggtaatggc ccttgcagcg cgactgaggg cgatccgaag gagattgtca
agaataagcc 1260ggatgctagg gttacgttct caaacattag gattggtgag gttggtagca
cgtatgctcc 1320gggtgggaag tgcggtgtta agagcagggt tgctaggggg cttactgctt
cttaaggggg 1380gtgtgaagag aggaggaggt gttgttgggg gttggagatg ataattgggc
gagatggtgt 1440agagcgggtt ggttggatat gaatacgttg aattggatgt
148092440PRTCladorrhinum foecundissimum 92Met Val His Lys Phe
Ala Leu Leu Thr Gly Leu Ala Ala Ser Leu Ala1 5
10 15Ser Ala Gln Gln Ile Gly Thr Val Val Pro Glu
Ser His Pro Lys Leu 20 25
30Pro Thr Lys Arg Cys Thr Leu Ala Gly Gly Cys Gln Thr Val Asp Thr
35 40 45Ser Ile Val Ile Asp Ala Phe Gln
Arg Pro Leu His Lys Ile Gly Asp 50 55
60Pro Ser Thr Pro Cys Val Val Gly Gly Pro Leu Cys Pro Asp Ala Lys65
70 75 80Ser Cys Ala Glu Asn
Cys Ala Leu Glu Gly Val Asp Tyr Ala Ser Trp 85
90 95Gly Ile Lys Thr Glu Gly Asp Ala Leu Thr Leu
Asn Gln Trp Met Pro 100 105
110Asp Pro Ala Asn Pro Gly Gln Tyr Lys Thr Thr Thr Pro Arg Thr Tyr
115 120 125Leu Val Ala Glu Asp Gly Lys
Asn Tyr Glu Asp Val Lys Leu Leu Ala 130 135
140Lys Glu Ile Ser Phe Asp Ala Asp Val Ser Asn Leu Pro Cys Gly
Met145 150 155 160Asn Gly
Ala Phe Tyr Leu Ser Glu Met Leu Met Asp Gly Gly Arg Gly
165 170 175Asp Leu Asn Pro Ala Gly Ala
Glu Tyr Gly Thr Gly Tyr Cys Asp Ala 180 185
190Gln Cys Phe Lys Leu Asp Phe Ile Asn Gly Glu Ala Asn Ile
Asp Gln 195 200 205Lys His Gly Ala
Cys Cys Asn Glu Met Asp Ile Phe Glu Ser Asn Ser 210
215 220Arg Ala Lys Thr Phe Val Pro His Pro Cys Asn Ile
Thr Gln Val Tyr225 230 235
240Lys Cys Glu Gly Glu Asp Glu Cys Gly Gln Pro Val Gly Val Cys Asp
245 250 255Lys Trp Gly Cys Gly
Phe Asn Glu Tyr Lys Trp Gly Val Glu Ser Phe 260
265 270Tyr Gly Arg Gly Ser Gln Phe Ala Ile Asp Ser Ser
Lys Lys Phe Thr 275 280 285Val Thr
Thr Gln Phe Leu Thr Asp Asn Gly Lys Glu Asp Gly Val Leu 290
295 300Val Glu Ile Arg Arg Leu Trp His Gln Asp Gly
Lys Leu Ile Lys Asn305 310 315
320Thr Ala Ile Gln Val Glu Glu Asn Tyr Ser Thr Asp Ser Val Ser Thr
325 330 335Glu Phe Cys Glu
Lys Thr Ala Ser Phe Thr Met Gln Arg Gly Gly Leu 340
345 350Lys Ala Met Gly Glu Ala Ile Gly Arg Gly Met
Val Leu Val Phe Ser 355 360 365Ile
Trp Ala Asp Asp Ser Gly Phe Met Asn Trp Leu Asp Ala Glu Gly 370
375 380Asn Gly Pro Cys Ser Ala Thr Glu Gly Asp
Pro Lys Glu Ile Val Lys385 390 395
400Asn Lys Pro Asp Ala Arg Val Thr Phe Ser Asn Ile Arg Ile Gly
Glu 405 410 415Val Gly Ser
Thr Tyr Ala Pro Gly Gly Lys Cys Gly Val Lys Ser Arg 420
425 430Val Ala Arg Gly Leu Thr Ala Ser
435 440931380DNATrichoderma reesei 93atggcgccct
cagttacact gccgttgacc acggccatcc tggccattgc ccggctcgtc 60gccgcccagc
aaccgggtac cagcaccccc gaggtccatc ccaagttgac aacctacaag 120tgtacaaagt
ccggggggtg cgtggcccag gacacctcgg tggtccttga ctggaactac 180cgctggatgc
acgacgcaaa ctacaactcg tgcaccgtca acggcggcgt caacaccacg 240ctctgccctg
acgaggcgac ctgtggcaag aactgcttca tcgagggcgt cgactacgcc 300gcctcgggcg
tcacgacctc gggcagcagc ctcaccatga accagtacat gcccagcagc 360tctggcggct
acagcagcgt ctctcctcgg ctgtatctcc tggactctga cggtgagtac 420gtgatgctga
agctcaacgg ccaggagctg agcttcgacg tcgacctctc tgctctgccg 480tgtggagaga
acggctcgct ctacctgtct cagatggacg agaacggggg cgccaaccag 540tataacacgg
ccggtgccaa ctacgggagc ggctactgcg atgctcagtg ccccgtccag 600acatggagga
acggcaccct caacactagc caccagggct tctgctgcaa cgagatggat 660atcctggagg
gcaactcgag ggcgaatgcc ttgacccctc actcttgcac ggccacggcc 720tgcgactctg
ccggttgcgg cttcaacccc tatggcagcg gctacaaaag ctactacggc 780cccggagata
ccgttgacac ctccaagacc ttcaccatca tcacccagtt caacacggac 840aacggctcgc
cctcgggcaa ccttgtgagc atcacccgca agtaccagca aaacggcgtc 900gacatcccca
gcgcccagcc cggcggcgac accatctcgt cctgcccgtc cgcctcagcc 960tacggcggcc
tcgccaccat gggcaaggcc ctgagcagcg gcatggtgct cgtgttcagc 1020atttggaacg
acaacagcca gtacatgaac tggctcgaca gcggcaacgc cggcccctgc 1080agcagcaccg
agggcaaccc atccaacatc ctggccaaca accccaacac gcacgtcgtc 1140ttctccaaca
tccgctgggg agacattggg tctactacga actcgactgc gcccccgccc 1200ccgcctgcgt
ccagcacgac gttttcgact acacggagga gctcgacgac ttcgagcagc 1260ccgagctgca
cgcagactca ctgggggcag tgcggtggca ttgggtacag cgggtgcaag 1320acgtgcacgt
cgggcactac gtgccagtat agcaacgact actactcgca atgcctttag
138094459PRTTrichoderma reesei 94Met Ala Pro Ser Val Thr Leu Pro Leu Thr
Thr Ala Ile Leu Ala Ile1 5 10
15Ala Arg Leu Val Ala Ala Gln Gln Pro Gly Thr Ser Thr Pro Glu Val
20 25 30His Pro Lys Leu Thr Thr
Tyr Lys Cys Thr Lys Ser Gly Gly Cys Val 35 40
45Ala Gln Asp Thr Ser Val Val Leu Asp Trp Asn Tyr Arg Trp
Met His 50 55 60Asp Ala Asn Tyr Asn
Ser Cys Thr Val Asn Gly Gly Val Asn Thr Thr65 70
75 80Leu Cys Pro Asp Glu Ala Thr Cys Gly Lys
Asn Cys Phe Ile Glu Gly 85 90
95Val Asp Tyr Ala Ala Ser Gly Val Thr Thr Ser Gly Ser Ser Leu Thr
100 105 110Met Asn Gln Tyr Met
Pro Ser Ser Ser Gly Gly Tyr Ser Ser Val Ser 115
120 125Pro Arg Leu Tyr Leu Leu Asp Ser Asp Gly Glu Tyr
Val Met Leu Lys 130 135 140Leu Asn Gly
Gln Glu Leu Ser Phe Asp Val Asp Leu Ser Ala Leu Pro145
150 155 160Cys Gly Glu Asn Gly Ser Leu
Tyr Leu Ser Gln Met Asp Glu Asn Gly 165
170 175Gly Ala Asn Gln Tyr Asn Thr Ala Gly Ala Asn Tyr
Gly Ser Gly Tyr 180 185 190Cys
Asp Ala Gln Cys Pro Val Gln Thr Trp Arg Asn Gly Thr Leu Asn 195
200 205Thr Ser His Gln Gly Phe Cys Cys Asn
Glu Met Asp Ile Leu Glu Gly 210 215
220Asn Ser Arg Ala Asn Ala Leu Thr Pro His Ser Cys Thr Ala Thr Ala225
230 235 240Cys Asp Ser Ala
Gly Cys Gly Phe Asn Pro Tyr Gly Ser Gly Tyr Lys 245
250 255Ser Tyr Tyr Gly Pro Gly Asp Thr Val Asp
Thr Ser Lys Thr Phe Thr 260 265
270Ile Ile Thr Gln Phe Asn Thr Asp Asn Gly Ser Pro Ser Gly Asn Leu
275 280 285Val Ser Ile Thr Arg Lys Tyr
Gln Gln Asn Gly Val Asp Ile Pro Ser 290 295
300Ala Gln Pro Gly Gly Asp Thr Ile Ser Ser Cys Pro Ser Ala Ser
Ala305 310 315 320Tyr Gly
Gly Leu Ala Thr Met Gly Lys Ala Leu Ser Ser Gly Met Val
325 330 335Leu Val Phe Ser Ile Trp Asn
Asp Asn Ser Gln Tyr Met Asn Trp Leu 340 345
350Asp Ser Gly Asn Ala Gly Pro Cys Ser Ser Thr Glu Gly Asn
Pro Ser 355 360 365Asn Ile Leu Ala
Asn Asn Pro Asn Thr His Val Val Phe Ser Asn Ile 370
375 380Arg Trp Gly Asp Ile Gly Ser Thr Thr Asn Ser Thr
Ala Pro Pro Pro385 390 395
400Pro Pro Ala Ser Ser Thr Thr Phe Ser Thr Thr Arg Arg Ser Ser Thr
405 410 415Thr Ser Ser Ser Pro
Ser Cys Thr Gln Thr His Trp Gly Gln Cys Gly 420
425 430Gly Ile Gly Tyr Ser Gly Cys Lys Thr Cys Thr Ser
Gly Thr Thr Cys 435 440 445Gln Tyr
Ser Asn Asp Tyr Tyr Ser Gln Cys Leu 450
455951545DNATrichoderma reesei 95atgtatcgga agttggccgt catctcggcc
ttcttggcca cagctcgtgc tcagtcggcc 60tgcactctcc aatcggagac tcacccgcct
ctgacatggc agaaatgctc gtctggtggc 120acgtgcactc aacagacagg ctccgtggtc
atcgacgcca actggcgctg gactcacgct 180acgaacagca gcacgaactg ctacgatggc
aacacttgga gctcgaccct atgtcctgac 240aacgagacct gcgcgaagaa ctgctgtctg
gacggtgccg cctacgcgtc cacgtacgga 300gttaccacga gcggtaacag cctctccatt
ggctttgtca cccagtctgc gcagaagaac 360gttggcgctc gcctttacct tatggcgagc
gacacgacct accaggaatt caccctgctt 420ggcaacgagt tctctttcga tgttgatgtt
tcgcagctgc cgtgcggctt gaacggagct 480ctctacttcg tgtccatgga cgcggatggt
ggcgtgagca agtatcccac caacaccgct 540ggcgccaagt acggcacggg gtactgtgac
agccagtgtc cccgcgatct gaagttcatc 600aatggccagg ccaacgttga gggctgggag
ccgtcatcca acaacgcgaa cacgggcatt 660ggaggacacg gaagctgctg ctctgagatg
gatatctggg aggccaactc catctccgag 720gctcttaccc cccacccttg cacgactgtc
ggccaggaga tctgcgaggg tgatgggtgc 780ggcggaactt actccgataa cagatatggc
ggcacttgcg atcccgatgg ctgcgactgg 840aacccatacc gcctgggcaa caccagcttc
tacggccctg gctcaagctt taccctcgat 900accaccaaga aattgaccgt tgtcacccag
ttcgagacgt cgggtgccat caaccgatac 960tatgtccaga atggcgtcac tttccagcag
cccaacgccg agcttggtag ttactctggc 1020aacgagctca acgatgatta ctgcacagct
gaggaggcag aattcggcgg atcctctttc 1080tcagacaagg gcggcctgac tcagttcaag
aaggctacct ctggcggcat ggttctggtc 1140atgagtctgt gggatgatta ctacgccaac
atgctgtggc tggactccac ctacccgaca 1200aacgagacct cctccacacc cggtgccgtg
cgcggaagct gctccaccag ctccggtgtc 1260cctgctcagg tcgaatctca gtctcccaac
gccaaggtca ccttctccaa catcaagttc 1320ggacccattg gcagcaccgg caaccctagc
ggcggcaacc ctcccggcgg aaacccgcct 1380ggcaccacca ccacccgccg cccagccact
accactggaa gctctcccgg acctacccag 1440tctcactacg gccagtgcgg cggtattggc
tacagcggcc ccacggtctg cgccagcggc 1500acaacttgcc aggtcctgaa cccttactac
tctcagtgcc tgtaa 154596514PRTTrichoderma reesei 96Met
Tyr Arg Lys Leu Ala Val Ile Ser Ala Phe Leu Ala Thr Ala Arg1
5 10 15Ala Gln Ser Ala Cys Thr Leu
Gln Ser Glu Thr His Pro Pro Leu Thr 20 25
30Trp Gln Lys Cys Ser Ser Gly Gly Thr Cys Thr Gln Gln Thr
Gly Ser 35 40 45Val Val Ile Asp
Ala Asn Trp Arg Trp Thr His Ala Thr Asn Ser Ser 50 55
60Thr Asn Cys Tyr Asp Gly Asn Thr Trp Ser Ser Thr Leu
Cys Pro Asp65 70 75
80Asn Glu Thr Cys Ala Lys Asn Cys Cys Leu Asp Gly Ala Ala Tyr Ala
85 90 95Ser Thr Tyr Gly Val Thr
Thr Ser Gly Asn Ser Leu Ser Ile Gly Phe 100
105 110Val Thr Gln Ser Ala Gln Lys Asn Val Gly Ala Arg
Leu Tyr Leu Met 115 120 125Ala Ser
Asp Thr Thr Tyr Gln Glu Phe Thr Leu Leu Gly Asn Glu Phe 130
135 140Ser Phe Asp Val Asp Val Ser Gln Leu Pro Cys
Gly Leu Asn Gly Ala145 150 155
160Leu Tyr Phe Val Ser Met Asp Ala Asp Gly Gly Val Ser Lys Tyr Pro
165 170 175Thr Asn Thr Ala
Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln 180
185 190Cys Pro Arg Asp Leu Lys Phe Ile Asn Gly Gln
Ala Asn Val Glu Gly 195 200 205Trp
Glu Pro Ser Ser Asn Asn Ala Asn Thr Gly Ile Gly Gly His Gly 210
215 220Ser Cys Cys Ser Glu Met Asp Ile Trp Glu
Ala Asn Ser Ile Ser Glu225 230 235
240Ala Leu Thr Pro His Pro Cys Thr Thr Val Gly Gln Glu Ile Cys
Glu 245 250 255Gly Asp Gly
Cys Gly Gly Thr Tyr Ser Asp Asn Arg Tyr Gly Gly Thr 260
265 270Cys Asp Pro Asp Gly Cys Asp Trp Asn Pro
Tyr Arg Leu Gly Asn Thr 275 280
285Ser Phe Tyr Gly Pro Gly Ser Ser Phe Thr Leu Asp Thr Thr Lys Lys 290
295 300Leu Thr Val Val Thr Gln Phe Glu
Thr Ser Gly Ala Ile Asn Arg Tyr305 310
315 320Tyr Val Gln Asn Gly Val Thr Phe Gln Gln Pro Asn
Ala Glu Leu Gly 325 330
335Ser Tyr Ser Gly Asn Glu Leu Asn Asp Asp Tyr Cys Thr Ala Glu Glu
340 345 350Ala Glu Phe Gly Gly Ser
Ser Phe Ser Asp Lys Gly Gly Leu Thr Gln 355 360
365Phe Lys Lys Ala Thr Ser Gly Gly Met Val Leu Val Met Ser
Leu Trp 370 375 380Asp Asp Tyr Tyr Ala
Asn Met Leu Trp Leu Asp Ser Thr Tyr Pro Thr385 390
395 400Asn Glu Thr Ser Ser Thr Pro Gly Ala Val
Arg Gly Ser Cys Ser Thr 405 410
415Ser Ser Gly Val Pro Ala Gln Val Glu Ser Gln Ser Pro Asn Ala Lys
420 425 430Val Thr Phe Ser Asn
Ile Lys Phe Gly Pro Ile Gly Ser Thr Gly Asn 435
440 445Pro Ser Gly Gly Asn Pro Pro Gly Gly Asn Pro Pro
Gly Thr Thr Thr 450 455 460Thr Arg Arg
Pro Ala Thr Thr Thr Gly Ser Ser Pro Gly Pro Thr Gln465
470 475 480Ser His Tyr Gly Gln Cys Gly
Gly Ile Gly Tyr Ser Gly Pro Thr Val 485
490 495Cys Ala Ser Gly Thr Thr Cys Gln Val Leu Asn Pro
Tyr Tyr Ser Gln 500 505 510Cys
Leu971611DNATrichoderma reesei 97atgattgtcg gcattctcac cacgctggct
acgctggcca cactcgcagc tagtgtgcct 60ctagaggagc ggcaagcttg ctcaagcgtc
tggtaattat gtgaaccctc tcaagagacc 120caaatactga gatatgtcaa ggggccaatg
tggtggccag aattggtcgg gtccgacttg 180ctgtgcttcc ggaagcacat gcgtctactc
caacgactat tactcccagt gtcttcccgg 240cgctgcaagc tcaagctcgt ccacgcgcgc
cgcgtcgacg acttctcgag tatcccccac 300aacatcccgg tcgagctccg cgacgcctcc
acctggttct actactacca gagtacctcc 360agtcggatcg ggaaccgcta cgtattcagg
caaccctttt gttggggtca ctccttgggc 420caatgcatat tacgcctctg aagttagcag
cctcgctatt cctagcttga ctggagccat 480ggccactgct gcagcagctg tcgcaaaggt
tccctctttt atgtggctgt aggtcctccc 540ggaaccaagg caatctgtta ctgaaggctc
atcattcact gcagagatac tcttgacaag 600acccctctca tggagcaaac cttggccgac
atccgcaccg ccaacaagaa tggcggtaac 660tatgccggac agtttgtggt gtatgacttg
ccggatcgcg attgcgctgc ccttgcctcg 720aatggcgaat actctattgc cgatggtggc
gtcgccaaat ataagaacta tatcgacacc 780attcgtcaaa ttgtcgtgga atattccgat
atccggaccc tcctggttat tggtatgagt 840ttaaacacct gcctcccccc ccccttccct
tcctttcccg ccggcatctt gtcgttgtgc 900taactattgt tccctcttcc agagcctgac
tctcttgcca acctggtgac caacctcggt 960actccaaagt gtgccaatgc tcagtcagcc
taccttgagt gcatcaacta cgccgtcaca 1020cagctgaacc ttccaaatgt tgcgatgtat
ttggacgctg gccatgcagg atggcttggc 1080tggccggcaa accaagaccc ggccgctcag
ctatttgcaa atgtttacaa gaatgcatcg 1140tctccgagag ctcttcgcgg attggcaacc
aatgtcgcca actacaacgg gtggaacatt 1200accagccccc catcgtacac gcaaggcaac
gctgtctaca acgagaagct gtacatccac 1260gctattggac gtcttcttgc caatcacggc
tggtccaacg ccttcttcat cactgatcaa 1320ggtcgatcgg gaaagcagcc taccggacag
caacagtggg gagactggtg caatgtgatc 1380ggcaccggat ttggtattcg cccatccgca
aacactgggg actcgttgct ggattcgttt 1440gtctgggtca agccaggcgg cgagtgtgac
ggcaccagcg acagcagtgc gccacgattt 1500gactcccact gtgcgctccc agatgccttg
caaccggcgc ctcaagctgg tgcttggttc 1560caagcctact ttgtgcagct tctcacaaac
gcaaacccat cgttcctgta a 161198471PRTTrichoderma reesei 98Met
Ile Val Gly Ile Leu Thr Thr Leu Ala Thr Leu Ala Thr Leu Ala1
5 10 15Ala Ser Val Pro Leu Glu Glu
Arg Gln Ala Cys Ser Ser Val Trp Gly 20 25
30Gln Cys Gly Gly Gln Asn Trp Ser Gly Pro Thr Cys Cys Ala
Ser Gly 35 40 45Ser Thr Cys Val
Tyr Ser Asn Asp Tyr Tyr Ser Gln Cys Leu Pro Gly 50 55
60Ala Ala Ser Ser Ser Ser Ser Thr Arg Ala Ala Ser Thr
Thr Ser Arg65 70 75
80Val Ser Pro Thr Thr Ser Arg Ser Ser Ser Ala Thr Pro Pro Pro Gly
85 90 95Ser Thr Thr Thr Arg Val
Pro Pro Val Gly Ser Gly Thr Ala Thr Tyr 100
105 110Ser Gly Asn Pro Phe Val Gly Val Thr Pro Trp Ala
Asn Ala Tyr Tyr 115 120 125Ala Ser
Glu Val Ser Ser Leu Ala Ile Pro Ser Leu Thr Gly Ala Met 130
135 140Ala Thr Ala Ala Ala Ala Val Ala Lys Val Pro
Ser Phe Met Trp Leu145 150 155
160Asp Thr Leu Asp Lys Thr Pro Leu Met Glu Gln Thr Leu Ala Asp Ile
165 170 175Arg Thr Ala Asn
Lys Asn Gly Gly Asn Tyr Ala Gly Gln Phe Val Val 180
185 190Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Leu
Ala Ser Asn Gly Glu 195 200 205Tyr
Ser Ile Ala Asp Gly Gly Val Ala Lys Tyr Lys Asn Tyr Ile Asp 210
215 220Thr Ile Arg Gln Ile Val Val Glu Tyr Ser
Asp Ile Arg Thr Leu Leu225 230 235
240Val Ile Glu Pro Asp Ser Leu Ala Asn Leu Val Thr Asn Leu Gly
Thr 245 250 255Pro Lys Cys
Ala Asn Ala Gln Ser Ala Tyr Leu Glu Cys Ile Asn Tyr 260
265 270Ala Val Thr Gln Leu Asn Leu Pro Asn Val
Ala Met Tyr Leu Asp Ala 275 280
285Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Gln Asp Pro Ala Ala 290
295 300Gln Leu Phe Ala Asn Val Tyr Lys
Asn Ala Ser Ser Pro Arg Ala Leu305 310
315 320Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Gly
Trp Asn Ile Thr 325 330
335Ser Pro Pro Ser Tyr Thr Gln Gly Asn Ala Val Tyr Asn Glu Lys Leu
340 345 350Tyr Ile His Ala Ile Gly
Arg Leu Leu Ala Asn His Gly Trp Ser Asn 355 360
365Ala Phe Phe Ile Thr Asp Gln Gly Arg Ser Gly Lys Gln Pro
Thr Gly 370 375 380Gln Gln Gln Trp Gly
Asp Trp Cys Asn Val Ile Gly Thr Gly Phe Gly385 390
395 400Ile Arg Pro Ser Ala Asn Thr Gly Asp Ser
Leu Leu Asp Ser Phe Val 405 410
415Trp Val Lys Pro Gly Gly Glu Cys Asp Gly Thr Ser Asp Ser Ser Ala
420 425 430Pro Arg Phe Asp Ser
His Cys Ala Leu Pro Asp Ala Leu Gln Pro Ala 435
440 445Pro Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val
Gln Leu Leu Thr 450 455 460Asn Ala Asn
Pro Ser Phe Leu465 470992046DNAHumicola insolens
99gccgtgacct tgcgcgcttt gggtggcggt ggcgagtcgt ggacggtgct tgctggtcgc
60cggccttccc ggcgatccgc gtgatgagag ggccaccaac ggcgggatga tgctccatgg
120ggaacttccc catggagaag agagagaaac ttgcggagcc gtgatctggg gaaagatgct
180ccgtgtctcg tctatataac tcgagtctcc ccgagccctc aacaccacca gctctgatct
240caccatcccc atcgacaatc acgcaaacac agcagttgtc gggccattcc ttcagacaca
300tcagtcaccc tccttcaaaa tgcgtaccgc caagttcgcc accctcgccg cccttgtggc
360ctcggccgcc gcccagcagg cgtgcagtct caccaccgag aggcaccctt ccctctcttg
420gaacaagtgc accgccggcg gccagtgcca gaccgtccag gcttccatca ctctcgactc
480caactggcgc tggactcacc aggtgtctgg ctccaccaac tgctacacgg gcaacaagtg
540ggatactagc atctgcactg atgccaagtc gtgcgctcag aactgctgcg tcgatggtgc
600cgactacacc agcacctatg gcatcaccac caacggtgat tccctgagcc tcaagttcgt
660caccaagggc cagcactcga ccaacgtcgg ctcgcgtacc tacctgatgg acggcgagga
720caagtatcag agtacgttct atcttcagcc ttctcgcgcc ttgaatcctg gctaacgttt
780acacttcaca gccttcgagc tcctcggcaa cgagttcacc ttcgatgtcg atgtctccaa
840catcggctgc ggtctcaacg gcgccctgta cttcgtctcc atggacgccg atggtggtct
900cagccgctat cctggcaaca aggctggtgc caagtacggt accggctact gcgatgctca
960gtgcccccgt gacatcaagt tcatcaacgg cgaggccaac attgagggct ggaccggctc
1020caccaacgac cccaacgccg gcgcgggccg ctatggtacc tgctgctctg agatggatat
1080ctgggaagcc aacaacatgg ctactgcctt cactcctcac ccttgcacca tcattggcca
1140gagccgctgc gagggcgact cgtgcggtgg cacctacagc aacgagcgct acgccggcgt
1200ctgcgacccc gatggctgcg acttcaactc gtaccgccag ggcaacaaga ccttctacgg
1260caagggcatg accgtcgaca ccaccaagaa gatcactgtc gtcacccagt tcctcaagga
1320tgccaacggc gatctcggcg agatcaagcg cttctacgtc caggatggca agatcatccc
1380caactccgag tccaccatcc ccggcgtcga gggcaattcc atcacccagg actggtgcga
1440ccgccagaag gttgcctttg gcgacattga cgacttcaac cgcaagggcg gcatgaagca
1500gatgggcaag gccctcgccg gccccatggt cctggtcatg tccatctggg atgaccacgc
1560ctccaacatg ctctggctcg actcgacctt ccctgtcgat gccgctggca agcccggcgc
1620cgagcgcggt gcctgcccga ccacctcggg tgtccctgct gaggttgagg ccgaggcccc
1680caacagcaac gtcgtcttct ccaacatccg cttcggcccc atcggctcga ccgttgctgg
1740tctccccggc gcgggcaacg gcggcaacaa cggcggcaac cccccgcccc ccaccaccac
1800cacctcctcg gctccggcca ccaccaccac cgccagcgct ggccccaagg ctggccgctg
1860gcagcagtgc ggcggcatcg gcttcactgg cccgacccag tgcgaggagc cctacatttg
1920caccaagctc aacgactggt actctcagtg cctgtaaatt ctgagtcgct gactcgacga
1980tcacggccgg tttttgcatg aaaggaaaca aacgaccgcg ataaaaatgg agggtaatga
2040gatgtc
2046100525PRTHumicola insolens 100Met Arg Thr Ala Lys Phe Ala Thr Leu Ala
Ala Leu Val Ala Ser Ala1 5 10
15Ala Ala Gln Gln Ala Cys Ser Leu Thr Thr Glu Arg His Pro Ser Leu
20 25 30Ser Trp Asn Lys Cys Thr
Ala Gly Gly Gln Cys Gln Thr Val Gln Ala 35 40
45Ser Ile Thr Leu Asp Ser Asn Trp Arg Trp Thr His Gln Val
Ser Gly 50 55 60Ser Thr Asn Cys Tyr
Thr Gly Asn Lys Trp Asp Thr Ser Ile Cys Thr65 70
75 80Asp Ala Lys Ser Cys Ala Gln Asn Cys Cys
Val Asp Gly Ala Asp Tyr 85 90
95Thr Ser Thr Tyr Gly Ile Thr Thr Asn Gly Asp Ser Leu Ser Leu Lys
100 105 110Phe Val Thr Lys Gly
Gln His Ser Thr Asn Val Gly Ser Arg Thr Tyr 115
120 125Leu Met Asp Gly Glu Asp Lys Tyr Gln Thr Phe Glu
Leu Leu Gly Asn 130 135 140Glu Phe Thr
Phe Asp Val Asp Val Ser Asn Ile Gly Cys Gly Leu Asn145
150 155 160Gly Ala Leu Tyr Phe Val Ser
Met Asp Ala Asp Gly Gly Leu Ser Arg 165
170 175Tyr Pro Gly Asn Lys Ala Gly Ala Lys Tyr Gly Thr
Gly Tyr Cys Asp 180 185 190Ala
Gln Cys Pro Arg Asp Ile Lys Phe Ile Asn Gly Glu Ala Asn Ile 195
200 205Glu Gly Trp Thr Gly Ser Thr Asn Asp
Pro Asn Ala Gly Ala Gly Arg 210 215
220Tyr Gly Thr Cys Cys Ser Glu Met Asp Ile Trp Glu Ala Asn Asn Met225
230 235 240Ala Thr Ala Phe
Thr Pro His Pro Cys Thr Ile Ile Gly Gln Ser Arg 245
250 255Cys Glu Gly Asp Ser Cys Gly Gly Thr Tyr
Ser Asn Glu Arg Tyr Ala 260 265
270Gly Val Cys Asp Pro Asp Gly Cys Asp Phe Asn Ser Tyr Arg Gln Gly
275 280 285Asn Lys Thr Phe Tyr Gly Lys
Gly Met Thr Val Asp Thr Thr Lys Lys 290 295
300Ile Thr Val Val Thr Gln Phe Leu Lys Asp Ala Asn Gly Asp Leu
Gly305 310 315 320Glu Ile
Lys Arg Phe Tyr Val Gln Asp Gly Lys Ile Ile Pro Asn Ser
325 330 335Glu Ser Thr Ile Pro Gly Val
Glu Gly Asn Ser Ile Thr Gln Asp Trp 340 345
350Cys Asp Arg Gln Lys Val Ala Phe Gly Asp Ile Asp Asp Phe
Asn Arg 355 360 365Lys Gly Gly Met
Lys Gln Met Gly Lys Ala Leu Ala Gly Pro Met Val 370
375 380Leu Val Met Ser Ile Trp Asp Asp His Ala Ser Asn
Met Leu Trp Leu385 390 395
400Asp Ser Thr Phe Pro Val Asp Ala Ala Gly Lys Pro Gly Ala Glu Arg
405 410 415Gly Ala Cys Pro Thr
Thr Ser Gly Val Pro Ala Glu Val Glu Ala Glu 420
425 430Ala Pro Asn Ser Asn Val Val Phe Ser Asn Ile Arg
Phe Gly Pro Ile 435 440 445Gly Ser
Thr Val Ala Gly Leu Pro Gly Ala Gly Asn Gly Gly Asn Asn 450
455 460Gly Gly Asn Pro Pro Pro Pro Thr Thr Thr Thr
Ser Ser Ala Pro Ala465 470 475
480Thr Thr Thr Thr Ala Ser Ala Gly Pro Lys Ala Gly Arg Trp Gln Gln
485 490 495Cys Gly Gly Ile
Gly Phe Thr Gly Pro Thr Gln Cys Glu Glu Pro Tyr 500
505 510Ile Cys Thr Lys Leu Asn Asp Trp Tyr Ser Gln
Cys Leu 515 520
5251011812DNAMyceliophthora thermophila 101atggccaaga agcttttcat
caccgccgcc cttgcggctg ccgtgttggc ggcccccgtc 60attgaggagc gccagaactg
cggcgctgtg tggtaagaaa gcccggtctg agtttcccat 120gactttctca tcgagtaatg
gcataaggcc caccccttcg actgactgtg agaatcgatc 180aaatccagga ctcaatgcgg
cggcaacggg tggcagggtc ccacatgctg cgcctcgggc 240tcgacctgcg ttgcgcagaa
cgagtggtac tctcagtgcc tgcccaacaa tcaggtgacg 300agttccaaca ctccgtcgtc
gacttccacc tcgcagcgca gcagcagcac ctccagcagc 360agcaccagga gcggcagctc
ctcctcctcc accaccacgc cccctcccgt ctccagcccc 420gtgactagca ttcccggcgg
tgcgaccacc acggcgagct actctggcaa ccccttctcg 480ggcgtccggc tcttcgccaa
cgactactac aggtccgagg tccacaatct cgccattcct 540agcatgaccg gtactctggc
ggccaaggct tccgccgtcg ccgaagtccc tagcttccag 600tggctcgacc ggaacgtcac
catcgacacc ctgatggtcc agactctgtc ccagatccgg 660gctgccaata atgccggtgc
caatcctccc tatgctggtg agttacatgg cggcgacttg 720ccttctcgtc ccccaccttt
cttgacggga tcggttacct gacctggagg caaaacaaaa 780ccagcccaac ttgtcgtcta
cgacctcccc gaccgtgact gcgccgccgc tgcgtccaac 840ggcgagtttt cgattgcaaa
cggcggcgcc gccaactaca ggagctacat cgacgctatc 900cgcaagcaca tcattgagta
ctcggacatc cggatcatcc tggttatcga gcccgactcg 960atggccaaca tggtgaccaa
catgaacgtg gccaagtgca gcaacgccgc gtcgacgtac 1020cacgagttga ccgtgtacgc
gctcaagcag ctgaacctgc ccaacgtcgc catgtatctc 1080gacgccggcc acgccggctg
gctcggctgg cccgccaaca tccagcccgc cgccgacctg 1140tttgccggca tctacaatga
cgccggcaag ccggctgccg tccgcggcct ggccactaac 1200gtcgccaact acaacgcctg
gagtatcgct tcggccccgt cgtacacgtc ccctaaccct 1260aactacgacg agaagcacta
catcgaggcc ttcagcccgc tcctgaacgc ggccggcttc 1320cccgcacgct tcattgtcga
cactggccgc aacggcaaac aacctaccgg tatggttttt 1380ttcttttttt ttctctgttc
ccctccccct tccccttcag ttggcgtcca caaggtctct 1440tagtcttgct tcttctcgga
ccaaccttcc cccaccccca aaacgcaccg cccacaaccg 1500ttcgactcta tactcttggg
aatgggcgcc gaaactgacc gttcgacagg ccaacaacag 1560tggggtgact ggtgcaatgt
caagggcact ggctttggcg tgcgcccgac ggccaacacg 1620ggccacgacc tggtcgatgc
ctttgtctgg gtcaagcccg gcggcgagtc cgacggcaca 1680agcgacacca gcgccgcccg
ctacgactac cactgcggcc tgtccgatgc cctgcagcct 1740gctccggagg ctggacagtg
gttccaggcc tacttcgagc agctgctcac caacgccaac 1800ccgcccttct aa
1812102482PRTMyceliophthora
thermophila 102Met Ala Lys Lys Leu Phe Ile Thr Ala Ala Leu Ala Ala Ala
Val Leu1 5 10 15Ala Ala
Pro Val Ile Glu Glu Arg Gln Asn Cys Gly Ala Val Trp Thr 20
25 30Gln Cys Gly Gly Asn Gly Trp Gln Gly
Pro Thr Cys Cys Ala Ser Gly 35 40
45Ser Thr Cys Val Ala Gln Asn Glu Trp Tyr Ser Gln Cys Leu Pro Asn 50
55 60Asn Gln Val Thr Ser Ser Asn Thr Pro
Ser Ser Thr Ser Thr Ser Gln65 70 75
80Arg Ser Ser Ser Thr Ser Ser Ser Ser Thr Arg Ser Gly Ser
Ser Ser 85 90 95Ser Ser
Thr Thr Thr Pro Pro Pro Val Ser Ser Pro Val Thr Ser Ile 100
105 110Pro Gly Gly Ala Thr Thr Thr Ala Ser
Tyr Ser Gly Asn Pro Phe Ser 115 120
125Gly Val Arg Leu Phe Ala Asn Asp Tyr Tyr Arg Ser Glu Val His Asn
130 135 140Leu Ala Ile Pro Ser Met Thr
Gly Thr Leu Ala Ala Lys Ala Ser Ala145 150
155 160Val Ala Glu Val Pro Ser Phe Gln Trp Leu Asp Arg
Asn Val Thr Ile 165 170
175Asp Thr Leu Met Val Gln Thr Leu Ser Gln Ile Arg Ala Ala Asn Asn
180 185 190Ala Gly Ala Asn Pro Pro
Tyr Ala Ala Gln Leu Val Val Tyr Asp Leu 195 200
205Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn Gly Glu Phe
Ser Ile 210 215 220Ala Asn Gly Gly Ala
Ala Asn Tyr Arg Ser Tyr Ile Asp Ala Ile Arg225 230
235 240Lys His Ile Ile Glu Tyr Ser Asp Ile Arg
Ile Ile Leu Val Ile Glu 245 250
255Pro Asp Ser Met Ala Asn Met Val Thr Asn Met Asn Val Ala Lys Cys
260 265 270Ser Asn Ala Ala Ser
Thr Tyr His Glu Leu Thr Val Tyr Ala Leu Lys 275
280 285Gln Leu Asn Leu Pro Asn Val Ala Met Tyr Leu Asp
Ala Gly His Ala 290 295 300Gly Trp Leu
Gly Trp Pro Ala Asn Ile Gln Pro Ala Ala Asp Leu Phe305
310 315 320Ala Gly Ile Tyr Asn Asp Ala
Gly Lys Pro Ala Ala Val Arg Gly Leu 325
330 335Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser Ile
Ala Ser Ala Pro 340 345 350Ser
Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu Lys His Tyr Ile Glu 355
360 365Ala Phe Ser Pro Leu Leu Asn Ala Ala
Gly Phe Pro Ala Arg Phe Ile 370 375
380Val Asp Thr Gly Arg Asn Gly Lys Gln Pro Thr Gly Gln Gln Gln Trp385
390 395 400Gly Asp Trp Cys
Asn Val Lys Gly Thr Gly Phe Gly Val Arg Pro Thr 405
410 415Ala Asn Thr Gly His Asp Leu Val Asp Ala
Phe Val Trp Val Lys Pro 420 425
430Gly Gly Glu Ser Asp Gly Thr Ser Asp Thr Ser Ala Ala Arg Tyr Asp
435 440 445Tyr His Cys Gly Leu Ser Asp
Ala Leu Gln Pro Ala Pro Glu Ala Gly 450 455
460Gln Trp Phe Gln Ala Tyr Phe Glu Gln Leu Leu Thr Asn Ala Asn
Pro465 470 475 480Pro
Phe1031802DNAMyceliophthora thermophila 103atggccaaga agcttttcat
caccgccgcg cttgcggctg ccgtgttggc ggcccccgtc 60attgaggagc gccagaactg
cggcgctgtg tggtaagaaa gcccggtccg agtctcccat 120gattttctcg tcgagtaatg
gcataagggc caccccttcg actgaccgtg agaatcgatc 180aaatccagga ctcaatgcgg
cggtaacggg tggcaaggtc ccacatgctg cgcctcgggc 240tcgacctgcg ttgcgcagaa
cgagtggtac tctcagtgcc tgcccaacag ccaggtgacg 300agttccacca ctccgtcgtc
gacttccacc tcgcagcgca gcaccagcac ctccagcagc 360accaccagga gcggcagctc
ctcctcctcc tccaccacgc ccccgcccgt ctccagcccc 420gtgaccagca ttcccggcgg
tgcgacctcc acggcgagct actctggcaa ccccttctcg 480ggcgtccggc tcttcgccaa
cgactactac aggtccgagg tccacaatct cgccattcct 540agcatgactg gtactctggc
ggccaaggct tccgccgtcg ccgaagtccc tagcttccag 600tggctcgacc ggaacgtcac
catcgacacc ctgatggtcc agactctgtc ccaggtccgg 660gctctcaata aggccggtgc
caatcctccc tatgctggtg agttacatgg cgacttgcct 720tctcgtcccc tacctttctt
gacgggatcg gttacctgac ctggaggcaa aacaacaaca 780gcccaactcg tcgtctacga
cctccccgac cgtgactgtg ccgccgctgc gtccaacggc 840gagttttcga ttgcaaacgg
cggcgccgcc aactacagga gctacatcga cgctatccgc 900aagcacatca ttgagtactc
ggacatccgg atcatcctgg ttatcgagcc cgactcgatg 960gccaacatgg tgaccaacat
gaacgtggcc aagtgcagca acgccgcgtc gacgtaccac 1020gagttgaccg tgtacgcgct
caagcagctg aacctgccca acgtcgccat gtatctcgac 1080gccggccacg ccggctggct
cggctggccc gccaacatcc agcccgccgc cgagctgttt 1140gccggcatct acaatgatgc
cggcaagccg gctgccgtcc gcggcctggc cactaacgtc 1200gccaactaca acgcctggag
catcgcttcg gccccgtcgt acacgtcgcc taaccctaac 1260tacgacgaga agcactacat
cgaggccttc agcccgctct tgaactcggc cggcttcccc 1320gcacgcttca ttgtcgacac
tggccgcaac ggcaaacaac ctaccggtat gttttttttt 1380cttttgtctc tgtccccccc
ttttctcccc cttcagttgg cgtccacaag gtctcttagt 1440cctgcttcat ctgtgaccaa
cctccccccc cccggcaccg cccacaaccg tttgactcta 1500tactcttggg aatgggcgcc
gaaactgacc gttccacagg ccaacaacag tggggtgact 1560ggtgcaatgt caagggcacc
ggctttggcg tgcgcccgac ggccaacacg ggccacgagc 1620tggtcgatgc ctttgtctgg
gtcaagcccg gcggcgagtc cgacggcaca agcgacacca 1680gcgccgcccg ctacgactac
cactgcggcc tgtccgatgc cctgcagcct gcccccgagg 1740ctggacagtg gttccaggcc
tacttcgagc agctgctcac caacgccaac ccgcccttct 1800aa
1802104481PRTMyceliophthora
thermophila 104Met Ala Lys Lys Leu Phe Ile Thr Ala Ala Leu Ala Ala Ala
Val Leu1 5 10 15Ala Ala
Pro Val Ile Glu Glu Arg Gln Asn Cys Gly Ala Val Trp Thr 20
25 30Gln Cys Gly Gly Asn Gly Trp Gln Gly
Pro Thr Cys Cys Ala Ser Gly 35 40
45Ser Thr Cys Val Ala Gln Asn Glu Trp Tyr Ser Gln Cys Leu Pro Asn 50
55 60Ser Gln Val Thr Ser Ser Thr Thr Pro
Ser Ser Thr Ser Thr Ser Gln65 70 75
80Arg Ser Thr Ser Thr Ser Ser Ser Thr Thr Arg Ser Gly Ser
Ser Ser 85 90 95Ser Ser
Ser Thr Thr Pro Pro Pro Val Ser Ser Pro Val Thr Ser Ile 100
105 110Pro Gly Gly Ala Thr Ser Thr Ala Ser
Tyr Ser Gly Asn Pro Phe Ser 115 120
125Gly Val Arg Leu Phe Ala Asn Asp Tyr Tyr Arg Ser Glu Val His Asn
130 135 140Leu Ala Ile Pro Ser Met Thr
Gly Thr Leu Ala Ala Lys Ala Ser Ala145 150
155 160Val Ala Glu Val Pro Ser Phe Gln Trp Leu Asp Arg
Asn Val Thr Ile 165 170
175Asp Thr Leu Met Val Gln Thr Leu Ser Gln Val Arg Ala Leu Asn Lys
180 185 190Ala Gly Ala Asn Pro Pro
Tyr Ala Ala Gln Leu Val Val Tyr Asp Leu 195 200
205Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn Gly Glu Phe
Ser Ile 210 215 220Ala Asn Gly Gly Ala
Ala Asn Tyr Arg Ser Tyr Ile Asp Ala Ile Arg225 230
235 240Lys His Ile Ile Glu Tyr Ser Asp Ile Arg
Ile Ile Leu Val Ile Glu 245 250
255Pro Asp Ser Met Ala Asn Met Val Thr Asn Met Asn Val Ala Lys Cys
260 265 270Ser Asn Ala Ala Ser
Thr Tyr His Glu Leu Thr Val Tyr Ala Leu Lys 275
280 285Gln Leu Asn Leu Pro Asn Val Ala Met Tyr Leu Asp
Ala Gly His Ala 290 295 300Gly Trp Leu
Gly Trp Pro Ala Asn Ile Gln Pro Ala Ala Glu Leu Phe305
310 315 320Ala Gly Ile Tyr Asn Asp Ala
Gly Lys Pro Ala Ala Val Arg Gly Leu 325
330 335Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser Ile
Ala Ser Ala Pro 340 345 350Ser
Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu Lys His Tyr Ile Glu 355
360 365Ala Phe Ser Pro Leu Leu Asn Ser Ala
Gly Phe Pro Ala Arg Phe Ile 370 375
380Val Asp Thr Gly Arg Asn Gly Lys Gln Pro Thr Gly Gln Gln Gln Trp385
390 395 400Gly Asp Trp Cys
Asn Val Lys Gly Thr Gly Phe Gly Val Arg Pro Thr 405
410 415Ala Asn Thr Gly His Glu Leu Val Asp Ala
Phe Val Trp Val Lys Pro 420 425
430Gly Gly Glu Ser Asp Gly Thr Ser Asp Thr Ser Ala Ala Arg Tyr Asp
435 440 445Tyr His Cys Gly Leu Ser Asp
Ala Leu Gln Pro Ala Pro Glu Ala Gly 450 455
460Gln Trp Phe Gln Ala Tyr Phe Glu Gln Leu Leu Thr Asn Ala Asn
Pro465 470 475
480Pro1051446DNAThielavia terrestris 105atggctcaga agctccttct cgccgccgcc
cttgcggcca gcgccctcgc tgctcccgtc 60gtcgaggagc gccagaactg cggttccgtc
tggagccaat gcggcggcat tggctggtcc 120ggcgcgacct gctgcgcttc gggcaatacc
tgcgttgagc tgaacccgta ctactcgcag 180tgcctgccca acagccaggt gactacctcg
accagcaaga ccacctccac caccaccagg 240agcagcacca ccagccacag cagcggtccc
accagcacga gcaccaccac caccagcagt 300cccgtggtca ctaccccgcc gagtacctcc
atccccggcg gtgcctcgtc aacggccagc 360tggtccggca acccgttctc gggcgtgcag
atgtgggcca acgactacta cgcctccgag 420gtctcgtcgc tggccatccc cagcatgacg
ggcgccatgg ccaccaaggc ggccgaggtg 480gccaaggtgc ccagcttcca gtggcttgac
cgcaacgtca ccatcgacac gctgttcgcc 540cacacgctgt cgcagatccg cgcggccaac
cagaaaggcg ccaacccgcc ctacgcgggc 600atcttcgtgg tctacgacct tccggaccgc
gactgcgccg ccgccgcgtc caacggcgag 660ttctccatcg cgaacaacgg ggcggccaac
tacaagacgt acatcgacgc gatccggagc 720ctcgtcatcc agtactcaga catccgcatc
atcttcgtca tcgagcccga ctcgctggcc 780aacatggtga ccaacctgaa cgtggccaag
tgcgccaacg ccgagtcgac ctacaaggag 840ttgaccgtct acgcgctgca gcagctgaac
ctgcccaacg tggccatgta cctggacgcc 900ggccacgccg gctggctcgg ctggcccgcc
aacatccagc cggccgccaa cctcttcgcc 960gagatctaca cgagcgccgg caagccggcc
gccgtgcgcg gcctcgccac caacgtggcc 1020aactacaacg gctggagcct ggccacgccg
ccctcgtaca cccagggcga ccccaactac 1080gacgagagcc actacgtcca ggccctcgcc
ccgctgctca ccgccaacgg cttccccgcc 1140cacttcatca ccgacaccgg ccgcaacggc
aagcagccga ccggacaacg gcaatgggga 1200gactggtgca acgttatcgg aactggcttc
ggcgtgcgcc cgacgacaaa caccggcctc 1260gacatcgagg acgccttcgt ctgggtcaag
cccggcggcg agtgcgacgg cacgagcaac 1320acgacctctc cccgctacga ctaccactgc
ggcctgtcgg acgcgctgca gcctgctccg 1380gaggccggca cttggttcca ggcctacttc
gagcagctcc tgaccaacgc caacccgccc 1440ttttaa
1446106481PRTThielavia terrestris 106Met
Ala Gln Lys Leu Leu Leu Ala Ala Ala Leu Ala Ala Ser Ala Leu1
5 10 15Ala Ala Pro Val Val Glu Glu
Arg Gln Asn Cys Gly Ser Val Trp Ser 20 25
30Gln Cys Gly Gly Ile Gly Trp Ser Gly Ala Thr Cys Cys Ala
Ser Gly 35 40 45Asn Thr Cys Val
Glu Leu Asn Pro Tyr Tyr Ser Gln Cys Leu Pro Asn 50 55
60Ser Gln Val Thr Thr Ser Thr Ser Lys Thr Thr Ser Thr
Thr Thr Arg65 70 75
80Ser Ser Thr Thr Ser His Ser Ser Gly Pro Thr Ser Thr Ser Thr Thr
85 90 95Thr Thr Ser Ser Pro Val
Val Thr Thr Pro Pro Ser Thr Ser Ile Pro 100
105 110Gly Gly Ala Ser Ser Thr Ala Ser Trp Ser Gly Asn
Pro Phe Ser Gly 115 120 125Val Gln
Met Trp Ala Asn Asp Tyr Tyr Ala Ser Glu Val Ser Ser Leu 130
135 140Ala Ile Pro Ser Met Thr Gly Ala Met Ala Thr
Lys Ala Ala Glu Val145 150 155
160Ala Lys Val Pro Ser Phe Gln Trp Leu Asp Arg Asn Val Thr Ile Asp
165 170 175Thr Leu Phe Ala
His Thr Leu Ser Gln Ile Arg Ala Ala Asn Gln Lys 180
185 190Gly Ala Asn Pro Pro Tyr Ala Gly Ile Phe Val
Val Tyr Asp Leu Pro 195 200 205Asp
Arg Asp Cys Ala Ala Ala Ala Ser Asn Gly Glu Phe Ser Ile Ala 210
215 220Asn Asn Gly Ala Ala Asn Tyr Lys Thr Tyr
Ile Asp Ala Ile Arg Ser225 230 235
240Leu Val Ile Gln Tyr Ser Asp Ile Arg Ile Ile Phe Val Ile Glu
Pro 245 250 255Asp Ser Leu
Ala Asn Met Val Thr Asn Leu Asn Val Ala Lys Cys Ala 260
265 270Asn Ala Glu Ser Thr Tyr Lys Glu Leu Thr
Val Tyr Ala Leu Gln Gln 275 280
285Leu Asn Leu Pro Asn Val Ala Met Tyr Leu Asp Ala Gly His Ala Gly 290
295 300Trp Leu Gly Trp Pro Ala Asn Ile
Gln Pro Ala Ala Asn Leu Phe Ala305 310
315 320Glu Ile Tyr Thr Ser Ala Gly Lys Pro Ala Ala Val
Arg Gly Leu Ala 325 330
335Thr Asn Val Ala Asn Tyr Asn Gly Trp Ser Leu Ala Thr Pro Pro Ser
340 345 350Tyr Thr Gln Gly Asp Pro
Asn Tyr Asp Glu Ser His Tyr Val Gln Ala 355 360
365Leu Ala Pro Leu Leu Thr Ala Asn Gly Phe Pro Ala His Phe
Ile Thr 370 375 380Asp Thr Gly Arg Asn
Gly Lys Gln Pro Thr Gly Gln Arg Gln Trp Gly385 390
395 400Asp Trp Cys Asn Val Ile Gly Thr Gly Phe
Gly Val Arg Pro Thr Thr 405 410
415Asn Thr Gly Leu Asp Ile Glu Asp Ala Phe Val Trp Val Lys Pro Gly
420 425 430Gly Glu Cys Asp Gly
Thr Ser Asn Thr Thr Ser Pro Arg Tyr Asp Tyr 435
440 445His Cys Gly Leu Ser Asp Ala Leu Gln Pro Ala Pro
Glu Ala Gly Thr 450 455 460Trp Phe Gln
Ala Tyr Phe Glu Gln Leu Leu Thr Asn Ala Asn Pro Pro465
470 475 480Phe1071593DNAChaetomium
thermophilum 107atgatgtaca agaagttcgc cgctctcgcc gccctcgtgg ctggcgccgc
cgcccagcag 60gcttgctccc tcaccactga gacccacccc agactcactt ggaagcgctg
cacctctggc 120ggcaactgct cgaccgtgaa cggcgccgtc accatcgatg ccaactggcg
ctggactcac 180actgtttccg gctcgaccaa ctgctacacc ggcaacgagt gggatacctc
catctgctct 240gatggcaaga gctgcgccca gacctgctgc gtcgacggcg ctgactactc
ttcgacctat 300ggtatcacca ccagcggtga ctccctgaac ctcaagttcg tcaccaagca
ccagcacggc 360accaatgtcg gctctcgtgt ctacctgatg gagaacgaca ccaagtacca
gatgttcgag 420ctcctcggca acgagttcac cttcgatgtc gatgtctcta acctgggctg
cggtctcaac 480ggcgccctct acttcgtctc catggacgct gatggtggta tgagcaagta
ctctggcaac 540aaggctggcg ccaagtacgg taccggctac tgcgatgctc agtgcccgcg
cgaccttaag 600ttcatcaacg gcgaggccaa cattgagaac tggacccctt cgaccaatga
tgccaacgcc 660ggtttcggcc gctatggcag ctgctgctct gagatggata tctgggatgc
caacaacatg 720gctactgcct tcactcctca cccttgcacc attatcggcc agagccgctg
cgagggcaac 780agctgcggtg gcacctacag ctctgagcgc tatgctggtg tttgcgatcc
tgatggctgc 840gacttcaacg cctaccgcca gggcgacaag accttctacg gcaagggcat
gaccgtcgac 900accaccaaga agatgaccgt cgtcacccag ttccacaaga actcggctgg
cgtcctcagc 960gagatcaagc gcttctacgt tcaggacggc aagatcattg ccaacgccga
gtccaagatc 1020cccggcaacc ccggcaactc catcacccag gagtggtgcg atgcccagaa
ggtcgccttc 1080ggtgacatcg atgacttcaa ccgcaagggc ggtatggctc agatgagcaa
ggccctcgag 1140ggccctatgg tcctggtcat gtccgtctgg gatgaccact acgccaacat
gctctggctc 1200gactcgacct accccattga caaggccggc acccccggcg ccgagcgcgg
tgcttgcccg 1260accacctccg gtgtccctgc cgagattgag gcccaggtcc ccaacagcaa
cgttatcttc 1320tccaacatcc gcttcggccc catcggctcg accgtccctg gcctcgacgg
cagcaccccc 1380agcaacccga ccgccaccgt tgctcctccc acttctacca ccaccagcgt
gagaagcagc 1440actactcaga tttccacccc gactagccag cccggcggct gcaccaccca
gaagtggggc 1500cagtgcggtg gtatcggcta caccggctgc actaactgcg ttgctggcac
tacctgcact 1560gagctcaacc cctggtacag ccagtgcctg taa
1593108530PRTChaetomium thermophilum 108Met Met Tyr Lys Lys
Phe Ala Ala Leu Ala Ala Leu Val Ala Gly Ala1 5
10 15Ala Ala Gln Gln Ala Cys Ser Leu Thr Thr Glu
Thr His Pro Arg Leu 20 25
30Thr Trp Lys Arg Cys Thr Ser Gly Gly Asn Cys Ser Thr Val Asn Gly
35 40 45Ala Val Thr Ile Asp Ala Asn Trp
Arg Trp Thr His Thr Val Ser Gly 50 55
60Ser Thr Asn Cys Tyr Thr Gly Asn Glu Trp Asp Thr Ser Ile Cys Ser65
70 75 80Asp Gly Lys Ser Cys
Ala Gln Thr Cys Cys Val Asp Gly Ala Asp Tyr 85
90 95Ser Ser Thr Tyr Gly Ile Thr Thr Ser Gly Asp
Ser Leu Asn Leu Lys 100 105
110Phe Val Thr Lys His Gln His Gly Thr Asn Val Gly Ser Arg Val Tyr
115 120 125Leu Met Glu Asn Asp Thr Lys
Tyr Gln Met Phe Glu Leu Leu Gly Asn 130 135
140Glu Phe Thr Phe Asp Val Asp Val Ser Asn Leu Gly Cys Gly Leu
Asn145 150 155 160Gly Ala
Leu Tyr Phe Val Ser Met Asp Ala Asp Gly Gly Met Ser Lys
165 170 175Tyr Ser Gly Asn Lys Ala Gly
Ala Lys Tyr Gly Thr Gly Tyr Cys Asp 180 185
190Ala Gln Cys Pro Arg Asp Leu Lys Phe Ile Asn Gly Glu Ala
Asn Ile 195 200 205Glu Asn Trp Thr
Pro Ser Thr Asn Asp Ala Asn Ala Gly Phe Gly Arg 210
215 220Tyr Gly Ser Cys Cys Ser Glu Met Asp Ile Trp Asp
Ala Asn Asn Met225 230 235
240Ala Thr Ala Phe Thr Pro His Pro Cys Thr Ile Ile Gly Gln Ser Arg
245 250 255Cys Glu Gly Asn Ser
Cys Gly Gly Thr Tyr Ser Ser Glu Arg Tyr Ala 260
265 270Gly Val Cys Asp Pro Asp Gly Cys Asp Phe Asn Ala
Tyr Arg Gln Gly 275 280 285Asp Lys
Thr Phe Tyr Gly Lys Gly Met Thr Val Asp Thr Thr Lys Lys 290
295 300Met Thr Val Val Thr Gln Phe His Lys Asn Ser
Ala Gly Val Leu Ser305 310 315
320Glu Ile Lys Arg Phe Tyr Val Gln Asp Gly Lys Ile Ile Ala Asn Ala
325 330 335Glu Ser Lys Ile
Pro Gly Asn Pro Gly Asn Ser Ile Thr Gln Glu Trp 340
345 350Cys Asp Ala Gln Lys Val Ala Phe Gly Asp Ile
Asp Asp Phe Asn Arg 355 360 365Lys
Gly Gly Met Ala Gln Met Ser Lys Ala Leu Glu Gly Pro Met Val 370
375 380Leu Val Met Ser Val Trp Asp Asp His Tyr
Ala Asn Met Leu Trp Leu385 390 395
400Asp Ser Thr Tyr Pro Ile Asp Lys Ala Gly Thr Pro Gly Ala Glu
Arg 405 410 415Gly Ala Cys
Pro Thr Thr Ser Gly Val Pro Ala Glu Ile Glu Ala Gln 420
425 430Val Pro Asn Ser Asn Val Ile Phe Ser Asn
Ile Arg Phe Gly Pro Ile 435 440
445Gly Ser Thr Val Pro Gly Leu Asp Gly Ser Thr Pro Ser Asn Pro Thr 450
455 460Ala Thr Val Ala Pro Pro Thr Ser
Thr Thr Thr Ser Val Arg Ser Ser465 470
475 480Thr Thr Gln Ile Ser Thr Pro Thr Ser Gln Pro Gly
Gly Cys Thr Thr 485 490
495Gln Lys Trp Gly Gln Cys Gly Gly Ile Gly Tyr Thr Gly Cys Thr Asn
500 505 510Cys Val Ala Gly Thr Thr
Cys Thr Glu Leu Asn Pro Trp Tyr Ser Gln 515 520
525Cys Leu 5301091434DNAChaetomium thermophilum
109atggctaagc agctgctgct cactgccgct cttgcggcca cttcgctggc tgcccctctc
60cttgaggagc gccagagctg ctcctccgtc tggggtcaat gcggtggcat caattacaac
120ggcccgacct gctgccagtc cggcagtgtt tgcacttacc tgaatgactg gtacagccag
180tgcattcccg gtcaggctca gcccggcacg actagcacca cggctcggac caccagcacc
240agcaccacca gcacttcgtc ggtccgcccg accacctcga atacccctgt gacgactgct
300cccccgacga ccaccatccc gggcggcgcc tcgagcacgg ccagctacaa cggcaacccg
360ttttcgggtg ttcaactttg ggccaacacc tactactcgt ccgaggtgca cactttggcc
420atccccagct tgtctcctga gctggctgcc aaggccgcca aggtcgctga ggttcccagc
480ttccagtggc tcgaccgcaa tgtgactgtt gacactctct tctccggcac tcttgccgaa
540atccgcgccg ccaaccagcg cggtgccaac ccgccttatg ccggcatttt cgtggtttat
600gacttaccag accgtgattg cgcggctgct gcttcgaacg gcgagtggtc tatcgccaac
660aatggtgcca acaactacaa gcgctacatc gaccggatcc gtgagctcct tatccagtac
720tccgatatcc gcactattct ggtcattgaa cctgattccc tggccaacat ggtcaccaac
780atgaacgtcc agaagtgctc gaacgctgcc tccacttaca aggagcttac tgtctatgcc
840ctcaaacagc tcaatcttcc tcacgttgcc atgtacatgg atgctggcca cgctggctgg
900cttggctggc ccgccaacat ccagcctgct gctgagctct ttgctcaaat ctaccgcgac
960gctggcaggc ccgctgctgt ccgcggtctt gcgaccaacg ttgccaacta caatgcttgg
1020tcgatcgcca gccctccgtc ctacacctct cctaacccga actacgacga gaagcactat
1080attgaggcct ttgctcctct tctccgcaac cagggcttcg acgcaaagtt catcgtcgac
1140accggccgta acggcaagca gcccactggc cagcttgaat ggggtcactg gtgcaatgtc
1200aagggaactg gcttcggtgt gcgccctact gctaacactg ggcatgaact tgttgatgct
1260ttcgtgtggg tcaagcccgg tggcgagtcc gacggcacca gtgcggacac cagcgctgct
1320cgttatgact atcactgcgg cctttccgac gcactgactc cggcgcctga ggctggccaa
1380tggttccagg cttatttcga acagctgctc atcaatgcca accctccgct ctga
1434110477PRTChaetomium thermophilum 110Met Ala Lys Gln Leu Leu Leu Thr
Ala Ala Leu Ala Ala Thr Ser Leu1 5 10
15Ala Ala Pro Leu Leu Glu Glu Arg Gln Ser Cys Ser Ser Val
Trp Gly 20 25 30Gln Cys Gly
Gly Ile Asn Tyr Asn Gly Pro Thr Cys Cys Gln Ser Gly 35
40 45Ser Val Cys Thr Tyr Leu Asn Asp Trp Tyr Ser
Gln Cys Ile Pro Gly 50 55 60Gln Ala
Gln Pro Gly Thr Thr Ser Thr Thr Ala Arg Thr Thr Ser Thr65
70 75 80Ser Thr Thr Ser Thr Ser Ser
Val Arg Pro Thr Thr Ser Asn Thr Pro 85 90
95Val Thr Thr Ala Pro Pro Thr Thr Thr Ile Pro Gly Gly
Ala Ser Ser 100 105 110Thr Ala
Ser Tyr Asn Gly Asn Pro Phe Ser Gly Val Gln Leu Trp Ala 115
120 125Asn Thr Tyr Tyr Ser Ser Glu Val His Thr
Leu Ala Ile Pro Ser Leu 130 135 140Ser
Pro Glu Leu Ala Ala Lys Ala Ala Lys Val Ala Glu Val Pro Ser145
150 155 160Phe Gln Trp Leu Asp Arg
Asn Val Thr Val Asp Thr Leu Phe Ser Gly 165
170 175Thr Leu Ala Glu Ile Arg Ala Ala Asn Gln Arg Gly
Ala Asn Pro Pro 180 185 190Tyr
Ala Gly Ile Phe Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala 195
200 205Ala Ala Ala Ser Asn Gly Glu Trp Ser
Ile Ala Asn Asn Gly Ala Asn 210 215
220Asn Tyr Lys Arg Tyr Ile Asp Arg Ile Arg Glu Leu Leu Ile Gln Tyr225
230 235 240Ser Asp Ile Arg
Thr Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn 245
250 255Met Val Thr Asn Met Asn Val Gln Lys Cys
Ser Asn Ala Ala Ser Thr 260 265
270Tyr Lys Glu Leu Thr Val Tyr Ala Leu Lys Gln Leu Asn Leu Pro His
275 280 285Val Ala Met Tyr Met Asp Ala
Gly His Ala Gly Trp Leu Gly Trp Pro 290 295
300Ala Asn Ile Gln Pro Ala Ala Glu Leu Phe Ala Gln Ile Tyr Arg
Asp305 310 315 320Ala Gly
Arg Pro Ala Ala Val Arg Gly Leu Ala Thr Asn Val Ala Asn
325 330 335Tyr Asn Ala Trp Ser Ile Ala
Ser Pro Pro Ser Tyr Thr Ser Pro Asn 340 345
350Pro Asn Tyr Asp Glu Lys His Tyr Ile Glu Ala Phe Ala Pro
Leu Leu 355 360 365Arg Asn Gln Gly
Phe Asp Ala Lys Phe Ile Val Asp Thr Gly Arg Asn 370
375 380Gly Lys Gln Pro Thr Gly Gln Leu Glu Trp Gly His
Trp Cys Asn Val385 390 395
400Lys Gly Thr Gly Phe Gly Val Arg Pro Thr Ala Asn Thr Gly His Glu
405 410 415Leu Val Asp Ala Phe
Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly 420
425 430Thr Ser Ala Asp Thr Ser Ala Ala Arg Tyr Asp Tyr
His Cys Gly Leu 435 440 445Ser Asp
Ala Leu Thr Pro Ala Pro Glu Ala Gly Gln Trp Phe Gln Ala 450
455 460Tyr Phe Glu Gln Leu Leu Ile Asn Ala Asn Pro
Pro Leu465 470 4751112586DNAAspergillus
oryzae 111atgaagcttg gttggatcga ggtggccgca ttggcggctg cctcagtagt
cagtgccaag 60gatgatctcg cgtactcccc tcctttctac ccttccccat gggcagatgg
tcagggtgaa 120tgggcggaag tatacaaacg cgctgtagac atagtttccc agatgacgtt
gacagagaaa 180gtcaacttaa cgactggaac aggatggcaa ctagagaggt gtgttggaca
aactggcagt 240gttcccagac tcaacatccc cagcttgtgt ttgcaggata gtcctcttgg
tattcgtttc 300tcggactaca attcagcttt ccctgcgggt gttaatgtcg ctgccacctg
ggacaagacg 360ctcgcctacc ttcgtggtca ggcaatgggt gaggagttca gtgataaggg
tattgacgtt 420cagctgggtc ctgctgctgg ccctctcggt gctcatccgg atggcggtag
aaactgggaa 480ggtttctcac cagatccagc cctcaccggt gtactttttg cggagacgat
taagggtatt 540caagatgctg gtgtcattgc gacagctaag cattatatca tgaacgaaca
agagcatttc 600cgccaacaac ccgaggctgc gggttacgga ttcaacgtaa gcgacagttt
gagttccaac 660gttgatgaca agactatgca tgaattgtac ctctggccct tcgcggatgc
agtacgcgct 720ggagtcggtg ctgtcatgtg ctcttacaac caaatcaaca acagctacgg
ttgcgagaat 780agcgaaactc tgaacaagct tttgaaggcg gagcttggtt tccaaggctt
cgtcatgagt 840gattggaccg ctcatcacag cggcgtaggc gctgctttag caggtctgga
tatgtcgatg 900cccggtgatg ttaccttcga tagtggtacg tctttctggg gtgcaaactt
gacggtcggt 960gtccttaacg gtacaatccc ccaatggcgt gttgatgaca tggctgtccg
tatcatggcc 1020gcttattaca aggttggccg cgacaccaaa tacacccctc ccaacttcag
ctcgtggacc 1080agggacgaat atggtttcgc gcataaccat gtttcggaag gtgcttacga
gagggtcaac 1140gaattcgtgg acgtgcaacg cgatcatgcc gacctaatcc gtcgcatcgg
cgcgcagagc 1200actgttctgc tgaagaacaa gggtgccttg cccttgagcc gcaaggaaaa
gctggtcgcc 1260cttctgggag aggatgcggg ttccaactcg tggggcgcta acggctgtga
tgaccgtggt 1320tgcgataacg gtacccttgc catggcctgg ggtagcggta ctgcgaattt
cccatacctc 1380gtgacaccag agcaggcgat tcagaacgaa gttcttcagg gccgtggtaa
tgtcttcgcc 1440gtgaccgaca gttgggcgct cgacaagatc gctgcggctg cccgccaggc
cagcgtatct 1500ctcgtgttcg tcaactccga ctcaggagaa ggctatctta gtgtggatgg
aaatgagggc 1560gatcgtaaca acatcactct gtggaagaac ggcgacaatg tggtcaagac
cgcagcgaat 1620aactgtaaca acaccgttgt catcatccac tccgtcggac cagttttgat
cgatgaatgg 1680tatgaccacc ccaatgtcac tggtattctc tgggctggtc tgccaggcca
ggagtctggt 1740aactccattg ccgatgtgct gtacggtcgt gtcaaccctg gcgccaagtc
tcctttcact 1800tggggcaaga cccgggagtc gtatggttct cccttggtca aggatgccaa
caatggcaac 1860ggagcgcccc agtctgattt cacccagggt gttttcatcg attaccgcca
tttcgataag 1920ttcaatgaga cccctatcta cgagtttggc tacggcttga gctacaccac
cttcgagctc 1980tccgacctcc atgttcagcc cctgaacgcg tcccgataca ctcccaccag
tggcatgact 2040gaagctgcaa agaactttgg tgaaattggc gatgcgtcgg agtacgtgta
tccggagggg 2100ctggaaagga tccatgagtt tatctatccc tggatcaact ctaccgacct
gaaggcatcg 2160tctgacgatt ctaactacgg ctgggaagac tccaagtata ttcccgaagg
cgccacggat 2220gggtctgccc agccccgttt gcccgctagt ggtggtgccg gaggaaaccc
cggtctgtac 2280gaggatcttt tccgcgtctc tgtgaaggtc aagaacacgg gcaatgtcgc
cggtgatgaa 2340gttcctcagc tgtacgtttc cctaggcggc ccgaatgagc ccaaggtggt
actgcgcaag 2400tttgagcgta ttcacttggc cccttcgcag gaggccgtgt ggacaacgac
ccttacccgt 2460cgtgaccttg caaactggga cgtttcggct caggactgga ccgtcactcc
ttaccccaag 2520acgatctacg ttggaaactc ctcacggaaa ctgccgctcc aggcctcgct
gcctaaggcc 2580cagtaa
2586112861PRTAspergillus oryzae 112Met Lys Leu Gly Trp Ile Glu
Val Ala Ala Leu Ala Ala Ala Ser Val1 5 10
15Val Ser Ala Lys Asp Asp Leu Ala Tyr Ser Pro Pro Phe
Tyr Pro Ser 20 25 30Pro Trp
Ala Asp Gly Gln Gly Glu Trp Ala Glu Val Tyr Lys Arg Ala 35
40 45Val Asp Ile Val Ser Gln Met Thr Leu Thr
Glu Lys Val Asn Leu Thr 50 55 60Thr
Gly Thr Gly Trp Gln Leu Glu Arg Cys Val Gly Gln Thr Gly Ser65
70 75 80Val Pro Arg Leu Asn Ile
Pro Ser Leu Cys Leu Gln Asp Ser Pro Leu 85
90 95Gly Ile Arg Phe Ser Asp Tyr Asn Ser Ala Phe Pro
Ala Gly Val Asn 100 105 110Val
Ala Ala Thr Trp Asp Lys Thr Leu Ala Tyr Leu Arg Gly Gln Ala 115
120 125Met Gly Glu Glu Phe Ser Asp Lys Gly
Ile Asp Val Gln Leu Gly Pro 130 135
140Ala Ala Gly Pro Leu Gly Ala His Pro Asp Gly Gly Arg Asn Trp Glu145
150 155 160Gly Phe Ser Pro
Asp Pro Ala Leu Thr Gly Val Leu Phe Ala Glu Thr 165
170 175Ile Lys Gly Ile Gln Asp Ala Gly Val Ile
Ala Thr Ala Lys His Tyr 180 185
190Ile Met Asn Glu Gln Glu His Phe Arg Gln Gln Pro Glu Ala Ala Gly
195 200 205Tyr Gly Phe Asn Val Ser Asp
Ser Leu Ser Ser Asn Val Asp Asp Lys 210 215
220Thr Met His Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg
Ala225 230 235 240Gly Val
Gly Ala Val Met Cys Ser Tyr Asn Gln Ile Asn Asn Ser Tyr
245 250 255Gly Cys Glu Asn Ser Glu Thr
Leu Asn Lys Leu Leu Lys Ala Glu Leu 260 265
270Gly Phe Gln Gly Phe Val Met Ser Asp Trp Thr Ala His His
Ser Gly 275 280 285Val Gly Ala Ala
Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Val 290
295 300Thr Phe Asp Ser Gly Thr Ser Phe Trp Gly Ala Asn
Leu Thr Val Gly305 310 315
320Val Leu Asn Gly Thr Ile Pro Gln Trp Arg Val Asp Asp Met Ala Val
325 330 335Arg Ile Met Ala Ala
Tyr Tyr Lys Val Gly Arg Asp Thr Lys Tyr Thr 340
345 350Pro Pro Asn Phe Ser Ser Trp Thr Arg Asp Glu Tyr
Gly Phe Ala His 355 360 365Asn His
Val Ser Glu Gly Ala Tyr Glu Arg Val Asn Glu Phe Val Asp 370
375 380Val Gln Arg Asp His Ala Asp Leu Ile Arg Arg
Ile Gly Ala Gln Ser385 390 395
400Thr Val Leu Leu Lys Asn Lys Gly Ala Leu Pro Leu Ser Arg Lys Glu
405 410 415Lys Leu Val Ala
Leu Leu Gly Glu Asp Ala Gly Ser Asn Ser Trp Gly 420
425 430Ala Asn Gly Cys Asp Asp Arg Gly Cys Asp Asn
Gly Thr Leu Ala Met 435 440 445Ala
Trp Gly Ser Gly Thr Ala Asn Phe Pro Tyr Leu Val Thr Pro Glu 450
455 460Gln Ala Ile Gln Asn Glu Val Leu Gln Gly
Arg Gly Asn Val Phe Ala465 470 475
480Val Thr Asp Ser Trp Ala Leu Asp Lys Ile Ala Ala Ala Ala Arg
Gln 485 490 495Ala Ser Val
Ser Leu Val Phe Val Asn Ser Asp Ser Gly Glu Gly Tyr 500
505 510Leu Ser Val Asp Gly Asn Glu Gly Asp Arg
Asn Asn Ile Thr Leu Trp 515 520
525Lys Asn Gly Asp Asn Val Val Lys Thr Ala Ala Asn Asn Cys Asn Asn 530
535 540Thr Val Val Ile Ile His Ser Val
Gly Pro Val Leu Ile Asp Glu Trp545 550
555 560Tyr Asp His Pro Asn Val Thr Gly Ile Leu Trp Ala
Gly Leu Pro Gly 565 570
575Gln Glu Ser Gly Asn Ser Ile Ala Asp Val Leu Tyr Gly Arg Val Asn
580 585 590Pro Gly Ala Lys Ser Pro
Phe Thr Trp Gly Lys Thr Arg Glu Ser Tyr 595 600
605Gly Ser Pro Leu Val Lys Asp Ala Asn Asn Gly Asn Gly Ala
Pro Gln 610 615 620Ser Asp Phe Thr Gln
Gly Val Phe Ile Asp Tyr Arg His Phe Asp Lys625 630
635 640Phe Asn Glu Thr Pro Ile Tyr Glu Phe Gly
Tyr Gly Leu Ser Tyr Thr 645 650
655Thr Phe Glu Leu Ser Asp Leu His Val Gln Pro Leu Asn Ala Ser Arg
660 665 670Tyr Thr Pro Thr Ser
Gly Met Thr Glu Ala Ala Lys Asn Phe Gly Glu 675
680 685Ile Gly Asp Ala Ser Glu Tyr Val Tyr Pro Glu Gly
Leu Glu Arg Ile 690 695 700His Glu Phe
Ile Tyr Pro Trp Ile Asn Ser Thr Asp Leu Lys Ala Ser705
710 715 720Ser Asp Asp Ser Asn Tyr Gly
Trp Glu Asp Ser Lys Tyr Ile Pro Glu 725
730 735Gly Ala Thr Asp Gly Ser Ala Gln Pro Arg Leu Pro
Ala Ser Gly Gly 740 745 750Ala
Gly Gly Asn Pro Gly Leu Tyr Glu Asp Leu Phe Arg Val Ser Val 755
760 765Lys Val Lys Asn Thr Gly Asn Val Ala
Gly Asp Glu Val Pro Gln Leu 770 775
780Tyr Val Ser Leu Gly Gly Pro Asn Glu Pro Lys Val Val Leu Arg Lys785
790 795 800Phe Glu Arg Ile
His Leu Ala Pro Ser Gln Glu Ala Val Trp Thr Thr 805
810 815Thr Leu Thr Arg Arg Asp Leu Ala Asn Trp
Asp Val Ser Ala Gln Asp 820 825
830Trp Thr Val Thr Pro Tyr Pro Lys Thr Ile Tyr Val Gly Asn Ser Ser
835 840 845Arg Lys Leu Pro Leu Gln Ala
Ser Leu Pro Lys Ala Gln 850 855
8601133060DNAAspergillus fumigatus 113atgagattcg gttggctcga ggtggccgct
ctgacggccg cttctgtagc caatgcccag 60gtttgtgatg ctttcccgtc attgtttcgg
atatagttga caatagtcat ggaaataatc 120aggaattggc tttctctcca ccattctacc
cttcgccttg ggctgatggc cagggagagt 180gggcagatgc ccatcgacgc gccgtcgaga
tcgtttctca gatgacactg gcggagaagg 240ttaaccttac aacgggtact gggtgggttg
cgactttttt gttgacagtg agctttcttc 300actgaccatc tacacagatg ggaaatggac
cgatgcgtcg gtcaaaccgg cagcgttccc 360aggtaagctt gcaattctgc aacaacgtgc
aagtgtagtt gctaaaacgc ggtggtgcag 420acttggtatc aactggggtc tttgtggcca
ggattcccct ttgggtatcc gtttctgtga 480gctatacccg cggagtcttt cagtccttgt
attatgtgct gatgattgtc tctgtatagc 540tgacctcaac tccgccttcc ctgctggtac
taatgtcgcc gcgacatggg acaagacact 600cgcctacctt cgtggcaagg ccatgggtga
ggaattcaac gacaagggcg tggacatttt 660gctggggcct gctgctggtc ctctcggcaa
atacccggac ggcggcagaa tctgggaagg 720cttctctcct gatccggttc tcactggtgt
acttttcgcc gaaactatca agggtatcca 780agacgcgggt gtgattgcta ctgccaagca
ttacattctg aatgaacagg agcatttccg 840acaggttggc gaggcccagg gatatggtta
caacatcacg gagacgatca gctccaacgt 900ggatgacaag accatgcacg agttgtacct
ttggtgagta gttgacactg caaatgagga 960ccttgattga tttgactgac ctggaatgca
ggccctttgc agatgctgtg cgcggtaaga 1020ttttccgtag acttgacctc gcgacgaaga
aatcgctgac gaaccatcgt agctggcgtt 1080ggcgctgtca tgtgttccta caatcaaatc
aacaacagct acggttgtca aaacagtcaa 1140actctcaaca agctcctcaa ggctgagctg
ggcttccaag gcttcgtcat gagtgactgg 1200agcgctcacc acagcggtgt cggcgctgcc
ctcgctgggt tggatatgtc gatgcctgga 1260gacatttcct tcgacgacgg actctccttc
tggggcacga acctaactgt cagtgttctt 1320aacggcaccg ttccagcctg gcgtgtcgat
gacatggctg ttcgtatcat gaccgcgtac 1380tacaaggttg gtcgtgaccg tcttcgtatt
ccccctaact tcagctcctg gacccgggat 1440gagtacggct gggagcattc tgctgtctcc
gagggagcct ggaccaaggt gaacgacttc 1500gtcaatgtgc agcgcagtca ctctcagatc
atccgtgaga ttggtgccgc tagtacagtg 1560ctcttgaaga acacgggtgc tcttcctttg
accggcaagg aggttaaagt gggtgttctc 1620ggtgaagacg ctggttccaa cccgtggggt
gctaacggct gccccgaccg cggctgtgat 1680aacggcactc ttgctatggc ctggggtagt
ggtactgcca acttccctta ccttgtcacc 1740cccgagcagg ctatccagcg agaggtcatc
agcaacggcg gcaatgtctt tgctgtgact 1800gataacgggg ctctcagcca gatggcagat
gttgcatctc aatccaggtg agtgcgggct 1860cttagaaaaa gaacgttctc tgaatgaagt
tttttaacca ttgcgaacag cgtgtctttg 1920gtgtttgtca acgccgactc tggagagggt
ttcatcagtg tcgacggcaa cgagggtgac 1980cgcaaaaatc tcactctgtg gaagaacggc
gaggccgtca ttgacactgt tgtcagccac 2040tgcaacaaca cgattgtggt tattcacagt
gttgggcccg tcttgatcga ccggtggtat 2100gataacccca acgtcactgc catcatctgg
gccggcttgc ccggtcagga gagtggcaac 2160tccctggtcg acgtgctcta tggccgcgtc
aaccccagcg ccaagacccc gttcacctgg 2220ggcaagactc gggagtctta cggggctccc
ttgctcaccg agcctaacaa tggcaatggt 2280gctccccagg atgatttcaa cgagggcgtc
ttcattgact accgtcactt tgacaagcgc 2340aatgagaccc ccatttatga gtttggccat
ggcttgagct acaccacctt tggttactct 2400caccttcggg ttcaggccct caatagttcg
agttcggcat atgtcccgac tagcggagag 2460accaagcctg cgccaaccta tggtgagatc
ggtagtgccg ccgactacct gtatcccgag 2520ggtctcaaaa gaattaccaa gtttatttac
ccttggctca actcgaccga cctcgaggat 2580tcttctgacg acccgaacta cggctgggag
gactcggagt acattcccga aggcgctagg 2640gatgggtctc ctcaacccct cctgaaggct
ggcggcgctc ctggtggtaa ccctaccctt 2700tatcaggatc ttgttagggt gtcggccacc
ataaccaaca ctggtaacgt cgccggttat 2760gaagtccctc aattggtgag tgacccgcat
gttccttgcg ttgcaatttg gctaactcgc 2820ttctagtatg tttcactggg cggaccgaac
gagcctcggg tcgttctgcg caagttcgac 2880cgaatcttcc tggctcctgg ggagcaaaag
gtttggacca cgactcttaa ccgtcgtgat 2940ctcgccaatt gggatgtgga ggctcaggac
tgggtcatca caaagtaccc caagaaagtg 3000cacgtcggca gctcctcgcg taagctgcct
ctgagagcgc ctctgccccg tgtctactag 3060114863PRTAspergillus fumigatus
114Met Arg Phe Gly Trp Leu Glu Val Ala Ala Leu Thr Ala Ala Ser Val1
5 10 15Ala Asn Ala Gln Glu Leu
Ala Phe Ser Pro Pro Phe Tyr Pro Ser Pro 20 25
30Trp Ala Asp Gly Gln Gly Glu Trp Ala Asp Ala His Arg
Arg Ala Val 35 40 45Glu Ile Val
Ser Gln Met Thr Leu Ala Glu Lys Val Asn Leu Thr Thr 50
55 60Gly Thr Gly Trp Glu Met Asp Arg Cys Val Gly Gln
Thr Gly Ser Val65 70 75
80Pro Arg Leu Gly Ile Asn Trp Gly Leu Cys Gly Gln Asp Ser Pro Leu
85 90 95Gly Ile Arg Phe Ser Asp
Leu Asn Ser Ala Phe Pro Ala Gly Thr Asn 100
105 110Val Ala Ala Thr Trp Asp Lys Thr Leu Ala Tyr Leu
Arg Gly Lys Ala 115 120 125Met Gly
Glu Glu Phe Asn Asp Lys Gly Val Asp Ile Leu Leu Gly Pro 130
135 140Ala Ala Gly Pro Leu Gly Lys Tyr Pro Asp Gly
Gly Arg Ile Trp Glu145 150 155
160Gly Phe Ser Pro Asp Pro Val Leu Thr Gly Val Leu Phe Ala Glu Thr
165 170 175Ile Lys Gly Ile
Gln Asp Ala Gly Val Ile Ala Thr Ala Lys His Tyr 180
185 190Ile Leu Asn Glu Gln Glu His Phe Arg Gln Val
Gly Glu Ala Gln Gly 195 200 205Tyr
Gly Tyr Asn Ile Thr Glu Thr Ile Ser Ser Asn Val Asp Asp Lys 210
215 220Thr Met His Glu Leu Tyr Leu Trp Pro Phe
Ala Asp Ala Val Arg Ala225 230 235
240Gly Val Gly Ala Val Met Cys Ser Tyr Asn Gln Ile Asn Asn Ser
Tyr 245 250 255Gly Cys Gln
Asn Ser Gln Thr Leu Asn Lys Leu Leu Lys Ala Glu Leu 260
265 270Gly Phe Gln Gly Phe Val Met Ser Asp Trp
Ser Ala His His Ser Gly 275 280
285Val Gly Ala Ala Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Ile 290
295 300Ser Phe Asp Asp Gly Leu Ser Phe
Trp Gly Thr Asn Leu Thr Val Ser305 310
315 320Val Leu Asn Gly Thr Val Pro Ala Trp Arg Val Asp
Asp Met Ala Val 325 330
335Arg Ile Met Thr Ala Tyr Tyr Lys Val Gly Arg Asp Arg Leu Arg Ile
340 345 350Pro Pro Asn Phe Ser Ser
Trp Thr Arg Asp Glu Tyr Gly Trp Glu His 355 360
365Ser Ala Val Ser Glu Gly Ala Trp Thr Lys Val Asn Asp Phe
Val Asn 370 375 380Val Gln Arg Ser His
Ser Gln Ile Ile Arg Glu Ile Gly Ala Ala Ser385 390
395 400Thr Val Leu Leu Lys Asn Thr Gly Ala Leu
Pro Leu Thr Gly Lys Glu 405 410
415Val Lys Val Gly Val Leu Gly Glu Asp Ala Gly Ser Asn Pro Trp Gly
420 425 430Ala Asn Gly Cys Pro
Asp Arg Gly Cys Asp Asn Gly Thr Leu Ala Met 435
440 445Ala Trp Gly Ser Gly Thr Ala Asn Phe Pro Tyr Leu
Val Thr Pro Glu 450 455 460Gln Ala Ile
Gln Arg Glu Val Ile Ser Asn Gly Gly Asn Val Phe Ala465
470 475 480Val Thr Asp Asn Gly Ala Leu
Ser Gln Met Ala Asp Val Ala Ser Gln 485
490 495Ser Ser Val Ser Leu Val Phe Val Asn Ala Asp Ser
Gly Glu Gly Phe 500 505 510Ile
Ser Val Asp Gly Asn Glu Gly Asp Arg Lys Asn Leu Thr Leu Trp 515
520 525Lys Asn Gly Glu Ala Val Ile Asp Thr
Val Val Ser His Cys Asn Asn 530 535
540Thr Ile Val Val Ile His Ser Val Gly Pro Val Leu Ile Asp Arg Trp545
550 555 560Tyr Asp Asn Pro
Asn Val Thr Ala Ile Ile Trp Ala Gly Leu Pro Gly 565
570 575Gln Glu Ser Gly Asn Ser Leu Val Asp Val
Leu Tyr Gly Arg Val Asn 580 585
590Pro Ser Ala Lys Thr Pro Phe Thr Trp Gly Lys Thr Arg Glu Ser Tyr
595 600 605Gly Ala Pro Leu Leu Thr Glu
Pro Asn Asn Gly Asn Gly Ala Pro Gln 610 615
620Asp Asp Phe Asn Glu Gly Val Phe Ile Asp Tyr Arg His Phe Asp
Lys625 630 635 640Arg Asn
Glu Thr Pro Ile Tyr Glu Phe Gly His Gly Leu Ser Tyr Thr
645 650 655Thr Phe Gly Tyr Ser His Leu
Arg Val Gln Ala Leu Asn Ser Ser Ser 660 665
670Ser Ala Tyr Val Pro Thr Ser Gly Glu Thr Lys Pro Ala Pro
Thr Tyr 675 680 685Gly Glu Ile Gly
Ser Ala Ala Asp Tyr Leu Tyr Pro Glu Gly Leu Lys 690
695 700Arg Ile Thr Lys Phe Ile Tyr Pro Trp Leu Asn Ser
Thr Asp Leu Glu705 710 715
720Asp Ser Ser Asp Asp Pro Asn Tyr Gly Trp Glu Asp Ser Glu Tyr Ile
725 730 735Pro Glu Gly Ala Arg
Asp Gly Ser Pro Gln Pro Leu Leu Lys Ala Gly 740
745 750Gly Ala Pro Gly Gly Asn Pro Thr Leu Tyr Gln Asp
Leu Val Arg Val 755 760 765Ser Ala
Thr Ile Thr Asn Thr Gly Asn Val Ala Gly Tyr Glu Val Pro 770
775 780Gln Leu Tyr Val Ser Leu Gly Gly Pro Asn Glu
Pro Arg Val Val Leu785 790 795
800Arg Lys Phe Asp Arg Ile Phe Leu Ala Pro Gly Glu Gln Lys Val Trp
805 810 815Thr Thr Thr Leu
Asn Arg Arg Asp Leu Ala Asn Trp Asp Val Glu Ala 820
825 830Gln Asp Trp Val Ile Thr Lys Tyr Pro Lys Lys
Val His Val Gly Ser 835 840 845Ser
Ser Arg Lys Leu Pro Leu Arg Ala Pro Leu Pro Arg Val Tyr 850
855 8601152800DNAPenicillium brasilianum
115tgaaaatgca gggttctaca atctttctgg ctttcgcctc atgggcgagc caggttgctg
60ccattgcgca gcccatacag aagcacgagg tttgttttat cttgctcatg gacgtgcttt
120gacttgacta attgttttac atacagcccg gatttctgca cgggccccaa gccatagaat
180cgttctcaga accgttctac ccgtcgccct ggatgaatcc tcacgccgag ggctgggagg
240ccgcatatca gaaagctcaa gattttgtct cgcaactcac tatcttggag aaaataaatc
300tgaccaccgg tgttgggtaa gtctctccga ctgcttctgg gtcacggtgc gacgagccac
360tgactttttg aagctgggaa aatgggccgt gtgtaggaaa cactggatca attcctcgtc
420tcggattcaa aggattttgt acccaggatt caccacaggg tgttcggttc gcagattatt
480cctccgcttt cacatctagc caaatggccg ccgcaacatt tgaccgctca attctttatc
540aacgaggcca agccatggca caggaacaca aggctaaggg tatcacaatt caattgggcc
600ctgttgccgg ccctctcggt cgcatccccg agggcggccg caactgggaa ggattctccc
660ctgatcctgt cttgactggt atagccatgg ctgagacaat taagggcatg caggatactg
720gagtgattgc ttgcgctaaa cattatattg gaaacgagca ggagcacttc cgtcaagtgg
780gtgaagctgc gggtcacgga tacactattt ccgatactat ttcatctaat attgacgacc
840gtgctatgca tgagctatac ttgtggccat ttgctgatgc cgttcgcgct ggtgtgggtt
900ctttcatgtg ctcatactct cagatcaaca actcctacgg atgccaaaac agtcagaccc
960tcaacaagct cctcaagagc gaattgggct tccaaggctt tgtcatgagc gattggggtg
1020cccatcactc tggagtgtca tcggcgctag ctggacttga tatgagcatg ccgggtgata
1080ccgaatttga ttctggcttg agcttctggg gctctaacct caccattgca attctgaacg
1140gcacggttcc cgaatggcgc ctggatgaca tggcgatgcg aattatggct gcatacttca
1200aagttggcct tactattgag gatcaaccag atgtcaactt caatgcctgg acccatgaca
1260cctacggata taaatacgct tatagcaagg aagattacga gcaggtcaac tggcatgtcg
1320atgttcgcag cgaccacaat aagctcattc gcgagactgc cgcgaagggt acagttctgc
1380tgaagaacaa ctttcatgct ctccctctga agcagcccag gttcgtggcc gtcgttggtc
1440aggatgccgg gccaaacccc aagggcccta acggctgcgc agaccgagga tgcgaccaag
1500gcactctcgc aatgggatgg ggctcagggt ctaccgaatt cccttacctg gtcactcctg
1560acactgctat tcagtcaaag gtcctcgaat acgggggtcg atacgagagt atttttgata
1620actatgacga caatgctatc ttgtcgcttg tctcacagcc tgatgcaacc tgtatcgttt
1680ttgcaaatgc cgattccggt gaaggctaca tcactgtcga caacaactgg ggtgaccgca
1740acaatctgac cctctggcaa aatgccgatc aagtgattag cactgtcagc tcgcgatgca
1800acaacacaat cgttgttctc cactctgtcg gaccagtgtt gctaaatggt atatatgagc
1860acccgaacat cacagctatt gtctgggcag ggatgccagg cgaagaatct ggcaatgctc
1920tcgtggatat tctttggggc aatgttaacc ctgccggtcg cactccgttc acctgggcca
1980aaagtcgaga ggactatggc actgatataa tgtacgagcc caacaacggc cagcgtgcgc
2040ctcagcagga tttcaccgag agcatctacc tcgactaccg ccatttcgac aaagctggta
2100tcgagccaat ttacgagttt ggattcggcc tctcctatac caccttcgaa tactctgacc
2160tccgtgttgt gaagaagtat gttcaaccat acagtcccac gaccggcacc ggtgctcaag
2220caccttccat cggacagcca cctagccaga acctggatac ctacaagttc cctgctacat
2280acaagtacat caaaaccttc atttatccct acctgaacag cactgtctcc ctccgcgctg
2340cttccaagga tcccgaatac ggtcgtacag actttatccc accccacgcg cgtgatggct
2400cccctcaacc tctcaacccc gctggagacc cagtggccag tggtggaaac aacatgctct
2460acgacgaact ttacgaggtc actgcacaga tcaaaaacac tggcgacgtg gccggcgacg
2520aagtcgtcca gctttacgta gatctcgggg gtgacaaccc gcctcgtcag ttgagaaact
2580ttgacaggtt ttatctgctg cccggtcaga gctcaacatt ccgggctaca ttgacgcgcc
2640gtgatttgag caactgggat attgaggcgc agaactggcg agttacggaa tcgcctaaga
2700gagtgtatgt tggacggtcg agtcgggatt tgccgctgag ctcacaattg gagtaatgat
2760catgtctacc aatagatgtt gaatgtctgg tgtggatatt
2800116878PRTPenicillium brasilianum 116Met Gln Gly Ser Thr Ile Phe Leu
Ala Phe Ala Ser Trp Ala Ser Gln1 5 10
15Val Ala Ala Ile Ala Gln Pro Ile Gln Lys His Glu Pro Gly
Phe Leu 20 25 30His Gly Pro
Gln Ala Ile Glu Ser Phe Ser Glu Pro Phe Tyr Pro Ser 35
40 45Pro Trp Met Asn Pro His Ala Glu Gly Trp Glu
Ala Ala Tyr Gln Lys 50 55 60Ala Gln
Asp Phe Val Ser Gln Leu Thr Ile Leu Glu Lys Ile Asn Leu65
70 75 80Thr Thr Gly Val Gly Trp Glu
Asn Gly Pro Cys Val Gly Asn Thr Gly 85 90
95Ser Ile Pro Arg Leu Gly Phe Lys Gly Phe Cys Thr Gln
Asp Ser Pro 100 105 110Gln Gly
Val Arg Phe Ala Asp Tyr Ser Ser Ala Phe Thr Ser Ser Gln 115
120 125Met Ala Ala Ala Thr Phe Asp Arg Ser Ile
Leu Tyr Gln Arg Gly Gln 130 135 140Ala
Met Ala Gln Glu His Lys Ala Lys Gly Ile Thr Ile Gln Leu Gly145
150 155 160Pro Val Ala Gly Pro Leu
Gly Arg Ile Pro Glu Gly Gly Arg Asn Trp 165
170 175Glu Gly Phe Ser Pro Asp Pro Val Leu Thr Gly Ile
Ala Met Ala Glu 180 185 190Thr
Ile Lys Gly Met Gln Asp Thr Gly Val Ile Ala Cys Ala Lys His 195
200 205Tyr Ile Gly Asn Glu Gln Glu His Phe
Arg Gln Val Gly Glu Ala Ala 210 215
220Gly His Gly Tyr Thr Ile Ser Asp Thr Ile Ser Ser Asn Ile Asp Asp225
230 235 240Arg Ala Met His
Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg 245
250 255Ala Gly Val Gly Ser Phe Met Cys Ser Tyr
Ser Gln Ile Asn Asn Ser 260 265
270Tyr Gly Cys Gln Asn Ser Gln Thr Leu Asn Lys Leu Leu Lys Ser Glu
275 280 285Leu Gly Phe Gln Gly Phe Val
Met Ser Asp Trp Gly Ala His His Ser 290 295
300Gly Val Ser Ser Ala Leu Ala Gly Leu Asp Met Ser Met Pro Gly
Asp305 310 315 320Thr Glu
Phe Asp Ser Gly Leu Ser Phe Trp Gly Ser Asn Leu Thr Ile
325 330 335Ala Ile Leu Asn Gly Thr Val
Pro Glu Trp Arg Leu Asp Asp Met Ala 340 345
350Met Arg Ile Met Ala Ala Tyr Phe Lys Val Gly Leu Thr Ile
Glu Asp 355 360 365Gln Pro Asp Val
Asn Phe Asn Ala Trp Thr His Asp Thr Tyr Gly Tyr 370
375 380Lys Tyr Ala Tyr Ser Lys Glu Asp Tyr Glu Gln Val
Asn Trp His Val385 390 395
400Asp Val Arg Ser Asp His Asn Lys Leu Ile Arg Glu Thr Ala Ala Lys
405 410 415Gly Thr Val Leu Leu
Lys Asn Asn Phe His Ala Leu Pro Leu Lys Gln 420
425 430Pro Arg Phe Val Ala Val Val Gly Gln Asp Ala Gly
Pro Asn Pro Lys 435 440 445Gly Pro
Asn Gly Cys Ala Asp Arg Gly Cys Asp Gln Gly Thr Leu Ala 450
455 460Met Gly Trp Gly Ser Gly Ser Thr Glu Phe Pro
Tyr Leu Val Thr Pro465 470 475
480Asp Thr Ala Ile Gln Ser Lys Val Leu Glu Tyr Gly Gly Arg Tyr Glu
485 490 495Ser Ile Phe Asp
Asn Tyr Asp Asp Asn Ala Ile Leu Ser Leu Val Ser 500
505 510Gln Pro Asp Ala Thr Cys Ile Val Phe Ala Asn
Ala Asp Ser Gly Glu 515 520 525Gly
Tyr Ile Thr Val Asp Asn Asn Trp Gly Asp Arg Asn Asn Leu Thr 530
535 540Leu Trp Gln Asn Ala Asp Gln Val Ile Ser
Thr Val Ser Ser Arg Cys545 550 555
560Asn Asn Thr Ile Val Val Leu His Ser Val Gly Pro Val Leu Leu
Asn 565 570 575Gly Ile Tyr
Glu His Pro Asn Ile Thr Ala Ile Val Trp Ala Gly Met 580
585 590Pro Gly Glu Glu Ser Gly Asn Ala Leu Val
Asp Ile Leu Trp Gly Asn 595 600
605Val Asn Pro Ala Gly Arg Thr Pro Phe Thr Trp Ala Lys Ser Arg Glu 610
615 620Asp Tyr Gly Thr Asp Ile Met Tyr
Glu Pro Asn Asn Gly Gln Arg Ala625 630
635 640Pro Gln Gln Asp Phe Thr Glu Ser Ile Tyr Leu Asp
Tyr Arg His Phe 645 650
655Asp Lys Ala Gly Ile Glu Pro Ile Tyr Glu Phe Gly Phe Gly Leu Ser
660 665 670Tyr Thr Thr Phe Glu Tyr
Ser Asp Leu Arg Val Val Lys Lys Tyr Val 675 680
685Gln Pro Tyr Ser Pro Thr Thr Gly Thr Gly Ala Gln Ala Pro
Ser Ile 690 695 700Gly Gln Pro Pro Ser
Gln Asn Leu Asp Thr Tyr Lys Phe Pro Ala Thr705 710
715 720Tyr Lys Tyr Ile Lys Thr Phe Ile Tyr Pro
Tyr Leu Asn Ser Thr Val 725 730
735Ser Leu Arg Ala Ala Ser Lys Asp Pro Glu Tyr Gly Arg Thr Asp Phe
740 745 750Ile Pro Pro His Ala
Arg Asp Gly Ser Pro Gln Pro Leu Asn Pro Ala 755
760 765Gly Asp Pro Val Ala Ser Gly Gly Asn Asn Met Leu
Tyr Asp Glu Leu 770 775 780Tyr Glu Val
Thr Ala Gln Ile Lys Asn Thr Gly Asp Val Ala Gly Asp785
790 795 800Glu Val Val Gln Leu Tyr Val
Asp Leu Gly Gly Asp Asn Pro Pro Arg 805
810 815Gln Leu Arg Asn Phe Asp Arg Phe Tyr Leu Leu Pro
Gly Gln Ser Ser 820 825 830Thr
Phe Arg Ala Thr Leu Thr Arg Arg Asp Leu Ser Asn Trp Asp Ile 835
840 845Glu Ala Gln Asn Trp Arg Val Thr Glu
Ser Pro Lys Arg Val Tyr Val 850 855
860Gly Arg Ser Ser Arg Asp Leu Pro Leu Ser Ser Gln Leu Glu865
870 8751172583DNAAspergillus niger 117atgaggttca
ctttgatcga ggcggtggct ctgactgccg tctcgctggc cagcgctgat 60gaattggcct
actccccacc gtattaccca tccccttggg ccaatggcca gggcgactgg 120gcgcaggcat
accagcgcgc tgttgatatt gtctcgcaaa tgacattgga tgagaaggtc 180aatctgacca
caggaactgg atgggaattg gaactatgtg ttggtcagac tggcggtgtt 240ccccgattgg
gagttccggg aatgtgttta caggatagcc ctctgggcgt tcgcgactcc 300gactacaact
ctgctttccc tgccggcatg aacgtggctg caacctggga caagaatctg 360gcataccttc
gcggcaaggc tatgggtcag gaatttagtg acaagggtgc cgatatccaa 420ttgggtccag
ctgccggccc tctcggtaga agtcccgacg gtggtcgtaa ctgggagggc 480ttctccccag
accctgccct aagtggtgtg ctctttgccg agaccatcaa gggtatccaa 540gatgctggtg
tggttgcgac ggctaagcac tacattgctt acgagcaaga gcatttccgt 600caggcgcctg
aagcccaagg ttttggattt aatatttccg agagtggaag tgcgaacctc 660gatgataaga
ctatgcacga gctgtacctc tggcccttcg cggatgccat ccgtgcaggt 720gctggcgctg
tgatgtgctc ctacaaccag atcaacaaca gttatggctg ccagaacagc 780tacactctga
acaagctgct caaggccgag ctgggcttcc agggctttgt catgagtgat 840tgggctgctc
accatgctgg tgtgagtggt gctttggcag gattggatat gtctatgcca 900ggagacgtcg
actacgacag tggtacgtct tactggggta caaacttgac cattagcgtg 960ctcaacggaa
cggtgcccca atggcgtgtt gatgacatgg ctgtccgcat catggccgcc 1020tactacaagg
tcggccgtga ccgtctgtgg actcctccca acttcagctc atggaccaga 1080gatgaatacg
gctacaagta ctactacgtg tcggagggac cgtacgagaa ggtcaaccag 1140tacgtgaatg
tgcaacgcaa ccacagcgaa ctgattcgcc gcattggagc ggacagcacg 1200gtgctcctca
agaacgacgg cgctctgcct ttgactggta aggagcgcct ggtcgcgctt 1260atcggagaag
atgcgggctc caacccttat ggtgccaacg gctgcagtga ccgtggatgc 1320gacaatggaa
cattggcgat gggctgggga agtggtactg ccaacttccc atacctggtg 1380acccccgagc
aggccatctc aaacgaggtg cttaagcaca agaatggtgt attcaccgcc 1440accgataact
gggctatcga tcagattgag gcgcttgcta agaccgccag tgtctctctt 1500gtctttgtca
acgccgactc tggtgagggt tacatcaatg tggacggaaa cctgggtgac 1560cgcaggaacc
tgaccctgtg gaggaacggc gataatgtga tcaaggctgc tgctagcaac 1620tgcaacaaca
caatcgttgt cattcactct gtcggaccag tcttggttaa cgagtggtac 1680gacaacccca
atgttaccgc tatcctctgg ggtggtttgc ccggtcagga gtctggcaac 1740tctcttgccg
acgtcctcta tggccgtgtc aaccccggtg ccaagtcgcc ctttacctgg 1800ggcaagactc
gtgaggccta ccaagactac ttggtcaccg agcccaacaa cggcaacgga 1860gcccctcagg
aagactttgt cgagggcgtc ttcattgact accgtggatt tgacaagcgc 1920aacgagaccc
cgatctacga gttcggctat ggtctgagct acaccacttt caactactcg 1980aaccttgagg
tgcaggtgct gagcgcccct gcatacgagc ctgcttcggg tgagaccgag 2040gcagcgccaa
ccttcggaga ggttggaaat gcgtcggatt acctctaccc cagcggattg 2100cagagaatta
ccaagttcat ctacccctgg ctcaacggta ccgatctcga ggcatcttcc 2160ggggatgcta
gctacgggca ggactcctcc gactatcttc ccgagggagc caccgatggc 2220tctgcgcaac
cgatcctgcc tgccggtggc ggtcctggcg gcaaccctcg cctgtacgac 2280gagctcatcc
gcgtgtcagt gaccatcaag aacaccggca aggttgctgg tgatgaagtt 2340ccccaactgt
atgtttccct tggcggtccc aatgagccca agatcgtgct gcgtcaattc 2400gagcgcatca
cgctgcagcc gtcggaggag acgaagtgga gcacgactct gacgcgccgt 2460gaccttgcaa
actggaatgt tgagaagcag gactgggaga ttacgtcgta tcccaagatg 2520gtgtttgtcg
gaagctcctc gcggaagctg ccgctccggg cgtctctgcc tactgttcac 2580taa
2583118860PRTAspergillus niger 118Met Arg Phe Thr Leu Ile Glu Ala Val Ala
Leu Thr Ala Val Ser Leu1 5 10
15Ala Ser Ala Asp Glu Leu Ala Tyr Ser Pro Pro Tyr Tyr Pro Ser Pro
20 25 30Trp Ala Asn Gly Gln Gly
Asp Trp Ala Gln Ala Tyr Gln Arg Ala Val 35 40
45Asp Ile Val Ser Gln Met Thr Leu Asp Glu Lys Val Asn Leu
Thr Thr 50 55 60Gly Thr Gly Trp Glu
Leu Glu Leu Cys Val Gly Gln Thr Gly Gly Val65 70
75 80Pro Arg Leu Gly Val Pro Gly Met Cys Leu
Gln Asp Ser Pro Leu Gly 85 90
95Val Arg Asp Ser Asp Tyr Asn Ser Ala Phe Pro Ala Gly Met Asn Val
100 105 110Ala Ala Thr Trp Asp
Lys Asn Leu Ala Tyr Leu Arg Gly Lys Ala Met 115
120 125Gly Gln Glu Phe Ser Asp Lys Gly Ala Asp Ile Gln
Leu Gly Pro Ala 130 135 140Ala Gly Pro
Leu Gly Arg Ser Pro Asp Gly Gly Arg Asn Trp Glu Gly145
150 155 160Phe Ser Pro Asp Pro Ala Leu
Ser Gly Val Leu Phe Ala Glu Thr Ile 165
170 175Lys Gly Ile Gln Asp Ala Gly Val Val Ala Thr Ala
Lys His Tyr Ile 180 185 190Ala
Tyr Glu Gln Glu His Phe Arg Gln Ala Pro Glu Ala Gln Gly Phe 195
200 205Gly Phe Asn Ile Ser Glu Ser Gly Ser
Ala Asn Leu Asp Asp Lys Thr 210 215
220Met His Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ala Ile Arg Ala Gly225
230 235 240Ala Gly Ala Val
Met Cys Ser Tyr Asn Gln Ile Asn Asn Ser Tyr Gly 245
250 255Cys Gln Asn Ser Tyr Thr Leu Asn Lys Leu
Leu Lys Ala Glu Leu Gly 260 265
270Phe Gln Gly Phe Val Met Ser Asp Trp Ala Ala His His Ala Gly Val
275 280 285Ser Gly Ala Leu Ala Gly Leu
Asp Met Ser Met Pro Gly Asp Val Asp 290 295
300Tyr Asp Ser Gly Thr Ser Tyr Trp Gly Thr Asn Leu Thr Ile Ser
Val305 310 315 320Leu Asn
Gly Thr Val Pro Gln Trp Arg Val Asp Asp Met Ala Val Arg
325 330 335Ile Met Ala Ala Tyr Tyr Lys
Val Gly Arg Asp Arg Leu Trp Thr Pro 340 345
350Pro Asn Phe Ser Ser Trp Thr Arg Asp Glu Tyr Gly Tyr Lys
Tyr Tyr 355 360 365Tyr Val Ser Glu
Gly Pro Tyr Glu Lys Val Asn Gln Tyr Val Asn Val 370
375 380Gln Arg Asn His Ser Glu Leu Ile Arg Arg Ile Gly
Ala Asp Ser Thr385 390 395
400Val Leu Leu Lys Asn Asp Gly Ala Leu Pro Leu Thr Gly Lys Glu Arg
405 410 415Leu Val Ala Leu Ile
Gly Glu Asp Ala Gly Ser Asn Pro Tyr Gly Ala 420
425 430Asn Gly Cys Ser Asp Arg Gly Cys Asp Asn Gly Thr
Leu Ala Met Gly 435 440 445Trp Gly
Ser Gly Thr Ala Asn Phe Pro Tyr Leu Val Thr Pro Glu Gln 450
455 460Ala Ile Ser Asn Glu Val Leu Lys His Lys Asn
Gly Val Phe Thr Ala465 470 475
480Thr Asp Asn Trp Ala Ile Asp Gln Ile Glu Ala Leu Ala Lys Thr Ala
485 490 495Ser Val Ser Leu
Val Phe Val Asn Ala Asp Ser Gly Glu Gly Tyr Ile 500
505 510Asn Val Asp Gly Asn Leu Gly Asp Arg Arg Asn
Leu Thr Leu Trp Arg 515 520 525Asn
Gly Asp Asn Val Ile Lys Ala Ala Ala Ser Asn Cys Asn Asn Thr 530
535 540Ile Val Val Ile His Ser Val Gly Pro Val
Leu Val Asn Glu Trp Tyr545 550 555
560Asp Asn Pro Asn Val Thr Ala Ile Leu Trp Gly Gly Leu Pro Gly
Gln 565 570 575Glu Ser Gly
Asn Ser Leu Ala Asp Val Leu Tyr Gly Arg Val Asn Pro 580
585 590Gly Ala Lys Ser Pro Phe Thr Trp Gly Lys
Thr Arg Glu Ala Tyr Gln 595 600
605Asp Tyr Leu Val Thr Glu Pro Asn Asn Gly Asn Gly Ala Pro Gln Glu 610
615 620Asp Phe Val Glu Gly Val Phe Ile
Asp Tyr Arg Gly Phe Asp Lys Arg625 630
635 640Asn Glu Thr Pro Ile Tyr Glu Phe Gly Tyr Gly Leu
Ser Tyr Thr Thr 645 650
655Phe Asn Tyr Ser Asn Leu Glu Val Gln Val Leu Ser Ala Pro Ala Tyr
660 665 670Glu Pro Ala Ser Gly Glu
Thr Glu Ala Ala Pro Thr Phe Gly Glu Val 675 680
685Gly Asn Ala Ser Asp Tyr Leu Tyr Pro Ser Gly Leu Gln Arg
Ile Thr 690 695 700Lys Phe Ile Tyr Pro
Trp Leu Asn Gly Thr Asp Leu Glu Ala Ser Ser705 710
715 720Gly Asp Ala Ser Tyr Gly Gln Asp Ser Ser
Asp Tyr Leu Pro Glu Gly 725 730
735Ala Thr Asp Gly Ser Ala Gln Pro Ile Leu Pro Ala Gly Gly Gly Pro
740 745 750Gly Gly Asn Pro Arg
Leu Tyr Asp Glu Leu Ile Arg Val Ser Val Thr 755
760 765Ile Lys Asn Thr Gly Lys Val Ala Gly Asp Glu Val
Pro Gln Leu Tyr 770 775 780Val Ser Leu
Gly Gly Pro Asn Glu Pro Lys Ile Val Leu Arg Gln Phe785
790 795 800Glu Arg Ile Thr Leu Gln Pro
Ser Glu Glu Thr Lys Trp Ser Thr Thr 805
810 815Leu Thr Arg Arg Asp Leu Ala Asn Trp Asn Val Glu
Lys Gln Asp Trp 820 825 830Glu
Ile Thr Ser Tyr Pro Lys Met Val Phe Val Gly Ser Ser Ser Arg 835
840 845Lys Leu Pro Leu Arg Ala Ser Leu Pro
Thr Val His 850 855
8601192583DNAAspergillus aculeatus 119atgaagctca gttggcttga ggcggctgcc
ttgacggctg cttcagtcgt cagcgctgat 60gaactggcgt tctctcctcc tttctacccc
tctccgtggg ccaatggcca gggagagtgg 120gcggaagcct accagcgtgc agtggccatt
gtatcccaga tgactctgga tgagaaggtc 180aacctgacca ccggaactgg atgggagctg
gagaagtgcg tcggtcagac tggtggtgtc 240ccaagactga acatcggtgg catgtgtctt
caggacagtc ccttgggaat tcgtgatagt 300gactacaatt cggctttccc tgctggtgtc
aacgttgctg cgacatggga caagaacctt 360gcttatctac gtggtcaggc tatgggtcaa
gagttcagtg acaaaggaat tgatgttcaa 420ttgggaccgg ccgcgggtcc cctcggcagg
agccctgatg gaggtcgcaa ctgggaaggt 480ttctctccag acccggctct tactggtgtg
ctctttgcgg agacgattaa gggtattcaa 540gacgctggtg tcgtggcgac agccaagcat
tacattctca atgagcaaga gcatttccgc 600caggtcgcag aggctgcggg ctacggattc
aatatctccg acacgatcag ctctaacgtt 660gatgacaaga ccattcatga aatgtacctc
tggcccttcg cggatgccgt tcgcgccggc 720gttggcgcca tcatgtgttc ctacaaccag
atcaacaaca gctacggttg ccagaacagt 780tacactctga acaagcttct gaaggccgag
ctcggcttcc agggctttgt gatgtctgac 840tggggtgctc accacagtgg tgttggctct
gctttggccg gcttggatat gtcaatgcct 900ggcgatatca ccttcgattc tgccactagt
ttctggggta ccaacctgac cattgctgtg 960ctcaacggta ccgtcccgca gtggcgcgtt
gacgacatgg ctgtccgtat catggctgcc 1020tactacaagg ttggccgcga ccgcctgtac
cagccgccta acttcagctc ctggactcgc 1080gatgaatacg gcttcaagta tttctacccc
caggaagggc cctatgagaa ggtcaatcac 1140tttgtcaatg tgcagcgcaa ccacagcgag
gttattcgca agttgggagc agacagtact 1200gttctactga agaacaacaa tgccctgccg
ctgaccggaa aggagcgcaa agttgcgatc 1260ctgggtgaag atgctggatc caactcgtac
ggtgccaatg gctgctctga ccgtggctgt 1320gacaacggta ctcttgctat ggcttggggt
agcggcactg ccgaattccc atatctcgtg 1380acccctgagc aggctattca agccgaggtg
ctcaagcata agggcagcgt ctacgccatc 1440acggacaact gggcgctgag ccaggtggag
accctcgcta aacaagccag tgtctctctt 1500gtatttgtca actcggacgc gggagagggc
tatatctccg tggacggaaa cgagggcgac 1560cgcaacaacc tcaccctctg gaagaacggc
gacaacctca tcaaggctgc tgcaaacaac 1620tgcaacaaca ccatcgttgt catccactcc
gttggacctg ttttggttga cgagtggtat 1680gaccacccca acgttactgc catcctctgg
gcgggcttgc ctggccagga gtctggcaac 1740tccttggctg acgtgctcta cggccgcgtc
aacccgggcg ccaaatctcc attcacctgg 1800ggcaagacga gggaggcgta cggggattac
cttgtccgtg agctcaacaa cggcaacgga 1860gctccccaag atgatttctc ggaaggtgtt
ttcattgact accgcggatt cgacaagcgc 1920aatgagaccc cgatctacga gttcggacat
ggtctgagct acaccacttt caactactct 1980ggccttcaca tccaggttct caacgcttcc
tccaacgctc aagtagccac tgagactggc 2040gccgctccca ccttcggaca agtcggcaat
gcctctgact acgtgtaccc tgagggattg 2100accagaatca gcaagttcat ctatccctgg
cttaattcca cagacctgaa ggcctcatct 2160ggcgacccgt actatggagt cgacaccgcg
gagcacgtgc ccgagggtgc tactgatggc 2220tctccgcagc ccgttctgcc tgccggtggt
ggctctggtg gtaacccgcg cctctacgat 2280gagttgatcc gtgtttcggt gacagtcaag
aacactggtc gtgttgccgg tgatgctgtg 2340cctcaattgt atgtttccct tggtggaccc
aatgagccca aggttgtgtt gcgcaaattc 2400gaccgcctca ccctcaagcc ctccgaggag
acggtgtgga cgactaccct gacccgccgc 2460gatctgtcta actgggacgt tgcggctcag
gactgggtca tcacttctta cccgaagaag 2520gtccatgttg gtagctcttc gcgtcagctg
ccccttcacg cggcgctccc gaaggtgcaa 2580tga
2583120860PRTAspergillus aculeatus
120Met Lys Leu Ser Trp Leu Glu Ala Ala Ala Leu Thr Ala Ala Ser Val1
5 10 15Val Ser Ala Asp Glu Leu
Ala Phe Ser Pro Pro Phe Tyr Pro Ser Pro 20 25
30Trp Ala Asn Gly Gln Gly Glu Trp Ala Glu Ala Tyr Gln
Arg Ala Val 35 40 45Ala Ile Val
Ser Gln Met Thr Leu Asp Glu Lys Val Asn Leu Thr Thr 50
55 60Gly Thr Gly Trp Glu Leu Glu Lys Cys Val Gly Gln
Thr Gly Gly Val65 70 75
80Pro Arg Leu Asn Ile Gly Gly Met Cys Leu Gln Asp Ser Pro Leu Gly
85 90 95Ile Arg Asp Ser Asp Tyr
Asn Ser Ala Phe Pro Ala Gly Val Asn Val 100
105 110Ala Ala Thr Trp Asp Lys Asn Leu Ala Tyr Leu Arg
Gly Gln Ala Met 115 120 125Gly Gln
Glu Phe Ser Asp Lys Gly Ile Asp Val Gln Leu Gly Pro Ala 130
135 140Ala Gly Pro Leu Gly Arg Ser Pro Asp Gly Gly
Arg Asn Trp Glu Gly145 150 155
160Phe Ser Pro Asp Pro Ala Leu Thr Gly Val Leu Phe Ala Glu Thr Ile
165 170 175Lys Gly Ile Gln
Asp Ala Gly Val Val Ala Thr Ala Lys His Tyr Ile 180
185 190Leu Asn Glu Gln Glu His Phe Arg Gln Val Ala
Glu Ala Ala Gly Tyr 195 200 205Gly
Phe Asn Ile Ser Asp Thr Ile Ser Ser Asn Val Asp Asp Lys Thr 210
215 220Ile His Glu Met Tyr Leu Trp Pro Phe Ala
Asp Ala Val Arg Ala Gly225 230 235
240Val Gly Ala Ile Met Cys Ser Tyr Asn Gln Ile Asn Asn Ser Tyr
Gly 245 250 255Cys Gln Asn
Ser Tyr Thr Leu Asn Lys Leu Leu Lys Ala Glu Leu Gly 260
265 270Phe Gln Gly Phe Val Met Ser Asp Trp Gly
Ala His His Ser Gly Val 275 280
285Gly Ser Ala Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Ile Thr 290
295 300Phe Asp Ser Ala Thr Ser Phe Trp
Gly Thr Asn Leu Thr Ile Ala Val305 310
315 320Leu Asn Gly Thr Val Pro Gln Trp Arg Val Asp Asp
Met Ala Val Arg 325 330
335Ile Met Ala Ala Tyr Tyr Lys Val Gly Arg Asp Arg Leu Tyr Gln Pro
340 345 350Pro Asn Phe Ser Ser Trp
Thr Arg Asp Glu Tyr Gly Phe Lys Tyr Phe 355 360
365Tyr Pro Gln Glu Gly Pro Tyr Glu Lys Val Asn His Phe Val
Asn Val 370 375 380Gln Arg Asn His Ser
Glu Val Ile Arg Lys Leu Gly Ala Asp Ser Thr385 390
395 400Val Leu Leu Lys Asn Asn Asn Ala Leu Pro
Leu Thr Gly Lys Glu Arg 405 410
415Lys Val Ala Ile Leu Gly Glu Asp Ala Gly Ser Asn Ser Tyr Gly Ala
420 425 430Asn Gly Cys Ser Asp
Arg Gly Cys Asp Asn Gly Thr Leu Ala Met Ala 435
440 445Trp Gly Ser Gly Thr Ala Glu Phe Pro Tyr Leu Val
Thr Pro Glu Gln 450 455 460Ala Ile Gln
Ala Glu Val Leu Lys His Lys Gly Ser Val Tyr Ala Ile465
470 475 480Thr Asp Asn Trp Ala Leu Ser
Gln Val Glu Thr Leu Ala Lys Gln Ala 485
490 495Ser Val Ser Leu Val Phe Val Asn Ser Asp Ala Gly
Glu Gly Tyr Ile 500 505 510Ser
Val Asp Gly Asn Glu Gly Asp Arg Asn Asn Leu Thr Leu Trp Lys 515
520 525Asn Gly Asp Asn Leu Ile Lys Ala Ala
Ala Asn Asn Cys Asn Asn Thr 530 535
540Ile Val Val Ile His Ser Val Gly Pro Val Leu Val Asp Glu Trp Tyr545
550 555 560Asp His Pro Asn
Val Thr Ala Ile Leu Trp Ala Gly Leu Pro Gly Gln 565
570 575Glu Ser Gly Asn Ser Leu Ala Asp Val Leu
Tyr Gly Arg Val Asn Pro 580 585
590Gly Ala Lys Ser Pro Phe Thr Trp Gly Lys Thr Arg Glu Ala Tyr Gly
595 600 605Asp Tyr Leu Val Arg Glu Leu
Asn Asn Gly Asn Gly Ala Pro Gln Asp 610 615
620Asp Phe Ser Glu Gly Val Phe Ile Asp Tyr Arg Gly Phe Asp Lys
Arg625 630 635 640Asn Glu
Thr Pro Ile Tyr Glu Phe Gly His Gly Leu Ser Tyr Thr Thr
645 650 655Phe Asn Tyr Ser Gly Leu His
Ile Gln Val Leu Asn Ala Ser Ser Asn 660 665
670Ala Gln Val Ala Thr Glu Thr Gly Ala Ala Pro Thr Phe Gly
Gln Val 675 680 685Gly Asn Ala Ser
Asp Tyr Val Tyr Pro Glu Gly Leu Thr Arg Ile Ser 690
695 700Lys Phe Ile Tyr Pro Trp Leu Asn Ser Thr Asp Leu
Lys Ala Ser Ser705 710 715
720Gly Asp Pro Tyr Tyr Gly Val Asp Thr Ala Glu His Val Pro Glu Gly
725 730 735Ala Thr Asp Gly Ser
Pro Gln Pro Val Leu Pro Ala Gly Gly Gly Ser 740
745 750Gly Gly Asn Pro Arg Leu Tyr Asp Glu Leu Ile Arg
Val Ser Val Thr 755 760 765Val Lys
Asn Thr Gly Arg Val Ala Gly Asp Ala Val Pro Gln Leu Tyr 770
775 780Val Ser Leu Gly Gly Pro Asn Glu Pro Lys Val
Val Leu Arg Lys Phe785 790 795
800Asp Arg Leu Thr Leu Lys Pro Ser Glu Glu Thr Val Trp Thr Thr Thr
805 810 815Leu Thr Arg Arg
Asp Leu Ser Asn Trp Asp Val Ala Ala Gln Asp Trp 820
825 830Val Ile Thr Ser Tyr Pro Lys Lys Val His Val
Gly Ser Ser Ser Arg 835 840 845Gln
Leu Pro Leu His Ala Ala Leu Pro Lys Val Gln 850 855
8601213294DNAAspergillus oryzae 121atgcgttcct cccccctcct
ccgctccgcc gttgtggccg ccctgccggt gttggccctt 60gccgctgatg gcaggtccac
ccgctactgg gactgctgca agccttcgtg cggctgggcc 120aagaaggctc ccgtgaacca
gcctgtcttt tcctgcaacg ccaacttcca gcgtatcacg 180gacttcgacg ccaagtccgg
ctgcgagccg ggcggtgtcg cctactcgtg cgccgaccag 240accccatggg ctgtgaacga
cgacttcgcg ctcggttttg ctgccacctc tattgccggc 300agcaatgagg cgggctggtg
ctgcgcctgc tacgagctca ccttcacatc cggtcctgtt 360gctggcaaga agatggtcgt
ccagtccacc agcactggcg gtgatcttgg cagcaaccac 420ttcgatctca acatccccgg
cggcggcgtc ggcatcttcg acggatgcac tccccagttc 480ggtggtctgc ccggccagcg
ctacggcggc atctcgtccc gcaacgagtg cgatcggttc 540cccgacgccc tcaagcccgg
ctgctactgg cgcttcgact ggttcaagaa cgccgacaat 600ccgagcttca gcttccgtca
ggtccagtgc ccagccgagc tcgtcgctcg caccggatgc 660cgccgcaacg acgacggcaa
cttccctgcc gtccagatcc ccatgcgttc ctcccccctc 720ctccgctccg ccgttgtggc
cgccctgccg gtgttggccc ttgccaagga tgatctcgcg 780tactcccctc ctttctaccc
ttccccatgg gcagatggtc agggtgaatg ggcggaagta 840tacaaacgcg ctgtagacat
agtttcccag atgacgttga cagagaaagt caacttaacg 900actggaacag gatggcaact
agagaggtgt gttggacaaa ctggcagtgt tcccagactc 960aacatcccca gcttgtgttt
gcaggatagt cctcttggta ttcgtttctc ggactacaat 1020tcagctttcc ctgcgggtgt
taatgtcgct gccacctggg acaagacgct cgcctacctt 1080cgtggtcagg caatgggtga
ggagttcagt gataagggta ttgacgttca gctgggtcct 1140gctgctggcc ctctcggtgc
tcatccggat ggcggtagaa actgggaagg tttctcacca 1200gatccagccc tcaccggtgt
actttttgcg gagacgatta agggtattca agatgctggt 1260gtcattgcga cagctaagca
ttatatcatg aacgaacaag agcatttccg ccaacaaccc 1320gaggctgcgg gttacggatt
caacgtaagc gacagtttga gttccaacgt tgatgacaag 1380actatgcatg aattgtacct
ctggcccttc gcggatgcag tacgcgctgg agtcggtgct 1440gtcatgtgct cttacaacca
aatcaacaac agctacggtt gcgagaatag cgaaactctg 1500aacaagcttt tgaaggcgga
gcttggtttc caaggcttcg tcatgagtga ttggaccgct 1560catcacagcg gcgtaggcgc
tgctttagca ggtctggata tgtcgatgcc cggtgatgtt 1620accttcgata gtggtacgtc
tttctggggt gcaaacttga cggtcggtgt ccttaacggt 1680acaatccccc aatggcgtgt
tgatgacatg gctgtccgta tcatggccgc ttattacaag 1740gttggccgcg acaccaaata
cacccctccc aacttcagct cgtggaccag ggacgaatat 1800ggtttcgcgc ataaccatgt
ttcggaaggt gcttacgaga gggtcaacga attcgtggac 1860gtgcaacgcg atcatgccga
cctaatccgt cgcatcggcg cgcagagcac tgttctgctg 1920aagaacaagg gtgccttgcc
cttgagccgc aaggaaaagc tggtcgccct tctgggagag 1980gatgcgggtt ccaactcgtg
gggcgctaac ggctgtgatg accgtggttg cgataacggt 2040acccttgcca tggcctgggg
tagcggtact gcgaatttcc catacctcgt gacaccagag 2100caggcgattc agaacgaagt
tcttcagggc cgtggtaatg tcttcgccgt gaccgacagt 2160tgggcgctcg acaagatcgc
tgcggctgcc cgccaggcca gcgtatctct cgtgttcgtc 2220aactccgact caggagaagg
ctatcttagt gtggatggaa atgagggcga tcgtaacaac 2280atcactctgt ggaagaacgg
cgacaatgtg gtcaagaccg cagcgaataa ctgtaacaac 2340accgttgtca tcatccactc
cgtcggacca gttttgatcg atgaatggta tgaccacccc 2400aatgtcactg gtattctctg
ggctggtctg ccaggccagg agtctggtaa ctccattgcc 2460gatgtgctgt acggtcgtgt
caaccctggc gccaagtctc ctttcacttg gggcaagacc 2520cgggagtcgt atggttctcc
cttggtcaag gatgccaaca atggcaacgg agcgccccag 2580tctgatttca cccagggtgt
tttcatcgat taccgccatt tcgataagtt caatgagacc 2640cctatctacg agtttggcta
cggcttgagc tacaccacct tcgagctctc cgacctccat 2700gttcagcccc tgaacgcgtc
ccgatacact cccaccagtg gcatgactga agctgcaaag 2760aactttggtg aaattggcga
tgcgtcggag tacgtgtatc cggaggggct ggaaaggatc 2820catgagttta tctatccctg
gatcaactct accgacctga aggcatcgtc tgacgattct 2880aactacggct gggaagactc
caagtatatt cccgaaggcg ccacggatgg gtctgcccag 2940ccccgtttgc ccgctagtgg
tggtgccgga ggaaaccccg gtctgtacga ggatcttttc 3000cgcgtctctg tgaaggtcaa
gaacacgggc aatgtcgccg gtgatgaagt tcctcagctg 3060tacgtttccc taggcggccc
gaatgagccc aaggtggtac tgcgcaagtt tgagcgtatt 3120cacttggccc cttcgcagga
ggccgtgtgg acaacgaccc ttacccgtcg tgaccttgca 3180aactgggacg tttcggctca
ggactggacc gtcactcctt accccaagac gatctacgtt 3240ggaaactcct cacggaaact
gccgctccag gcctcgctgc ctaaggccca gtaa 32941221097PRTAspergillus
oryzae 122Met Arg Ser Ser Pro Leu Leu Arg Ser Ala Val Val Ala Ala Leu
Pro1 5 10 15Val Leu Ala
Leu Ala Ala Asp Gly Arg Ser Thr Arg Tyr Trp Asp Cys 20
25 30Cys Lys Pro Ser Cys Gly Trp Ala Lys Lys
Ala Pro Val Asn Gln Pro 35 40
45Val Phe Ser Cys Asn Ala Asn Phe Gln Arg Ile Thr Asp Phe Asp Ala 50
55 60Lys Ser Gly Cys Glu Pro Gly Gly Val
Ala Tyr Ser Cys Ala Asp Gln65 70 75
80Thr Pro Trp Ala Val Asn Asp Asp Phe Ala Leu Gly Phe Ala
Ala Thr 85 90 95Ser Ile
Ala Gly Ser Asn Glu Ala Gly Trp Cys Cys Ala Cys Tyr Glu 100
105 110Leu Thr Phe Thr Ser Gly Pro Val Ala
Gly Lys Lys Met Val Val Gln 115 120
125Ser Thr Ser Thr Gly Gly Asp Leu Gly Ser Asn His Phe Asp Leu Asn
130 135 140Ile Pro Gly Gly Gly Val Gly
Ile Phe Asp Gly Cys Thr Pro Gln Phe145 150
155 160Gly Gly Leu Pro Gly Gln Arg Tyr Gly Gly Ile Ser
Ser Arg Asn Glu 165 170
175Cys Asp Arg Phe Pro Asp Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe
180 185 190Asp Trp Phe Lys Asn Ala
Asp Asn Pro Ser Phe Ser Phe Arg Gln Val 195 200
205Gln Cys Pro Ala Glu Leu Val Ala Arg Thr Gly Cys Arg Arg
Asn Asp 210 215 220Asp Gly Asn Phe Pro
Ala Val Gln Ile Pro Met Arg Ser Ser Pro Leu225 230
235 240Leu Arg Ser Ala Val Val Ala Ala Leu Pro
Val Leu Ala Leu Ala Lys 245 250
255Asp Asp Leu Ala Tyr Ser Pro Pro Phe Tyr Pro Ser Pro Trp Ala Asp
260 265 270Gly Gln Gly Glu Trp
Ala Glu Val Tyr Lys Arg Ala Val Asp Ile Val 275
280 285Ser Gln Met Thr Leu Thr Glu Lys Val Asn Leu Thr
Thr Gly Thr Gly 290 295 300Trp Gln Leu
Glu Arg Cys Val Gly Gln Thr Gly Ser Val Pro Arg Leu305
310 315 320Asn Ile Pro Ser Leu Cys Leu
Gln Asp Ser Pro Leu Gly Ile Arg Phe 325
330 335Ser Asp Tyr Asn Ser Ala Phe Pro Ala Gly Val Asn
Val Ala Ala Thr 340 345 350Trp
Asp Lys Thr Leu Ala Tyr Leu Arg Gly Gln Ala Met Gly Glu Glu 355
360 365Phe Ser Asp Lys Gly Ile Asp Val Gln
Leu Gly Pro Ala Ala Gly Pro 370 375
380Leu Gly Ala His Pro Asp Gly Gly Arg Asn Trp Glu Gly Phe Ser Pro385
390 395 400Asp Pro Ala Leu
Thr Gly Val Leu Phe Ala Glu Thr Ile Lys Gly Ile 405
410 415Gln Asp Ala Gly Val Ile Ala Thr Ala Lys
His Tyr Ile Met Asn Glu 420 425
430Gln Glu His Phe Arg Gln Gln Pro Glu Ala Ala Gly Tyr Gly Phe Asn
435 440 445Val Ser Asp Ser Leu Ser Ser
Asn Val Asp Asp Lys Thr Met His Glu 450 455
460Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg Ala Gly Val Gly
Ala465 470 475 480Val Met
Cys Ser Tyr Asn Gln Ile Asn Asn Ser Tyr Gly Cys Glu Asn
485 490 495Ser Glu Thr Leu Asn Lys Leu
Leu Lys Ala Glu Leu Gly Phe Gln Gly 500 505
510Phe Val Met Ser Asp Trp Thr Ala His His Ser Gly Val Gly
Ala Ala 515 520 525Leu Ala Gly Leu
Asp Met Ser Met Pro Gly Asp Val Thr Phe Asp Ser 530
535 540Gly Thr Ser Phe Trp Gly Ala Asn Leu Thr Val Gly
Val Leu Asn Gly545 550 555
560Thr Ile Pro Gln Trp Arg Val Asp Asp Met Ala Val Arg Ile Met Ala
565 570 575Ala Tyr Tyr Lys Val
Gly Arg Asp Thr Lys Tyr Thr Pro Pro Asn Phe 580
585 590Ser Ser Trp Thr Arg Asp Glu Tyr Gly Phe Ala His
Asn His Val Ser 595 600 605Glu Gly
Ala Tyr Glu Arg Val Asn Glu Phe Val Asp Val Gln Arg Asp 610
615 620His Ala Asp Leu Ile Arg Arg Ile Gly Ala Gln
Ser Thr Val Leu Leu625 630 635
640Lys Asn Lys Gly Ala Leu Pro Leu Ser Arg Lys Glu Lys Leu Val Ala
645 650 655Leu Leu Gly Glu
Asp Ala Gly Ser Asn Ser Trp Gly Ala Asn Gly Cys 660
665 670Asp Asp Arg Gly Cys Asp Asn Gly Thr Leu Ala
Met Ala Trp Gly Ser 675 680 685Gly
Thr Ala Asn Phe Pro Tyr Leu Val Thr Pro Glu Gln Ala Ile Gln 690
695 700Asn Glu Val Leu Gln Gly Arg Gly Asn Val
Phe Ala Val Thr Asp Ser705 710 715
720Trp Ala Leu Asp Lys Ile Ala Ala Ala Ala Arg Gln Ala Ser Val
Ser 725 730 735Leu Val Phe
Val Asn Ser Asp Ser Gly Glu Gly Tyr Leu Ser Val Asp 740
745 750Gly Asn Glu Gly Asp Arg Asn Asn Ile Thr
Leu Trp Lys Asn Gly Asp 755 760
765Asn Val Val Lys Thr Ala Ala Asn Asn Cys Asn Asn Thr Val Val Ile 770
775 780Ile His Ser Val Gly Pro Val Leu
Ile Asp Glu Trp Tyr Asp His Pro785 790
795 800Asn Val Thr Gly Ile Leu Trp Ala Gly Leu Pro Gly
Gln Glu Ser Gly 805 810
815Asn Ser Ile Ala Asp Val Leu Tyr Gly Arg Val Asn Pro Gly Ala Lys
820 825 830Ser Pro Phe Thr Trp Gly
Lys Thr Arg Glu Ser Tyr Gly Ser Pro Leu 835 840
845Val Lys Asp Ala Asn Asn Gly Asn Gly Ala Pro Gln Ser Asp
Phe Thr 850 855 860Gln Gly Val Phe Ile
Asp Tyr Arg His Phe Asp Lys Phe Asn Glu Thr865 870
875 880Pro Ile Tyr Glu Phe Gly Tyr Gly Leu Ser
Tyr Thr Thr Phe Glu Leu 885 890
895Ser Asp Leu His Val Gln Pro Leu Asn Ala Ser Arg Tyr Thr Pro Thr
900 905 910Ser Gly Met Thr Glu
Ala Ala Lys Asn Phe Gly Glu Ile Gly Asp Ala 915
920 925Ser Glu Tyr Val Tyr Pro Glu Gly Leu Glu Arg Ile
His Glu Phe Ile 930 935 940Tyr Pro Trp
Ile Asn Ser Thr Asp Leu Lys Ala Ser Ser Asp Asp Ser945
950 955 960Asn Tyr Gly Trp Glu Asp Ser
Lys Tyr Ile Pro Glu Gly Ala Thr Asp 965
970 975Gly Ser Ala Gln Pro Arg Leu Pro Ala Ser Gly Gly
Ala Gly Gly Asn 980 985 990Pro
Gly Leu Tyr Glu Asp Leu Phe Arg Val Ser Val Lys Val Lys Asn 995
1000 1005Thr Gly Asn Val Ala Gly Asp Glu
Val Pro Gln Leu Tyr Val Ser 1010 1015
1020Leu Gly Gly Pro Asn Glu Pro Lys Val Val Leu Arg Lys Phe Glu
1025 1030 1035Arg Ile His Leu Ala Pro
Ser Gln Glu Ala Val Trp Thr Thr Thr 1040 1045
1050Leu Thr Arg Arg Asp Leu Ala Asn Trp Asp Val Ser Ala Gln
Asp 1055 1060 1065Trp Thr Val Thr Pro
Tyr Pro Lys Thr Ile Tyr Val Gly Asn Ser 1070 1075
1080Ser Arg Lys Leu Pro Leu Gln Ala Ser Leu Pro Lys Ala
Gln 1085 1090
10951233294DNAAspergillus oryzae 123atgcgttcct cccccctcct ccgctccgcc
gttgtggccg ccctgccggt gttggccctt 60gccgctgatg gcaggtccac ccgctactgg
gactgctgca agccttcgtg cggctgggcc 120aagaaggctc ccgtgaacca gcctgtcttt
tcctgcaacg ccaacttcca gcgtatcacg 180gacttcgacg ccaagtccgg ctgcgagccg
ggcggtgtcg cctactcgtg cgccgaccag 240accccatggg ctgtgaacga cgacttcgcg
ctcggttttg ctgccacctc tattgccggc 300agcaatgagg cgggctggtg ctgcgcctgc
tacgagctca ccttcacatc cggtcctgtt 360gctggcaaga agatggtcgt ccagtccacc
agcactggcg gtgatcttgg cagcaaccac 420ttcgatctca acatccccgg cggcggcgtc
ggcatcttcg acggatgcac tccccagttc 480ggtggtctgc ccggccagcg ctacggcggc
atctcgtccc gcaacgagtg cgatcggttc 540cccgacgccc tcaagcccgg ctgctactgg
cgcttcgact ggttcaagaa cgccgacaat 600ccgagcttca gcttccgtca ggtccagtgc
ccagccgagc tcgtcgctcg caccggatgc 660cgccgcaacg acgacggcaa cttccctgcc
gtccagatcc ccatgcgttc ctcccccctc 720ctccgctccg ccgttgtggc cgccctgccg
gtgttggccc ttgccaagga tgatctcgcg 780tactcccctc ctttctaccc ttccccatgg
gcagatggtc agggtgaatg ggcggaagta 840tacaaacgcg ctgtagacat agtttcccag
atgacgttga cagagaaagt caacttaacg 900actggaacag gatggcaact agagaggtgt
gttggacaaa ctggcagtgt tcccagactc 960aacatcccca gcttgtgttt gcaggatagt
cctcttggta ttcgtttctc ggactacaat 1020tcagctttcc ctgcgggtgt taatgtcgct
gccacctggg acaagacgct cgcctacctt 1080cgtggtcagg caatgggtga ggagttcagt
gataagggta ttgacgttca gctgggtcct 1140gctgctggcc ctctcggtgc tcatccggat
ggcggtagaa actgggaaag tttctcacca 1200gatccagccc tcaccggtgt actttttgcg
gagacgatta agggtattca agatgctggt 1260gtcattgcga cagctaagca ttatatcatg
aacgaacaag agcatttccg ccaacaaccc 1320gaggctgcgg gttacggatt caacgtaagc
gacagtttga gttccaacgt tgatgacaag 1380actatgcatg aattgtacct ctggcccttc
gcggatgcag tacgcgctgg agtcggtgct 1440gttatgtgct cttacaacca aatcaacaac
agctacggtt gcgagaatag cgaaactctg 1500aacaagcttt tgaaggcgga gcttggtttc
caaggcttcg tcatgagtga ttggaccgct 1560caacacagcg gcgtaggcgc tgctttagca
ggtctggata tgtcgatgcc cggtgatgtt 1620accttcgata gtggtacgtc tttctggggt
gcaaacttga cggtcggtgt ccttaacggt 1680acaatccccc aatggcgtgt tgatgacatg
gctgtccgta tcatggccgc ttattacaag 1740gttggccgcg acaccaaata cacccctccc
aacttcagct cgtggaccag ggacgaatat 1800ggtttcgcgc ataaccatgt ttcggaaggt
gcttacgaga gggtcaacga attcgtggac 1860gtgcaacgcg atcatgccga cctaatccgt
cgcatcggcg cgcagagcac tgttctgctg 1920aagaacaagg gtgccttgcc cttgagccgc
aaggaaaagc tggtcgccct tctgggagag 1980gatgcgggtt ccaactcgtg gggcgctaac
ggctgtgatg accgtggttg cgataacggt 2040acccttgcca tggcctgggg tagcggtact
gcgaatttcc catacctcgt gacaccagag 2100caggcgattc agaacgaagt tcttcagggc
cgtggtaatg tcttcgccgt gaccgacagt 2160tgggcgctcg acaagatcgc tgcggctgcc
cgccaggcca gcgtatctct cgtgttcgtc 2220aactccgact caggagaagg ctatcttagt
gtggatggaa atgagggcga tcgtaacaac 2280atcactctgt ggaagaacgg cgacaatgtg
gtcaagaccg cagcgaataa ctgtaacaac 2340accgttgtca tcatccactc cgtcggacca
gttttgatcg atgaatggta tgaccacccc 2400aatgtcactg gtattctctg ggctggtctg
ccaggccagg agtctggtaa ctccattgcc 2460gatgtgctgt acggtcgtgt caaccctggc
gccaagtctc ctttcacttg gggcaagacc 2520cgggagtcgt atggttctcc cttggtcaag
gatgccaaca atggcaacgg agcgccccag 2580tctgatttca cccagggtgt tttcatcgat
taccgccatt tcgataagtt caatgagacc 2640cctatctacg agtttggcta cggcttgagc
tacaccacct tcgagctctc cgacctccat 2700gttcagcccc tgaacgcgtc ccgatacact
cccaccagtg gcatgactga agctgcaaag 2760aactttggtg aaattggcga tgcgtcggag
tacgtgtatc cggaggggct ggaaaggatc 2820catgagttta tctatccctg gatcaactct
accgacctga aggcatcgtc tgacgattct 2880aactacggct gggaagactc caagtatatt
cccgaaggcg ccacggatgg gtctgcccag 2940ccccgtttgc ccgctagtgg tggtgccgga
ggaaaccccg gtctgtacga ggatcttttc 3000cgcgtctctg tgaaggtcaa gaacacgggc
aatgtcgccg gtgatgaagt tcctcagctg 3060tacgtttccc taggcggccc gaatgagccc
aaggtggtac tgcgcaagtt tgagcgtatt 3120cacttggccc cttcgcagga ggccgtgtgg
acaacgaccc ttacccgtcg tgaccttgca 3180aactgggacg tttcggctca ggactggacc
gtcactcctt accccaagac gatctacgtt 3240ggaaactcct cacggaaact gccgctccag
gcctcgctgc ctaaggccca gtaa 32941241097PRTAspergillus oryzae
124Met Arg Ser Ser Pro Leu Leu Arg Ser Ala Val Val Ala Ala Leu Pro1
5 10 15Val Leu Ala Leu Ala Ala
Asp Gly Arg Ser Thr Arg Tyr Trp Asp Cys 20 25
30Cys Lys Pro Ser Cys Gly Trp Ala Lys Lys Ala Pro Val
Asn Gln Pro 35 40 45Val Phe Ser
Cys Asn Ala Asn Phe Gln Arg Ile Thr Asp Phe Asp Ala 50
55 60Lys Ser Gly Cys Glu Pro Gly Gly Val Ala Tyr Ser
Cys Ala Asp Gln65 70 75
80Thr Pro Trp Ala Val Asn Asp Asp Phe Ala Leu Gly Phe Ala Ala Thr
85 90 95Ser Ile Ala Gly Ser Asn
Glu Ala Gly Trp Cys Cys Ala Cys Tyr Glu 100
105 110Leu Thr Phe Thr Ser Gly Pro Val Ala Gly Lys Lys
Met Val Val Gln 115 120 125Ser Thr
Ser Thr Gly Gly Asp Leu Gly Ser Asn His Phe Asp Leu Asn 130
135 140Ile Pro Gly Gly Gly Val Gly Ile Phe Asp Gly
Cys Thr Pro Gln Phe145 150 155
160Gly Gly Leu Pro Gly Gln Arg Tyr Gly Gly Ile Ser Ser Arg Asn Glu
165 170 175Cys Asp Arg Phe
Pro Asp Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe 180
185 190Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe
Ser Phe Arg Gln Val 195 200 205Gln
Cys Pro Ala Glu Leu Val Ala Arg Thr Gly Cys Arg Arg Asn Asp 210
215 220Asp Gly Asn Phe Pro Ala Val Gln Ile Pro
Met Arg Ser Ser Pro Leu225 230 235
240Leu Arg Ser Ala Val Val Ala Ala Leu Pro Val Leu Ala Leu Ala
Lys 245 250 255Asp Asp Leu
Ala Tyr Ser Pro Pro Phe Tyr Pro Ser Pro Trp Ala Asp 260
265 270Gly Gln Gly Glu Trp Ala Glu Val Tyr Lys
Arg Ala Val Asp Ile Val 275 280
285Ser Gln Met Thr Leu Thr Glu Lys Val Asn Leu Thr Thr Gly Thr Gly 290
295 300Trp Gln Leu Glu Arg Cys Val Gly
Gln Thr Gly Ser Val Pro Arg Leu305 310
315 320Asn Ile Pro Ser Leu Cys Leu Gln Asp Ser Pro Leu
Gly Ile Arg Phe 325 330
335Ser Asp Tyr Asn Ser Ala Phe Pro Ala Gly Val Asn Val Ala Ala Thr
340 345 350Trp Asp Lys Thr Leu Ala
Tyr Leu Arg Gly Gln Ala Met Gly Glu Glu 355 360
365Phe Ser Asp Lys Gly Ile Asp Val Gln Leu Gly Pro Ala Ala
Gly Pro 370 375 380Leu Gly Ala His Pro
Asp Gly Gly Arg Asn Trp Glu Ser Phe Ser Pro385 390
395 400Asp Pro Ala Leu Thr Gly Val Leu Phe Ala
Glu Thr Ile Lys Gly Ile 405 410
415Gln Asp Ala Gly Val Ile Ala Thr Ala Lys His Tyr Ile Met Asn Glu
420 425 430Gln Glu His Phe Arg
Gln Gln Pro Glu Ala Ala Gly Tyr Gly Phe Asn 435
440 445Val Ser Asp Ser Leu Ser Ser Asn Val Asp Asp Lys
Thr Met His Glu 450 455 460Leu Tyr Leu
Trp Pro Phe Ala Asp Ala Val Arg Ala Gly Val Gly Ala465
470 475 480Val Met Cys Ser Tyr Asn Gln
Ile Asn Asn Ser Tyr Gly Cys Glu Asn 485
490 495Ser Glu Thr Leu Asn Lys Leu Leu Lys Ala Glu Leu
Gly Phe Gln Gly 500 505 510Phe
Val Met Ser Asp Trp Thr Ala Gln His Ser Gly Val Gly Ala Ala 515
520 525Leu Ala Gly Leu Asp Met Ser Met Pro
Gly Asp Val Thr Phe Asp Ser 530 535
540Gly Thr Ser Phe Trp Gly Ala Asn Leu Thr Val Gly Val Leu Asn Gly545
550 555 560Thr Ile Pro Gln
Trp Arg Val Asp Asp Met Ala Val Arg Ile Met Ala 565
570 575Ala Tyr Tyr Lys Val Gly Arg Asp Thr Lys
Tyr Thr Pro Pro Asn Phe 580 585
590Ser Ser Trp Thr Arg Asp Glu Tyr Gly Phe Ala His Asn His Val Ser
595 600 605Glu Gly Ala Tyr Glu Arg Val
Asn Glu Phe Val Asp Val Gln Arg Asp 610 615
620His Ala Asp Leu Ile Arg Arg Ile Gly Ala Gln Ser Thr Val Leu
Leu625 630 635 640Lys Asn
Lys Gly Ala Leu Pro Leu Ser Arg Lys Glu Lys Leu Val Ala
645 650 655Leu Leu Gly Glu Asp Ala Gly
Ser Asn Ser Trp Gly Ala Asn Gly Cys 660 665
670Asp Asp Arg Gly Cys Asp Asn Gly Thr Leu Ala Met Ala Trp
Gly Ser 675 680 685Gly Thr Ala Asn
Phe Pro Tyr Leu Val Thr Pro Glu Gln Ala Ile Gln 690
695 700Asn Glu Val Leu Gln Gly Arg Gly Asn Val Phe Ala
Val Thr Asp Ser705 710 715
720Trp Ala Leu Asp Lys Ile Ala Ala Ala Ala Arg Gln Ala Ser Val Ser
725 730 735Leu Val Phe Val Asn
Ser Asp Ser Gly Glu Gly Tyr Leu Ser Val Asp 740
745 750Gly Asn Glu Gly Asp Arg Asn Asn Ile Thr Leu Trp
Lys Asn Gly Asp 755 760 765Asn Val
Val Lys Thr Ala Ala Asn Asn Cys Asn Asn Thr Val Val Ile 770
775 780Ile His Ser Val Gly Pro Val Leu Ile Asp Glu
Trp Tyr Asp His Pro785 790 795
800Asn Val Thr Gly Ile Leu Trp Ala Gly Leu Pro Gly Gln Glu Ser Gly
805 810 815Asn Ser Ile Ala
Asp Val Leu Tyr Gly Arg Val Asn Pro Gly Ala Lys 820
825 830Ser Pro Phe Thr Trp Gly Lys Thr Arg Glu Ser
Tyr Gly Ser Pro Leu 835 840 845Val
Lys Asp Ala Asn Asn Gly Asn Gly Ala Pro Gln Ser Asp Phe Thr 850
855 860Gln Gly Val Phe Ile Asp Tyr Arg His Phe
Asp Lys Phe Asn Glu Thr865 870 875
880Pro Ile Tyr Glu Phe Gly Tyr Gly Leu Ser Tyr Thr Thr Phe Glu
Leu 885 890 895Ser Asp Leu
His Val Gln Pro Leu Asn Ala Ser Arg Tyr Thr Pro Thr 900
905 910Ser Gly Met Thr Glu Ala Ala Lys Asn Phe
Gly Glu Ile Gly Asp Ala 915 920
925Ser Glu Tyr Val Tyr Pro Glu Gly Leu Glu Arg Ile His Glu Phe Ile 930
935 940Tyr Pro Trp Ile Asn Ser Thr Asp
Leu Lys Ala Ser Ser Asp Asp Ser945 950
955 960Asn Tyr Gly Trp Glu Asp Ser Lys Tyr Ile Pro Glu
Gly Ala Thr Asp 965 970
975Gly Ser Ala Gln Pro Arg Leu Pro Ala Ser Gly Gly Ala Gly Gly Asn
980 985 990Pro Gly Leu Tyr Glu Asp
Leu Phe Arg Val Ser Val Lys Val Lys Asn 995 1000
1005Thr Gly Asn Val Ala Gly Asp Glu Val Pro Gln Leu
Tyr Val Ser 1010 1015 1020Leu Gly Gly
Pro Asn Glu Pro Lys Val Val Leu Arg Lys Phe Glu 1025
1030 1035Arg Ile His Leu Ala Pro Ser Gln Glu Ala Val
Trp Thr Thr Thr 1040 1045 1050Leu Thr
Arg Arg Asp Leu Ala Asn Trp Asp Val Ser Ala Gln Asp 1055
1060 1065Trp Thr Val Thr Pro Tyr Pro Lys Thr Ile
Tyr Val Gly Asn Ser 1070 1075 1080Ser
Arg Lys Leu Pro Leu Gln Ala Ser Leu Pro Lys Ala Gln 1085
1090 109512538DNATrichoderma reesei 125actggattta
ccatgaacaa gtccgtggct ccattgct
3812638DNATrichoderma reesei 126tcacctctag ttaattaact actttcttgc gagacacg
3812719PRTThielavia
terrestrisMISC_FEATURE(1)..(1)X=I,L,M, OR Vmisc_feature(3)..(6)Xaa can be
any naturally occurring amino acidmisc_feature(8)..(8)Xaa can be any
naturally occurring amino acidMISC_FEATURE(10)..(10)X=I,L,M, OR
Vmisc_feature(11)..(11)Xaa can be any naturally occurring amino
acidmisc_feature(13)..(13)Xaa can be any naturally occurring amino
acidMISC_FEATURE(14)..(14)X=E OR Qmisc_feature(15)..(18)Xaa can be any
naturally occurring amino acidMISC_FEATURE(19)..(19)X=H,N, OR Q 127Xaa
Pro Xaa Xaa Xaa Xaa Gly Xaa Tyr Xaa Xaa Arg Xaa Xaa Xaa Xaa1
5 10 15Xaa Xaa Xaa12820PRTThielavia
terrestrisMISC_FEATURE(1)..(1)X=I,L,M, OR Vmisc_feature(3)..(7)Xaa can be
any naturally occurring amino acidmisc_feature(9)..(9)Xaa can be any
naturally occurring amino acidMISC_FEATURE(11)..(11)X=I,L,M, OR
Vmisc_feature(12)..(12)Xaa can be any naturally occurring amino
acidmisc_feature(14)..(14)Xaa can be any naturally occurring amino
acidMISC_FEATURE(15)..(15)X=E OR Qmisc_feature(16)..(19)Xaa can be any
naturally occurring amino acidMISC_FEATURE(20)..(20)X=H,N, OR Q 128Xaa
Pro Xaa Xaa Xaa Xaa Xaa Gly Xaa Tyr Xaa Xaa Arg Xaa Xaa Xaa1
5 10 15Xaa Xaa Xaa Xaa
201299PRTThielavia terrestrismisc_feature(2)..(2)Xaa can be any naturally
occurring amino acidmisc_feature(5)..(7)Xaa can be any naturally
occurring amino acidMISC_FEATURE(8)..(8)X= Y OR WMISC_FEATURE(9)..(9)X=
A,I,L,M OR V 129His Xaa Gly Pro Xaa Xaa Xaa Xaa Xaa1
513010PRTThielavia terrestrismisc_feature(2)..(3)Xaa can be any naturally
occurring amino acidmisc_feature(6)..(8)Xaa can be any naturally
occurring amino acidMISC_FEATURE(9)..(9)X= Y OR WMISC_FEATURE(10)..(10)X=
Y OR W 130His Xaa Xaa Gly Pro Xaa Xaa Xaa Xaa Xaa1 5
1013111PRTThielavia terrestrisMISC_FEATURE(1)..(1)X= E OR
Qmisc_feature(2)..(2)Xaa can be any naturally occurring amino
acidmisc_feature(4)..(5)Xaa can be any naturally occurring amino
acidmisc_feature(7)..(7)Xaa can be any naturally occurring amino
acidMISC_FEATURE(8)..(8)X= E,H,Q OR NMISC_FEATURE(9)..(9)X=F,I,L, OR
Vmisc_feature(10)..(10)Xaa can be any naturally occurring amino
acidMISC_FEATURE(11)..(11)X=I,L,OR V 131Xaa Xaa Tyr Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa1 5 101329PRTThielavia
terrestrismisc_feature(2)..(2)Xaa can be any naturally occurring amino
acidmisc_feature(5)..(7)Xaa can be any naturally occurring amino
acidMISC_FEATURE(8)..(8)X= Y OR WMISC_FEATURE(9)..(9)X= A,I,L,M OR V
132His Xaa Gly Pro Xaa Xaa Xaa Xaa Xaa1 513310PRTThielavia
terrestrismisc_feature(2)..(3)Xaa can be any naturally occurring amino
acidmisc_feature(6)..(8)Xaa can be any naturally occurring amino
acidMISC_FEATURE(9)..(9)X= Y OR WMISC_FEATURE(10)..(10)X= A,I,L,M OR V
133His Xaa Xaa Gly Pro Xaa Xaa Xaa Xaa Xaa1 5
1013411PRTThielavia terrestrisMISC_FEATURE(1)..(1)X= E OR
Qmisc_feature(2)..(2)Xaa can be any naturally occurring amino
acidmisc_feature(4)..(5)Xaa can be any naturally occurring amino
acidmisc_feature(7)..(7)Xaa can be any naturally occurring amino
acidMISC_FEATURE(8)..(8)X= E,H,Q OR NMISC_FEATURE(9)..(9)X=F,I,L, OR
Vmisc_feature(10)..(10)Xaa can be any naturally occurring amino
acidMISC_FEATURE(11)..(11)X=I,L,OR V 134Xaa Xaa Tyr Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa1 5 101359PRTThielavia
terrestrismisc_feature(2)..(2)Xaa can be any naturally occurring amino
acidmisc_feature(5)..(7)Xaa can be any naturally occurring amino
acidMISC_FEATURE(8)..(8)X= Y OR WMISC_FEATURE(9)..(9)X= A,I,L,M OR V
135His Xaa Gly Pro Xaa Xaa Xaa Xaa Xaa1 513610PRTThielavia
terrestrismisc_feature(2)..(3)Xaa can be any naturally occurring amino
acidmisc_feature(6)..(8)Xaa can be any naturally occurring amino
acidMISC_FEATURE(9)..(9)X= Y OR WMISC_FEATURE(10)..(10)X= A,I,L,M OR V
136His Xaa Xaa Gly Pro Xaa Xaa Xaa Xaa Xaa1 5
1013711PRTThielavia terrestrisMISC_FEATURE(1)..(1)X= E OR
Qmisc_feature(2)..(2)Xaa can be any naturally occurring amino
acidmisc_feature(4)..(5)Xaa can be any naturally occurring amino
acidmisc_feature(7)..(7)Xaa can be any naturally occurring amino
acidMISC_FEATURE(8)..(8)X= E,H,Q OR NMISC_FEATURE(9)..(9)X=F,I,L, OR
Vmisc_feature(10)..(10)Xaa can be any naturally occurring amino
acidMISC_FEATURE(11)..(11)X=I,L,OR V 137Xaa Xaa Tyr Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa1 5 101389PRTThielavia
terrestrismisc_feature(2)..(2)Xaa can be any naturally occurring amino
acidmisc_feature(5)..(7)Xaa can be any naturally occurring amino
acidMISC_FEATURE(8)..(8)X= Y OR WMISC_FEATURE(9)..(9)X= A,I,L,M OR V
138His Xaa Gly Pro Xaa Xaa Xaa Xaa Xaa1 513910PRTThielavia
terrestrismisc_feature(2)..(3)Xaa can be any naturally occurring amino
acidmisc_feature(6)..(8)Xaa can be any naturally occurring amino
acidMISC_FEATURE(9)..(9)X= Y OR WMISC_FEATURE(10)..(10)X= A,I,L,M OR V
139His Xaa Xaa Gly Pro Xaa Xaa Xaa Xaa Xaa1 5
1014011PRTThielavia terrestrisMISC_FEATURE(1)..(1)X= E OR
Qmisc_feature(2)..(2)Xaa can be any naturally occurring amino
acidmisc_feature(4)..(5)Xaa can be any naturally occurring amino
acidmisc_feature(7)..(7)Xaa can be any naturally occurring amino
acidMISC_FEATURE(8)..(8)X= E,H,Q OR NMISC_FEATURE(9)..(9)X=F,I,L, OR
Vmisc_feature(10)..(10)Xaa can be any naturally occurring amino
acidMISC_FEATURE(11)..(11)X=I,L,OR V 140Xaa Xaa Tyr Xaa Xaa Cys Xaa Xaa
Xaa Xaa Xaa1 5 1014119PRTThielavia
terrestrisMISC_FEATURE(1)..(1)X=I,L,M OR Vmisc_feature(3)..(6)Xaa can be
any naturally occurring amino acidmisc_feature(8)..(8)Xaa can be any
naturally occurring amino acidMISC_FEATURE(10)..(10)X=I,L,M OR
Vmisc_feature(11)..(11)Xaa can be any naturally occurring amino
acidmisc_feature(13)..(13)Xaa can be any naturally occurring amino
acidMISC_FEATURE(14)..(14)X= E OR Qmisc_feature(15)..(17)Xaa can be any
naturally occurring amino acidMISC_FEATURE(19)..(19)X= H,N, OR Q 141Xaa
Pro Xaa Xaa Xaa Xaa Gly Xaa Tyr Xaa Xaa Arg Xaa Xaa Xaa Xaa1
5 10 15Xaa Ala Xaa14220PRTThielavia
terrestrisMISC_FEATURE(1)..(1)X=I,L,M OR Vmisc_feature(3)..(7)Xaa can be
any naturally occurring amino acidmisc_feature(9)..(9)Xaa can be any
naturally occurring amino acidMISC_FEATURE(11)..(11)X=I,L,M OR
Vmisc_feature(12)..(12)Xaa can be any naturally occurring amino
acidmisc_feature(14)..(14)Xaa can be any naturally occurring amino
acidMISC_FEATURE(15)..(15)X=E OR Qmisc_feature(16)..(18)Xaa can be any
naturally occurring amino acidMISC_FEATURE(20)..(20)X= H,N, OR Q 142Xaa
Pro Xaa Xaa Xaa Xaa Xaa Gly Xaa Tyr Xaa Xaa Arg Xaa Xaa Xaa1
5 10 15Xaa Xaa Ala Xaa
201431035DNAAspergillus aculeatus 143atgaagtata ttcctctcgt tattgcagtt
gctgccggcc tggcacgtcc ggctactgcc 60cactacatct tcagcaagct cgtgctgaac
ggagaggcat ctgcggactg gcaatacatc 120cgcgagacta ctcgcagcat agtctatgag
ccgaccaagt acacctctac cttcgataac 180ctaacaccca gcgatagcga cttccgctgt
aatctcggtt ccttcagcaa tgctgcgaag 240accgaggtcg ctgaggttgc ggcaggcgat
accatcgcaa tgaagctatt ctacgacacc 300agtattgcgc atcctggccc gggacaagtt
tatatgtcca aggcaccgac cggcaatgtt 360caggaatacc aaggagacgg ggattggttc
aaaatctggg aaaagaccct ttgcaacacg 420gatggtgatc tgactacaga ggcctggtgc
acctggggca tgtcacagtt tgaatttcaa 480atcccagctg cgaccccggc aggagagtac
ctagtgcgcg ccgagcatat aggcctgcat 540ggcgctcaag cgaacgaggc cgaattcttc
tacagctgtg cgcagatcaa ggttacaggc 600tcgggaactg gatctcccag tctcacgtat
caaattcctg gtctctataa cgacactatg 660accctgttca atggcctcaa tctttggact
gattcagccg agaaggtgca gctggatttc 720ctggagacgc caattgggga cgacgtgtgg
agcggagcag gctcggggag cccatctgct 780gccacctctt cgaccagcgg tgcaactctt
gcagctcagg gtacaactac ctctgccgcg 840catgctcagg cccagaccac cattaccacc
agcaccagca ccatcacgtc tctcgaatca 900gccagctcaa ccgatctcgt tgcgcagtat
ggtcagtgcg gaggccttaa ctggtccggt 960ccaaccgagt gtgagacacc ttatacctgt
gtgcagcaga acccttacta ccatcaatgc 1020gtgaattcgt gctga
1035144344PRTAspergillus aculeatus
144Met Lys Tyr Ile Pro Leu Val Ile Ala Val Ala Ala Gly Leu Ala Arg1
5 10 15Pro Ala Thr Ala His Tyr
Ile Phe Ser Lys Leu Val Leu Asn Gly Glu 20 25
30Ala Ser Ala Asp Trp Gln Tyr Ile Arg Glu Thr Thr Arg
Ser Ile Val 35 40 45Tyr Glu Pro
Thr Lys Tyr Thr Ser Thr Phe Asp Asn Leu Thr Pro Ser 50
55 60Asp Ser Asp Phe Arg Cys Asn Leu Gly Ser Phe Ser
Asn Ala Ala Lys65 70 75
80Thr Glu Val Ala Glu Val Ala Ala Gly Asp Thr Ile Ala Met Lys Leu
85 90 95Phe Tyr Asp Thr Ser Ile
Ala His Pro Gly Pro Gly Gln Val Tyr Met 100
105 110Ser Lys Ala Pro Thr Gly Asn Val Gln Glu Tyr Gln
Gly Asp Gly Asp 115 120 125Trp Phe
Lys Ile Trp Glu Lys Thr Leu Cys Asn Thr Asp Gly Asp Leu 130
135 140Thr Thr Glu Ala Trp Cys Thr Trp Gly Met Ser
Gln Phe Glu Phe Gln145 150 155
160Ile Pro Ala Ala Thr Pro Ala Gly Glu Tyr Leu Val Arg Ala Glu His
165 170 175Ile Gly Leu His
Gly Ala Gln Ala Asn Glu Ala Glu Phe Phe Tyr Ser 180
185 190Cys Ala Gln Ile Lys Val Thr Gly Ser Gly Thr
Gly Ser Pro Ser Leu 195 200 205Thr
Tyr Gln Ile Pro Gly Leu Tyr Asn Asp Thr Met Thr Leu Phe Asn 210
215 220Gly Leu Asn Leu Trp Thr Asp Ser Ala Glu
Lys Val Gln Leu Asp Phe225 230 235
240Leu Glu Thr Pro Ile Gly Asp Asp Val Trp Ser Gly Ala Gly Ser
Gly 245 250 255Ser Pro Ser
Ala Ala Thr Ser Ser Thr Ser Gly Ala Thr Leu Ala Ala 260
265 270Gln Gly Thr Thr Thr Ser Ala Ala His Ala
Gln Ala Gln Thr Thr Ile 275 280
285Thr Thr Ser Thr Ser Thr Ile Thr Ser Leu Glu Ser Ala Ser Ser Thr 290
295 300Asp Leu Val Ala Gln Tyr Gly Gln
Cys Gly Gly Leu Asn Trp Ser Gly305 310
315 320Pro Thr Glu Cys Glu Thr Pro Tyr Thr Cys Val Gln
Gln Asn Pro Tyr 325 330
335Tyr His Gln Cys Val Asn Ser Cys 3401451170DNAAspergillus
aculeatus 145atgaagtcct ctactttcgg tatgctcgct ctggcagcag cagccaagat
ggtcgatgcc 60cacaccaccg tcttcgccgt ctggatcaac ggcgaggacc agggtctggg
caacagtgcc 120agtggctaca tccggtctcc ccccagcaac agccccgtca aggacgtgac
ctcgaccgac 180atcacctgca acgtcaacgg cgaccaggcg gcggctaaga ccctctccgt
caagggcggc 240gacgtcgtca ccttcgagtg gcaccacgac agccgggacg cctccgacga
catcatcgcc 300tcctcccaca agggccccgt catggtctac atggccccga ccaccgccgg
cagcagcggc 360aagaactggg tcaagatcgc cgaggacgga tactccgacg gcacctgggc
cgtcgacacc 420ctgatcgcca acagcggcaa gcacaacatc accgtccccg acgtccccgc
cggcgactac 480ctcttccgcc cggagatcat cgccctccac gaggccgaga acgagggcgg
cgcccagttc 540tacatggagt gtgtccagtt caaggtcacc tccgacggtg ccaacactct
gcccgacggt 600gtcagcctgc ccggcgccta ctccgccact gaccccggta tcctcttcaa
catgtacggc 660tccttcgaca gctatcccat ccccggtccc tccgtctggg atggcactag
ctctggctct 720tcctcttctt cctcttcttc ctcttccagc tcttccgccg ccgctgccgt
tgttgccacc 780tcctcttcct cttcctctgc ttccatcgag gccgtgacca ccaagggtgc
cgtcgccgcc 840gtctccaccg ccgccgccgt ggctcctacc accaccaccg ctgcccccac
caccttcgcc 900acggccgtcg cctccaccaa gaaggccact gcctgccgca acaagaccaa
gtcctcctcc 960gctgccacca ccgccgccgc cgtcgccgag accacctctt ccaccgctgc
cgccaccgct 1020gctgcttcct ctgcctcttc cgcctccggc accgccggca agtacgagcg
ctgcggtggc 1080cagggctgga ccggtgccac cacctgcgtt gatggctgga cctgcaagca
gtggaaccct 1140tactactacc agtgcgttga gtctgcctag
1170146389PRTAspergillus aculeatus 146Met Lys Ser Ser Thr Phe
Gly Met Leu Ala Leu Ala Ala Ala Ala Lys1 5
10 15Met Val Asp Ala His Thr Thr Val Phe Ala Val Trp
Ile Asn Gly Glu 20 25 30Asp
Gln Gly Leu Gly Asn Ser Ala Ser Gly Tyr Ile Arg Ser Pro Pro 35
40 45Ser Asn Ser Pro Val Lys Asp Val Thr
Ser Thr Asp Ile Thr Cys Asn 50 55
60Val Asn Gly Asp Gln Ala Ala Ala Lys Thr Leu Ser Val Lys Gly Gly65
70 75 80Asp Val Val Thr Phe
Glu Trp His His Asp Ser Arg Asp Ala Ser Asp 85
90 95Asp Ile Ile Ala Ser Ser His Lys Gly Pro Val
Met Val Tyr Met Ala 100 105
110Pro Thr Thr Ala Gly Ser Ser Gly Lys Asn Trp Val Lys Ile Ala Glu
115 120 125Asp Gly Tyr Ser Asp Gly Thr
Trp Ala Val Asp Thr Leu Ile Ala Asn 130 135
140Ser Gly Lys His Asn Ile Thr Val Pro Asp Val Pro Ala Gly Asp
Tyr145 150 155 160Leu Phe
Arg Pro Glu Ile Ile Ala Leu His Glu Ala Glu Asn Glu Gly
165 170 175Gly Ala Gln Phe Tyr Met Glu
Cys Val Gln Phe Lys Val Thr Ser Asp 180 185
190Gly Ala Asn Thr Leu Pro Asp Gly Val Ser Leu Pro Gly Ala
Tyr Ser 195 200 205Ala Thr Asp Pro
Gly Ile Leu Phe Asn Met Tyr Gly Ser Phe Asp Ser 210
215 220Tyr Pro Ile Pro Gly Pro Ser Val Trp Asp Gly Thr
Ser Ser Gly Ser225 230 235
240Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ala Ala Ala Ala
245 250 255Val Val Ala Thr Ser
Ser Ser Ser Ser Ser Ala Ser Ile Glu Ala Val 260
265 270Thr Thr Lys Gly Ala Val Ala Ala Val Ser Thr Ala
Ala Ala Val Ala 275 280 285Pro Thr
Thr Thr Thr Ala Ala Pro Thr Thr Phe Ala Thr Ala Val Ala 290
295 300Ser Thr Lys Lys Ala Thr Ala Cys Arg Asn Lys
Thr Lys Ser Ser Ser305 310 315
320Ala Ala Thr Thr Ala Ala Ala Val Ala Glu Thr Thr Ser Ser Thr Ala
325 330 335Ala Ala Thr Ala
Ala Ala Ser Ser Ala Ser Ser Ala Ser Gly Thr Ala 340
345 350Gly Lys Tyr Glu Arg Cys Gly Gly Gln Gly Trp
Thr Gly Ala Thr Thr 355 360 365Cys
Val Asp Gly Trp Thr Cys Lys Gln Trp Asn Pro Tyr Tyr Tyr Gln 370
375 380Cys Val Glu Ser
Ala3851471221DNAAspergillus aculeatus 147atgcgtcagg ctcagtcttt gtccctcttg
acagctcttc tgtctgccac gcgtgtggct 60ggacacggtc acgtcactaa cgttgtcgtc
aacggtgttt actacgaggg cttcgatatc 120aacagcttcc cctacgagtc cgatccccct
aaggtggcgg cttggaccac tcctaacact 180ggcaacggtt tcatttcccc cagcgactac
ggtaccgatg acattatttg ccaccagaat 240gccaccaacg cccaggccca cattgttgtt
gcggctggtg acaagatcaa catccagtgg 300accgcgtggc ccgattccca ccacggtcct
gtccttgact acctcgctcg ctgcgacggt 360gagtgtgaga cggttgataa gaccactctt
gagtttttca agatcgacgg cgtcggtctc 420atcagtgaca ccgaagtgcc cggtacctgg
ggagatgacc agctgatcgc caacaacaac 480agctggttgg tcgagatccc cccgaccatt
gctcctggca actatgttct tcgccacgag 540cttatcgctc tccacagcgc cggcactgaa
gatggtgctc agaactaccc ccagtgtttc 600aacctccagg tcactggctc cggtactgac
gagcccgctg gtaccctcgg caccaagctc 660tacactgagg atgaggctgg tatcgttgtg
aacatctaca cctctctgtc ttcctatgcc 720gtccccggcc ccacccagta cagcggcgcc
gtctctgtca gccaatccac ttcggccatt 780acctccaccg gaactgctgt tgtcggtagc
ggcagcgctg ttgccacctc tgccgccgcg 840gctaccacca gcgctgctgc ttcttctgcc
gctgctgcta ccaccgctgc tgccgttacc 900agcgccaatg ccaacactca gattgcccag
cccagcagca gctcttctta ctcccagatc 960gccgtgcagg tgccctcctc ctggaccacc
cttgtgaccg tcactcctcc cgccgccgcc 1020gccaccaccc ctgctgccgt ccctgagcct
cagaccccct ctgccagctc tggagccacc 1080actaccagca gcagcagcgg cgccgcccag
tctctctacg gccagtgcgg tggtatcaac 1140tggaccggag ctacctcttg cgttgagggc
gctacttgct accagtacaa cccttactac 1200taccagtgca tctctgccta a
1221148406PRTAspergillus aculeatus
148Met Arg Gln Ala Gln Ser Leu Ser Leu Leu Thr Ala Leu Leu Ser Ala1
5 10 15Thr Arg Val Ala Gly His
Gly His Val Thr Asn Val Val Val Asn Gly 20 25
30Val Tyr Tyr Glu Gly Phe Asp Ile Asn Ser Phe Pro Tyr
Glu Ser Asp 35 40 45Pro Pro Lys
Val Ala Ala Trp Thr Thr Pro Asn Thr Gly Asn Gly Phe 50
55 60Ile Ser Pro Ser Asp Tyr Gly Thr Asp Asp Ile Ile
Cys His Gln Asn65 70 75
80Ala Thr Asn Ala Gln Ala His Ile Val Val Ala Ala Gly Asp Lys Ile
85 90 95Asn Ile Gln Trp Thr Ala
Trp Pro Asp Ser His His Gly Pro Val Leu 100
105 110Asp Tyr Leu Ala Arg Cys Asp Gly Glu Cys Glu Thr
Val Asp Lys Thr 115 120 125Thr Leu
Glu Phe Phe Lys Ile Asp Gly Val Gly Leu Ile Ser Asp Thr 130
135 140Glu Val Pro Gly Thr Trp Gly Asp Asp Gln Leu
Ile Ala Asn Asn Asn145 150 155
160Ser Trp Leu Val Glu Ile Pro Pro Thr Ile Ala Pro Gly Asn Tyr Val
165 170 175Leu Arg His Glu
Leu Ile Ala Leu His Ser Ala Gly Thr Glu Asp Gly 180
185 190Ala Gln Asn Tyr Pro Gln Cys Phe Asn Leu Gln
Val Thr Gly Ser Gly 195 200 205Thr
Asp Glu Pro Ala Gly Thr Leu Gly Thr Lys Leu Tyr Thr Glu Asp 210
215 220Glu Ala Gly Ile Val Val Asn Ile Tyr Thr
Ser Leu Ser Ser Tyr Ala225 230 235
240Val Pro Gly Pro Thr Gln Tyr Ser Gly Ala Val Ser Val Ser Gln
Ser 245 250 255Thr Ser Ala
Ile Thr Ser Thr Gly Thr Ala Val Val Gly Ser Gly Ser 260
265 270Ala Val Ala Thr Ser Ala Ala Ala Ala Thr
Thr Ser Ala Ala Ala Ser 275 280
285Ser Ala Ala Ala Ala Thr Thr Ala Ala Ala Val Thr Ser Ala Asn Ala 290
295 300Asn Thr Gln Ile Ala Gln Pro Ser
Ser Ser Ser Ser Tyr Ser Gln Ile305 310
315 320Ala Val Gln Val Pro Ser Ser Trp Thr Thr Leu Val
Thr Val Thr Pro 325 330
335Pro Ala Ala Ala Ala Thr Thr Pro Ala Ala Val Pro Glu Pro Gln Thr
340 345 350Pro Ser Ala Ser Ser Gly
Ala Thr Thr Thr Ser Ser Ser Ser Gly Ala 355 360
365Ala Gln Ser Leu Tyr Gly Gln Cys Gly Gly Ile Asn Trp Thr
Gly Ala 370 375 380Thr Ser Cys Val Glu
Gly Ala Thr Cys Tyr Gln Tyr Asn Pro Tyr Tyr385 390
395 400Tyr Gln Cys Ile Ser Ala
4051491284DNAAspergillus aculeatus 149atgtctcttt ccaagattgc cactcttctg
ctgggctcgg tctcgctggt cgctggtcat 60gggtatgtct cgagcatcga ggtggacggt
accacctatg gagggtactt ggtcgacact 120tattactacg aatccgaccc gcccgagtta
atcgcctggt ccacaaatgc cacggatgat 180ggctatgtat cgccctccga ctacgagagc
gtgaacatca tctgccacaa ggggtctgcg 240cccggcgcgt tgtcggcccc tgtcgcgccc
ggaggctggg tgcagatgac ctggaacacc 300tggcccaccg accatcacgg ccctgtcatc
acgtatatgg ccaattgcca cggttcttgc 360gcagatgtgg acaagaccac cctcgagttc
ttcaagatcg atgctggcgg cttgatcgat 420gacacggacg tgcctggaac ttgggcgacc
gatgagctca ttgaagatag ctatagtcgc 480aacatcacta tccccagcga tattgccccc
gggtactatg ttttgcgaca cgagatcatt 540gctctgcaca gcgccgagaa cctggacgga
gcccagaact acccccagtg catcaatctg 600gaagtcaccg gcagcgagac agcaaccccg
agtggcacct tgggcactgc tctgtacaag 660gagaccgacc ccggcatcta tgttgacatc
tggaacacgt tgagcacgta tactattccc 720ggccccgcgc tgtacactgc tggtagcact
gcgaccgcag ccgctgctgc cgataccacc 780actacttctg ctggcaccac cgctgaggcc
accaccgctg ccgccgccgt gagtaccacc 840gcggacgctg ttccgaccga gtcttcagct
ccttccgaga ccagcgcgac taccgcgaac 900cctgctcggc ccactgccgg cagcgacatc
cgcttccagc ccggtcaggt caaggctggt 960gcttcagtca acaactcggc tactgagact
tcctctggtg agtctgccac gacgaccaca 1020acatcagtgg ccactgcggc ttcgagcgcg
gattcgtcga cgacttctgg ggttttgagt 1080ggcgcctgca gccaggaggg ctactggtac
tgcaacgggg gcactgcgtt ccagcgctgt 1140gtcaacgggg aatgggatgc gtcccagagt
gtggctgcgg gcacggtctg caccgccggt 1200atctcggaga ccatcaccat ttcagccgcc
gccacgcgcc gggatgccat gcgtcgtcat 1260ctggcgcgtc ccaagcgtca ctga
1284150427PRTAspergillus aculeatus
150Met Ser Leu Ser Lys Ile Ala Thr Leu Leu Leu Gly Ser Val Ser Leu1
5 10 15Val Ala Gly His Gly Tyr
Val Ser Ser Ile Glu Val Asp Gly Thr Thr 20 25
30Tyr Gly Gly Tyr Leu Val Asp Thr Tyr Tyr Tyr Glu Ser
Asp Pro Pro 35 40 45Glu Leu Ile
Ala Trp Ser Thr Asn Ala Thr Asp Asp Gly Tyr Val Ser 50
55 60Pro Ser Asp Tyr Glu Ser Val Asn Ile Ile Cys His
Lys Gly Ser Ala65 70 75
80Pro Gly Ala Leu Ser Ala Pro Val Ala Pro Gly Gly Trp Val Gln Met
85 90 95Thr Trp Asn Thr Trp Pro
Thr Asp His His Gly Pro Val Ile Thr Tyr 100
105 110Met Ala Asn Cys His Gly Ser Cys Ala Asp Val Asp
Lys Thr Thr Leu 115 120 125Glu Phe
Phe Lys Ile Asp Ala Gly Gly Leu Ile Asp Asp Thr Asp Val 130
135 140Pro Gly Thr Trp Ala Thr Asp Glu Leu Ile Glu
Asp Ser Tyr Ser Arg145 150 155
160Asn Ile Thr Ile Pro Ser Asp Ile Ala Pro Gly Tyr Tyr Val Leu Arg
165 170 175His Glu Ile Ile
Ala Leu His Ser Ala Glu Asn Leu Asp Gly Ala Gln 180
185 190Asn Tyr Pro Gln Cys Ile Asn Leu Glu Val Thr
Gly Ser Glu Thr Ala 195 200 205Thr
Pro Ser Gly Thr Leu Gly Thr Ala Leu Tyr Lys Glu Thr Asp Pro 210
215 220Gly Ile Tyr Val Asp Ile Trp Asn Thr Leu
Ser Thr Tyr Thr Ile Pro225 230 235
240Gly Pro Ala Leu Tyr Thr Ala Gly Ser Thr Ala Thr Ala Ala Ala
Ala 245 250 255Ala Asp Thr
Thr Thr Thr Ser Ala Gly Thr Thr Ala Glu Ala Thr Thr 260
265 270Ala Ala Ala Ala Val Ser Thr Thr Ala Asp
Ala Val Pro Thr Glu Ser 275 280
285Ser Ala Pro Ser Glu Thr Ser Ala Thr Thr Ala Asn Pro Ala Arg Pro 290
295 300Thr Ala Gly Ser Asp Ile Arg Phe
Gln Pro Gly Gln Val Lys Ala Gly305 310
315 320Ala Ser Val Asn Asn Ser Ala Thr Glu Thr Ser Ser
Gly Glu Ser Ala 325 330
335Thr Thr Thr Thr Thr Ser Val Ala Thr Ala Ala Ser Ser Ala Asp Ser
340 345 350Ser Thr Thr Ser Gly Val
Leu Ser Gly Ala Cys Ser Gln Glu Gly Tyr 355 360
365Trp Tyr Cys Asn Gly Gly Thr Ala Phe Gln Arg Cys Val Asn
Gly Glu 370 375 380Trp Asp Ala Ser Gln
Ser Val Ala Ala Gly Thr Val Cys Thr Ala Gly385 390
395 400Ile Ser Glu Thr Ile Thr Ile Ser Ala Ala
Ala Thr Arg Arg Asp Ala 405 410
415Met Arg Arg His Leu Ala Arg Pro Lys Arg His 420
425151804DNAAspergillus aculeatus 151atgcttgtca aactcatctc
ttttctttca gctgctacca gcgtagctgc tcatggtcat 60gtgtcaaaca ttgtgatcaa
cggggtgtcc taccgcggat gggacatcaa ttcggaccct 120tacaattcca accctccggt
ggtggttgca tggcaaacac ccaacacagc taatggcttc 180atctcccctg atgcatacga
cacagatgat gttatttgcc atctgagcgc tacgaatgcc 240agaggccacg cagtcgtcgc
tgctggcgac aagatcagcc tccagtggac gacctggcct 300gacagtcacc atggccctgt
catcagctac ctagccaact gcggctccag ctgcgagaca 360gtcgataaga ccaccctcga
gttcttcaag atcgatggtg ttggcttggt ggatgagagc 420aatccccctg gtatctgggg
agacgatgag ctcattgcca acaacaactc ttggctggta 480gagattccag ctagtatcgc
gccaggatac tatgtgctgc gtcacgagtt gatcgctctg 540catggagcag ggagtgagaa
tggagcccag aattacatgc aatgtttcaa ccttcaggtt 600actgggactg gcacggtcca
gccttccggg gtcctgggca cggagctgta caaacccaca 660gacgctggaa ttcttgtcaa
tatctaccag tcgctctcca cctatgttgt tcctggcccg 720accctgatcc cccaggccgt
ttccctcgtt cagtcgagct ccaccattac cgcctcgggc 780acggcagtga caaccacggc
ttga 804152267PRTAspergillus
aculeatus 152Met Leu Val Lys Leu Ile Ser Phe Leu Ser Ala Ala Thr Ser Val
Ala1 5 10 15Ala His Gly
His Val Ser Asn Ile Val Ile Asn Gly Val Ser Tyr Arg 20
25 30Gly Trp Asp Ile Asn Ser Asp Pro Tyr Asn
Ser Asn Pro Pro Val Val 35 40
45Val Ala Trp Gln Thr Pro Asn Thr Ala Asn Gly Phe Ile Ser Pro Asp 50
55 60Ala Tyr Asp Thr Asp Asp Val Ile Cys
His Leu Ser Ala Thr Asn Ala65 70 75
80Arg Gly His Ala Val Val Ala Ala Gly Asp Lys Ile Ser Leu
Gln Trp 85 90 95Thr Thr
Trp Pro Asp Ser His His Gly Pro Val Ile Ser Tyr Leu Ala 100
105 110Asn Cys Gly Ser Ser Cys Glu Thr Val
Asp Lys Thr Thr Leu Glu Phe 115 120
125Phe Lys Ile Asp Gly Val Gly Leu Val Asp Glu Ser Asn Pro Pro Gly
130 135 140Ile Trp Gly Asp Asp Glu Leu
Ile Ala Asn Asn Asn Ser Trp Leu Val145 150
155 160Glu Ile Pro Ala Ser Ile Ala Pro Gly Tyr Tyr Val
Leu Arg His Glu 165 170
175Leu Ile Ala Leu His Gly Ala Gly Ser Glu Asn Gly Ala Gln Asn Tyr
180 185 190Met Gln Cys Phe Asn Leu
Gln Val Thr Gly Thr Gly Thr Val Gln Pro 195 200
205Ser Gly Val Leu Gly Thr Glu Leu Tyr Lys Pro Thr Asp Ala
Gly Ile 210 215 220Leu Val Asn Ile Tyr
Gln Ser Leu Ser Thr Tyr Val Val Pro Gly Pro225 230
235 240Thr Leu Ile Pro Gln Ala Val Ser Leu Val
Gln Ser Ser Ser Thr Ile 245 250
255Thr Ala Ser Gly Thr Ala Val Thr Thr Thr Ala 260
265153822DNAAspergillus aculeatus 153atgaagtatc ttgcgatctt
cgcggcagca gcagctggac tggcccgccc gacagcagcg 60cactacatct tcagcaagct
gattctggac ggcgaagtct ctgaggactg gcagtatatt 120cgtaaaacca cccgggagac
atgctatttg ccgaccaagt tcaccgacac cttcgacaac 180ttgactccga acgaccagga
tttccggtgc aatctcggct cgttcagcaa cgccgccaag 240accgaagtgg ccgaggtgga
agcgggctcc acgattggca tgcagctttt cgctggtagc 300cacatgcgtc acccgggacc
tgcgcaagtc ttcatgtcta aggccccgtc cggcaacgta 360cagagctacg agggtgacgg
ctcctggttc aagatctggg agcgtacact ctgcgacaaa 420agtggcgatc tgactggaga
tgcgtggtgt acatacggcc agaccgagat cgagtttcaa 480atccccgagg cgaccccgac
gggcgaatac ctggtccgag cggagcacat cggtcttcac 540cgcgcacaga gtaatcaagc
cgagttctac tacagctgcg cccaggtcaa ggtcacgggc 600aatggtaccg gggtgccgag
ccagacatat cagatccctg gcatgtacaa tgaccgctcg 660gagcttttca acgggctgaa
cttgtggtcc tactcggtgg agaacgtcga ggcagccatg 720aagaattcta tcgtgggtga
tgaaatttgg aatggaagtt ctgttccctc tgagtcccat 780gtcccgaagt ataagaagag
tcatgcttgt cgtgtttatt ga 822154273PRTAspergillus
aculeatus 154Met Lys Tyr Leu Ala Ile Phe Ala Ala Ala Ala Ala Gly Leu Ala
Arg1 5 10 15Pro Thr Ala
Ala His Tyr Ile Phe Ser Lys Leu Ile Leu Asp Gly Glu 20
25 30Val Ser Glu Asp Trp Gln Tyr Ile Arg Lys
Thr Thr Arg Glu Thr Cys 35 40
45Tyr Leu Pro Thr Lys Phe Thr Asp Thr Phe Asp Asn Leu Thr Pro Asn 50
55 60Asp Gln Asp Phe Arg Cys Asn Leu Gly
Ser Phe Ser Asn Ala Ala Lys65 70 75
80Thr Glu Val Ala Glu Val Glu Ala Gly Ser Thr Ile Gly Met
Gln Leu 85 90 95Phe Ala
Gly Ser His Met Arg His Pro Gly Pro Ala Gln Val Phe Met 100
105 110Ser Lys Ala Pro Ser Gly Asn Val Gln
Ser Tyr Glu Gly Asp Gly Ser 115 120
125Trp Phe Lys Ile Trp Glu Arg Thr Leu Cys Asp Lys Ser Gly Asp Leu
130 135 140Thr Gly Asp Ala Trp Cys Thr
Tyr Gly Gln Thr Glu Ile Glu Phe Gln145 150
155 160Ile Pro Glu Ala Thr Pro Thr Gly Glu Tyr Leu Val
Arg Ala Glu His 165 170
175Ile Gly Leu His Arg Ala Gln Ser Asn Gln Ala Glu Phe Tyr Tyr Ser
180 185 190Cys Ala Gln Val Lys Val
Thr Gly Asn Gly Thr Gly Val Pro Ser Gln 195 200
205Thr Tyr Gln Ile Pro Gly Met Tyr Asn Asp Arg Ser Glu Leu
Phe Asn 210 215 220Gly Leu Asn Leu Trp
Ser Tyr Ser Val Glu Asn Val Glu Ala Ala Met225 230
235 240Lys Asn Ser Ile Val Gly Asp Glu Ile Trp
Asn Gly Ser Ser Val Pro 245 250
255Ser Glu Ser His Val Pro Lys Tyr Lys Lys Ser His Ala Cys Arg Val
260 265
270Tyr155969DNAAurantiporus alborubescens 155atgcgaacca tcgccacgtt
tgttacgctt gtagcctcag ttctccctgc ggtcctcgca 60cacggaggtg tcctctccta
ttcsaacggg gggaattggt actggggatg gaagccttac 120aattcacctg acgggcagac
caccatccaa cgcccgtggg caacatacaa tccgatcact 180gatgcgacgg atcctaccat
tgcttgcaac aacgacggga catctggagc tctgcagttg 240actgcgacag tcgcggcggg
atctgccatc acggcgtatt ggaaccaggt gtggccgcat 300gataaagggc cgatgacgac
atacctcgca caatgccccg gcagtacctg cacaggagtc 360aacgcgaaga ctctgaaatg
gttcaagatc gatcacgccg ggttgctttc tggtactgtc 420tacagtggct cgtgggcatc
aggcaagatg attgcacaga actcgacctg gacaactacc 480attccagcga cggtgccttc
agggaactat ctgatacgtt tcgagactat tgccctgcac 540tctttgccag cgcaatttta
ccctgagtgc gcacaaattc aaatcacggg cggaggttcc 600cgtgctccaa ccgctgcaga
gcttgttagc ttccctggcg cgtacagcaa caatgatcct 660ggtgtcaaca ttgacatcta
ctccaatgcc gcgcagagtg caaccacata cgtaatacca 720ggacctccat tgtacggcgg
tgcttccgga tctggtccat cttccgcgcc tccatcaagt 780accccaggta gttcgtccac
ttcccacggt cccacgtccg tcagcacgtc cagcagtgct 840gcaccatcga cgacaggaac
cgtgacgcag tacggtcagt gcggtggcat tggttgggct 900ggagctaccg gctgtatctc
accattcaag tgcacggtca tcaacgatta ttactaccag 960tgcctctga
969156322PRTAurantiporus
alborubescens 156Met Arg Thr Ile Ala Thr Phe Val Thr Leu Val Ala Ser Val
Leu Pro1 5 10 15Ala Val
Leu Ala His Gly Gly Val Leu Ser Tyr Ser Asn Gly Gly Asn 20
25 30Trp Tyr Trp Gly Trp Lys Pro Tyr Asn
Ser Pro Asp Gly Gln Thr Thr 35 40
45Ile Gln Arg Pro Trp Ala Thr Tyr Asn Pro Ile Thr Asp Ala Thr Asp 50
55 60Pro Thr Ile Ala Cys Asn Asn Asp Gly
Thr Ser Gly Ala Leu Gln Leu65 70 75
80Thr Ala Thr Val Ala Ala Gly Ser Ala Ile Thr Ala Tyr Trp
Asn Gln 85 90 95Val Trp
Pro His Asp Lys Gly Pro Met Thr Thr Tyr Leu Ala Gln Cys 100
105 110Pro Gly Ser Thr Cys Thr Gly Val Asn
Ala Lys Thr Leu Lys Trp Phe 115 120
125Lys Ile Asp His Ala Gly Leu Leu Ser Gly Thr Val Tyr Ser Gly Ser
130 135 140Trp Ala Ser Gly Lys Met Ile
Ala Gln Asn Ser Thr Trp Thr Thr Thr145 150
155 160Ile Pro Ala Thr Val Pro Ser Gly Asn Tyr Leu Ile
Arg Phe Glu Thr 165 170
175Ile Ala Leu His Ser Leu Pro Ala Gln Phe Tyr Pro Glu Cys Ala Gln
180 185 190Ile Gln Ile Thr Gly Gly
Gly Ser Arg Ala Pro Thr Ala Ala Glu Leu 195 200
205Val Ser Phe Pro Gly Ala Tyr Ser Asn Asn Asp Pro Gly Val
Asn Ile 210 215 220Asp Ile Tyr Ser Asn
Ala Ala Gln Ser Ala Thr Thr Tyr Val Ile Pro225 230
235 240Gly Pro Pro Leu Tyr Gly Gly Ala Ser Gly
Ser Gly Pro Ser Ser Ala 245 250
255Pro Pro Ser Ser Thr Pro Gly Ser Ser Ser Thr Ser His Gly Pro Thr
260 265 270Ser Val Ser Thr Ser
Ser Ser Ala Ala Pro Ser Thr Thr Gly Thr Val 275
280 285Thr Gln Tyr Gly Gln Cys Gly Gly Ile Gly Trp Ala
Gly Ala Thr Gly 290 295 300Cys Ile Ser
Pro Phe Lys Cys Thr Val Ile Asn Asp Tyr Tyr Tyr Gln305
310 315 320Cys Leu157705DNAAurantiporus
alborubescens 157atgaaggcta tcttggctat tttctcggcc cttgctccac ttgccgctgc
gcattatacc 60ttccctgatt ttattgtcaa cggaacaaca actgccgatt gggtctacat
ccgagagacc 120gcgaaccact actcgaatgg tcctgtaacc aacgtgaacg atccagaatt
ccgatgctac 180gagctggacc tgcaaaacac ggcagcgagt accctcaccg ccacggtctc
tgcaggctcc 240agcgtcggct ttaaagctaa cagcgccctt taccatcctg gttatctcga
tgtgtatatg 300tccaaagcga ccccagctgc taattcaccc agtgctggaa cggaccaaag
ctggttcaag 360gtctatgaat ccgctccggt cttcgcgaat ggggccctaa gcttcccttc
ggagaacatc 420caatctttca cgttcacaat cccgaagtcc cttcccagtg gccaatatct
catccgtgtg 480gaacacatcg ctctccactc cgccagtagc tacggaggtg cacaattcta
catcagctgc 540gctcaagtca atgtcgtcaa cggcgggaac ggaaacccag gaccgttagt
caagattccc 600ggcgtttaca ctgggaacga gcctggcatc ctcatcaaca tctacagctt
cccaccgggt 660ttcagtggct accaatcccc gggacctgct gtgtggcgtg gttga
705158234PRTAurantiporus alborubescens 158Met Lys Ala Ile Leu
Ala Ile Phe Ser Ala Leu Ala Pro Leu Ala Ala1 5
10 15Ala His Tyr Thr Phe Pro Asp Phe Ile Val Asn
Gly Thr Thr Thr Ala 20 25
30Asp Trp Val Tyr Ile Arg Glu Thr Ala Asn His Tyr Ser Asn Gly Pro
35 40 45Val Thr Asn Val Asn Asp Pro Glu
Phe Arg Cys Tyr Glu Leu Asp Leu 50 55
60Gln Asn Thr Ala Ala Ser Thr Leu Thr Ala Thr Val Ser Ala Gly Ser65
70 75 80Ser Val Gly Phe Lys
Ala Asn Ser Ala Leu Tyr His Pro Gly Tyr Leu 85
90 95Asp Val Tyr Met Ser Lys Ala Thr Pro Ala Ala
Asn Ser Pro Ser Ala 100 105
110Gly Thr Asp Gln Ser Trp Phe Lys Val Tyr Glu Ser Ala Pro Val Phe
115 120 125Ala Asn Gly Ala Leu Ser Phe
Pro Ser Glu Asn Ile Gln Ser Phe Thr 130 135
140Phe Thr Ile Pro Lys Ser Leu Pro Ser Gly Gln Tyr Leu Ile Arg
Val145 150 155 160Glu His
Ile Ala Leu His Ser Ala Ser Ser Tyr Gly Gly Ala Gln Phe
165 170 175Tyr Ile Ser Cys Ala Gln Val
Asn Val Val Asn Gly Gly Asn Gly Asn 180 185
190Pro Gly Pro Leu Val Lys Ile Pro Gly Val Tyr Thr Gly Asn
Glu Pro 195 200 205Gly Ile Leu Ile
Asn Ile Tyr Ser Phe Pro Pro Gly Phe Ser Gly Tyr 210
215 220Gln Ser Pro Gly Pro Ala Val Trp Arg Gly225
230159702DNATrichophaea saccata 159atgacgcccc tgaaactccg
cccccttctc ctcctggtgc tttccacgac cctcagcctc 60gtgcacgcgc actatcgctt
ctacgaactg atcgccaacg gggccaccca cgcttccttc 120gaatacatcc gccaatgggt
gcccatctac agcaactctc ccgtaaccga cgtcaccagc 180gtcaacctcc gctgcaacgt
caacgccact cccgccgccg aggtgatcac cgttgctgcc 240ggtagcaccg tcggcttcgt
agcagacaca acagtaacgc accccggtgc gttcaccgcg 300tacatggcga aagcgcccga
agacatcacg gaatgggatg gcaacgggga ctggttcaag 360atctgggaga agggtccaac
gagtataacc agtagcggga taacctggga cgtcacggat 420acccaatgga ccttcaccat
cccttccgcg acaccaaacg gtcaatacct actccgcttc 480gagcacatag cgctccacgc
cgccagcacc gtggggggtg ctcaattcta catgtcgtgc 540gcgcagatac aagtaacgaa
cggcggcaac gggagtcccg ggcccaccat caagttcccg 600ggcggataca gcgccacaga
ccccggtatc ctgatcaata tctattatcc catccccact 660agttacacta ttcctggtcc
accggtttgg accggtaagt aa 702160233PRTTrichophaea
saccata 160Met Thr Pro Leu Lys Leu Arg Pro Leu Leu Leu Leu Val Leu Ser
Thr1 5 10 15Thr Leu Ser
Leu Val His Ala His Tyr Arg Phe Tyr Glu Leu Ile Ala 20
25 30Asn Gly Ala Thr His Ala Ser Phe Glu Tyr
Ile Arg Gln Trp Val Pro 35 40
45Ile Tyr Ser Asn Ser Pro Val Thr Asp Val Thr Ser Val Asn Leu Arg 50
55 60Cys Asn Val Asn Ala Thr Pro Ala Ala
Glu Val Ile Thr Val Ala Ala65 70 75
80Gly Ser Thr Val Gly Phe Val Ala Asp Thr Thr Val Thr His
Pro Gly 85 90 95Ala Phe
Thr Ala Tyr Met Ala Lys Ala Pro Glu Asp Ile Thr Glu Trp 100
105 110Asp Gly Asn Gly Asp Trp Phe Lys Ile
Trp Glu Lys Gly Pro Thr Ser 115 120
125Ile Thr Ser Ser Gly Ile Thr Trp Asp Val Thr Asp Thr Gln Trp Thr
130 135 140Phe Thr Ile Pro Ser Ala Thr
Pro Asn Gly Gln Tyr Leu Leu Arg Phe145 150
155 160Glu His Ile Ala Leu His Ala Ala Ser Thr Val Gly
Gly Ala Gln Phe 165 170
175Tyr Met Ser Cys Ala Gln Ile Gln Val Thr Asn Gly Gly Asn Gly Ser
180 185 190Pro Gly Pro Thr Ile Lys
Phe Pro Gly Gly Tyr Ser Ala Thr Asp Pro 195 200
205Gly Ile Leu Ile Asn Ile Tyr Tyr Pro Ile Pro Thr Ser Tyr
Thr Ile 210 215 220Pro Gly Pro Pro Val
Trp Thr Gly Lys225 230161714DNATrichophaea saccata
161atgaaatgcc ttctctccct ccttctcgcc gcgacagcgg tctccgctca cacgatcttc
60caagaaatcg gcataaacgg ggtgatgcaa gctcgctacg actacatgcg gctgccgtcc
120tacgacggtc ccattacgga cgtaacgagc acctacatgg cgtgcaacgg tggtcccaat
180ccattggtcc aaatctcgaa cgacgtcgct ttcgtaaaag ccggcgacag catcacgctg
240caatgggcgc aaacgttgac gacagatttc aacacggggc tgatcatcga tccatcgcac
300ttgggtcctg tgatggtcta catggccaaa gtaccctccg ccaccggtcc gatccccaac
360agcggctggt tcaaaatcta cgaagacggc tacgacccga caacaaagac atgggcggta
420accaagctca tcaacaacaa gggaaaagtg accgtcacca tcccatcgtg tctaccggca
480ggggactact tgctgcgcgg tgaaatcatt gccttgcacg cggctagtac ctatccaggc
540gcacagtttt acatggagtg tgcgcagttg cggcttacca gtggcggcac taagatgcct
600accacgtata acattccggg gatctattcg cccactgatc cgggtgttac gttcaatctt
660tacaatggat tcacgagtta taccattcct ggcccaaggc cgtttacatg ctag
714162237PRTTrichophaea saccata 162Met Lys Cys Leu Leu Ser Leu Leu Leu
Ala Ala Thr Ala Val Ser Ala1 5 10
15His Thr Ile Phe Gln Glu Ile Gly Ile Asn Gly Val Met Gln Ala
Arg 20 25 30Tyr Asp Tyr Met
Arg Leu Pro Ser Tyr Asp Gly Pro Ile Thr Asp Val 35
40 45Thr Ser Thr Tyr Met Ala Cys Asn Gly Gly Pro Asn
Pro Leu Val Gln 50 55 60Ile Ser Asn
Asp Val Ala Phe Val Lys Ala Gly Asp Ser Ile Thr Leu65 70
75 80Gln Trp Ala Gln Thr Leu Thr Thr
Asp Phe Asn Thr Gly Leu Ile Ile 85 90
95Asp Pro Ser His Leu Gly Pro Val Met Val Tyr Met Ala Lys
Val Pro 100 105 110Ser Ala Thr
Gly Pro Ile Pro Asn Ser Gly Trp Phe Lys Ile Tyr Glu 115
120 125Asp Gly Tyr Asp Pro Thr Thr Lys Thr Trp Ala
Val Thr Lys Leu Ile 130 135 140Asn Asn
Lys Gly Lys Val Thr Val Thr Ile Pro Ser Cys Leu Pro Ala145
150 155 160Gly Asp Tyr Leu Leu Arg Gly
Glu Ile Ile Ala Leu His Ala Ala Ser 165
170 175Thr Tyr Pro Gly Ala Gln Phe Tyr Met Glu Cys Ala
Gln Leu Arg Leu 180 185 190Thr
Ser Gly Gly Thr Lys Met Pro Thr Thr Tyr Asn Ile Pro Gly Ile 195
200 205Tyr Ser Pro Thr Asp Pro Gly Val Thr
Phe Asn Leu Tyr Asn Gly Phe 210 215
220Thr Ser Tyr Thr Ile Pro Gly Pro Arg Pro Phe Thr Cys225
230 2351631455DNAPenicillium thomii 163atgtctctgt
ctaagatttc tggattgatc ctcggatctg ctgccttggt ggctggccac 60ggttacgtga
gcggaatcgt cgttgacgat acctactatg gtggatacct tgtcacccag 120tacccttatg
agagtgacgc cccagagctc attgcctggt cggagcaaga gaccgatctg 180ggttacatcg
atggctctga gtatgccaac tccaacatca tctgtcacaa ggaggccaaa 240cctggtgctt
tggaagcacc cgttaaggct ggtggctccg tcgagctcca gtggaccact 300tggcctacca
gccaccacgg tcctgtcatt acctacatgg ccaactgtaa cggcgactgt 360gacgacgttg
acaagactac tttgcagttc ttcaagattg accagggtgg tttgatcagc 420gataccaccg
agcccggtac ctgggcaact gacaacctca tcgccaacaa caatagccgt 480actgtcaccg
tccccagcga cattgccgat ggaaactacg tcctccgtca cgagatcatt 540gccctccact
ccgccgggga gaccaacggt gcccagaact acccccaatg tatcaacttg 600aaggtcactg
gcggcggtag cgctactcct tctggtaccc tgggtaccgc cctgtacaag 660aacaccgacc
ccggtatcct gatcaacatc tacacttccc tcagcaccta cgatatcccc 720ggcccaaccc
tgtacactgc cggcgccgcc gctgctaccg ctgcctccac ggctgcctct 780tccaccgccg
ctgccgttac tactgccgac gccgtcacta ccgccgctgc cgtcaccagc 840agctctgcat
ccgtggaagt tgtgcccaca actactccca gctcatcaat cgtcagtgcc 900ttcccaacct
ggagcccctc ttctacccca cccttctcca actcttccaa cggatggcgt 960ccgtcattca
gccgcggacc tggtggcccc cgcttcacat ctgctcctgc tcctcagttc 1020tccgctccta
gcggcgctca gcagaagcag tctgccactg ctacccccat cgtggctacc 1080cctgtcgtga
tcaccatgac cgagaccagc acctcctggg tcaccgaaat ggttactctt 1140actgacaagt
ctgttgtgca gaccaccagc gctgtcccag tcgtcgtcgc cgccaccact 1200acccttaccg
agggaagcga gcctgctcag acagcctccc ccagcgttgt ctccggctcc 1260tctagctccg
gctctagctc ctcatctacc accaccacct caaagacctc aactggatcc 1320gactacgtct
ccagcgactg gatgtcttac ctcagctcct tgagcgctgc tgaggtcctc 1380cagatgctgc
gccagacctt ccgttggatg gtcagcaacg acaaggtgca cgctcgtgat 1440attaccatca
actag
1455164484PRTPenicillium thomii 164Met Ser Leu Ser Lys Ile Ser Gly Leu
Ile Leu Gly Ser Ala Ala Leu1 5 10
15Val Ala Gly His Gly Tyr Val Ser Gly Ile Val Val Asp Asp Thr
Tyr 20 25 30Tyr Gly Gly Tyr
Leu Val Thr Gln Tyr Pro Tyr Glu Ser Asp Ala Pro 35
40 45Glu Leu Ile Ala Trp Ser Glu Gln Glu Thr Asp Leu
Gly Tyr Ile Asp 50 55 60Gly Ser Glu
Tyr Ala Asn Ser Asn Ile Ile Cys His Lys Glu Ala Lys65 70
75 80Pro Gly Ala Leu Glu Ala Pro Val
Lys Ala Gly Gly Ser Val Glu Leu 85 90
95Gln Trp Thr Thr Trp Pro Thr Ser His His Gly Pro Val Ile
Thr Tyr 100 105 110Met Ala Asn
Cys Asn Gly Asp Cys Asp Asp Val Asp Lys Thr Thr Leu 115
120 125Gln Phe Phe Lys Ile Asp Gln Gly Gly Leu Ile
Ser Asp Thr Thr Glu 130 135 140Pro Gly
Thr Trp Ala Thr Asp Asn Leu Ile Ala Asn Asn Asn Ser Arg145
150 155 160Thr Val Thr Val Pro Ser Asp
Ile Ala Asp Gly Asn Tyr Val Leu Arg 165
170 175His Glu Ile Ile Ala Leu His Ser Ala Gly Glu Thr
Asn Gly Ala Gln 180 185 190Asn
Tyr Pro Gln Cys Ile Asn Leu Lys Val Thr Gly Gly Gly Ser Ala 195
200 205Thr Pro Ser Gly Thr Leu Gly Thr Ala
Leu Tyr Lys Asn Thr Asp Pro 210 215
220Gly Ile Leu Ile Asn Ile Tyr Thr Ser Leu Ser Thr Tyr Asp Ile Pro225
230 235 240Gly Pro Thr Leu
Tyr Thr Ala Gly Ala Ala Ala Ala Thr Ala Ala Ser 245
250 255Thr Ala Ala Ser Ser Thr Ala Ala Ala Val
Thr Thr Ala Asp Ala Val 260 265
270Thr Thr Ala Ala Ala Val Thr Ser Ser Ser Ala Ser Val Glu Val Val
275 280 285Pro Thr Thr Thr Pro Ser Ser
Ser Ile Val Ser Ala Phe Pro Thr Trp 290 295
300Ser Pro Ser Ser Thr Pro Pro Phe Ser Asn Ser Ser Asn Gly Trp
Arg305 310 315 320Pro Ser
Phe Ser Arg Gly Pro Gly Gly Pro Arg Phe Thr Ser Ala Pro
325 330 335Ala Pro Gln Phe Ser Ala Pro
Ser Gly Ala Gln Gln Lys Gln Ser Ala 340 345
350Thr Ala Thr Pro Ile Val Ala Thr Pro Val Val Ile Thr Met
Thr Glu 355 360 365Thr Ser Thr Ser
Trp Val Thr Glu Met Val Thr Leu Thr Asp Lys Ser 370
375 380Val Val Gln Thr Thr Ser Ala Val Pro Val Val Val
Ala Ala Thr Thr385 390 395
400Thr Leu Thr Glu Gly Ser Glu Pro Ala Gln Thr Ala Ser Pro Ser Val
405 410 415Val Ser Gly Ser Ser
Ser Ser Gly Ser Ser Ser Ser Ser Thr Thr Thr 420
425 430Thr Ser Lys Thr Ser Thr Gly Ser Asp Tyr Val Ser
Ser Asp Trp Met 435 440 445Ser Tyr
Leu Ser Ser Leu Ser Ala Ala Glu Val Leu Gln Met Leu Arg 450
455 460Gln Thr Phe Arg Trp Met Val Ser Asn Asp Lys
Val His Ala Arg Asp465 470 475
480Ile Thr Ile Asn1651021DNATalaromyces stipitatus 165atgccttcca
ctaaagttgc tgctctatct gccgtcctgg ctttggcctc cacggttgct 60ggccatggct
ttgtgcaaaa tattgtcatt gacggtaaat cgtaagtgac ttgcttttgt 120actatagagc
tagataaata cttatactaa ataattcagc tacactggct acctcgtgaa 180ccagtatcct
taccagtcca acccaccagc tgttattggg tggtcaacca ctgcaaccga 240cttgggattt
gtcgatggat ctggatacac caacccggat atcatctgcc acaaaaacgc 300caaacccggt
cagctttctg ctccggttgc cgcaggaggc aaggttgagc tcgaatggac 360aacatggccc
gagagccatc acggccctgt catcagctat ctcgccaatt gcaatggcga 420ttgtactacc
gtggataaga cgaagctcga atttgtcaaa atcgatcagc ggggtctgat 480cgacgacagc
aatcctcccg gtacatgggc cgccgaccag ctcatcgccg ccaacaacag 540ctggactgta
actattcccg agagcatcgc gcctggaaac tacgtccttc gccacgaaat 600catcgctctt
cactccgcca acaacgcaac cggagctcaa aactaccctc aatgcatcaa 660cttgcaaatc
actggcagcg ggacggccaa cccatctggt acccctggcg agaaactcta 720taccccaact
gacccaggta tcttggtcaa catctaccag tcattgtcgt cttatgttat 780tcccggtccg
actttgtgga gtggtgctgc agcgcacgtt gttgccactg cagccggttc 840tgctactggg
gttgcttctg ccaccgctac tccgaccact cttgtgactg ccgtttcatc 900gcctaccggt
gctccttcag tggtgactcc tgaggctcct tcagtaacct cgttcgcccc 960agtggtgact
gttactgatg tcgttactgt gactaccgtc atcactacta ctatctctta 1020g
1021166320PRTTalaromyces stipitatus 166Met Pro Ser Thr Lys Val Ala Ala
Leu Ser Ala Val Leu Ala Leu Ala1 5 10
15Ser Thr Val Ala Gly His Gly Phe Val Gln Asn Ile Val Ile
Asp Gly 20 25 30Lys Ser Tyr
Thr Gly Tyr Leu Val Asn Gln Tyr Pro Tyr Gln Ser Asn 35
40 45Pro Pro Ala Val Ile Gly Trp Ser Thr Thr Ala
Thr Asp Leu Gly Phe 50 55 60Val Asp
Gly Ser Gly Tyr Thr Asn Pro Asp Ile Ile Cys His Lys Asn65
70 75 80Ala Lys Pro Gly Gln Leu Ser
Ala Pro Val Ala Ala Gly Gly Lys Val 85 90
95Glu Leu Glu Trp Thr Thr Trp Pro Glu Ser His His Gly
Pro Val Ile 100 105 110Ser Tyr
Leu Ala Asn Cys Asn Gly Asp Cys Thr Thr Val Asp Lys Thr 115
120 125Lys Leu Glu Phe Val Lys Ile Asp Gln Arg
Gly Leu Ile Asp Asp Ser 130 135 140Asn
Pro Pro Gly Thr Trp Ala Ala Asp Gln Leu Ile Ala Ala Asn Asn145
150 155 160Ser Trp Thr Val Thr Ile
Pro Glu Ser Ile Ala Pro Gly Asn Tyr Val 165
170 175Leu Arg His Glu Ile Ile Ala Leu His Ser Ala Asn
Asn Ala Thr Gly 180 185 190Ala
Gln Asn Tyr Pro Gln Cys Ile Asn Leu Gln Ile Thr Gly Ser Gly 195
200 205Thr Ala Asn Pro Ser Gly Thr Pro Gly
Glu Lys Leu Tyr Thr Pro Thr 210 215
220Asp Pro Gly Ile Leu Val Asn Ile Tyr Gln Ser Leu Ser Ser Tyr Val225
230 235 240Ile Pro Gly Pro
Thr Leu Trp Ser Gly Ala Ala Ala His Val Val Ala 245
250 255Thr Ala Ala Gly Ser Ala Thr Gly Val Ala
Ser Ala Thr Ala Thr Pro 260 265
270Thr Thr Leu Val Thr Ala Val Ser Ser Pro Thr Gly Ala Pro Ser Val
275 280 285Val Thr Pro Glu Ala Pro Ser
Val Thr Ser Phe Ala Pro Val Val Thr 290 295
300Val Thr Asp Val Val Thr Val Thr Thr Val Ile Thr Thr Thr Ile
Ser305 310 315 320
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