PROCESSES FOR PRODUCING FERMENTATION PRODUCTS

Abstract

The present disclosure relates to processes for producing fermentation products from starch-containing material, wherein a thermostable phospholipase C is present and/or added during liquefaction to increase fermentation product yield, such as ethanol yield. The disclosure also relates to the use of a thermostable phospholipase C in processes of the disclosure, for example, to increase fermentation product yield, such as ethanol yield.

Description

FIELD OF THE INVENTION

[0001] The present disclosure relates to processes for producing fermentation products, especially ethanol, from starch-containing material. The disclosure also relates to use of a thermostable phospholipase C during liquefaction in a fermentation product production process of the disclosure to increase fermentation product yield, especially ethanol.

REFERENCE TO A SEQUENCE LISTING

[0002] This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0003] Production of fermentation products, such as ethanol, from starch-containing material is well-known in the art. Industrially two different kinds of processes are used today. The most commonly used process, often referred to as a "conventional process", includes liquefying gelatinized starch at high temperature using typically a bacterial alpha-amylase, followed by simultaneous saccharification and fermentation carried out in the presence of a glucoamylase and a fermentation organism. Another well-known process, often referred to as a "raw starch hydrolysis"-process (RSH process), includes simultaneously saccharifying and fermenting granular starch below the initial gelatinization temperature typically in the presence of at least a glucoamylase.

[0004] Despite significant improvement of fermentation product production processes over the past decade a significant amount of residual starch material is not converted into the desired fermentation product, such as ethanol.

[0005] Therefore, there is still a desire and need for providing processes for producing fermentation products, such as ethanol, from starch-containing material that can provide a higher fermentation product yield, or other advantages, compared to conventional processes.

SUMMARY OF THE INVENTION

[0006] The object of the present disclosure is to provide processes for producing fermentation products, such as ethanol, from starch-containing material that can provide a higher fermentation product yield, or other advantages, compared to conventional processes.

[0007] In the first aspect the present disclosure relates to a method of increasing fermentation product yield during fermentation product production process, wherein a phospholipase C is present and/or added during a liquefaction step of the fermentation product production process.

[0008] In another aspect the disclosure relates to processes of producing fermentation products, comprising:

(a) liquefying a starch-containing material using an alpha-amylase in the presence of a phospholipase C; (b) saccharifying the liquified starch-containing material using a carbohydrate-source generating enzyme to form fermentable sugars; and (c) fermenting the fermentable sugars using a fermenting organism to product the fermentation product.

[0009] In preferred embodiments the fermentation production product is ethanol and the ethanol yield is increased compared to performance of the method in the absence of using a phospholipase C.

[0010] In a preferred embodiment the phospholipase C is a thermostable phospholipase, preferably having a Melting Point (DSC) above 80.degree. C., such as above 82.degree. C., such as above 84.degree. C., such as above 86.degree. C., such as above 88.degree. C., such as above 88.degree. C., such as above 90.degree. C., such as above 92.degree. C., such as above 94.degree. C., such as above 96.degree. C., such as above 98.degree. C., such as above 100.degree. C., such as between 80.degree. C. and 110.degree. C., such as between 82.degree. C. and 110.degree. C., such as between 87.degree. C. and 110.degree. C.

[0011] Examples of thermostable phospholipase C of use herein include the phospholipase C shown in SEQ ID NO: 2 herein derived from a strain of Penicillium emersonii; or a polypeptide having phospholipase activity, preferably phospholipase C activity, having at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 2 herein; the phospholipase C shown in SEQ ID NO: 7 derived from a strain of Trichoderma harzanium; or a polypeptide having phospholipase activity, preferably phospholipase C activity, having at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 7 herein; the phospholipase C shown in SEQ ID NO: 8 derived from a strain of Trichoderma harzanium; or a polypeptide having phospholipase activity, preferably phospholipase C activity, having at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 8 herein; and the phospholipase C shown in SEQ ID NO: 9 derived from a strain of Rasamsonia eburnea or a polypeptide having phospholipase activity, preferably phospholipase C activity, having at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 9 herein.

[0012] Other enzymes such as endoglucanase, hemicellulases (e.g., xylanases, preferably a thermostable xylanase), carbohydrate source generating enzymes (e.g., glucoamylase, preferably a thermostable glucoamylase), proteases, pullulanases and phytases may also be used in the processes of the present disclosure.

BRIEF DESCRIPTION OF THE FIGURES

[0013] FIG. 1 shows the ethanol yield with Pe PLC (SEQ ID NO: 2) addition in liquefaction, and SSF with Glucoamylase SA (GSA).

DETAILED DESCRIPTION OF THE INVENTION

[0014] The object of the present disclosure is to provide processes for producing fermentation products, such as ethanol, from starch-containing material that can provide a higher fermentation product yield, or other advantages, compared to conventional processes.

[0015] The present disclosure relates to the use of a phospholipase C during the liquefaction step in a fermentation product production process. The use of phospholipase C provides a higher fermentation product yield, such as especially ethanol.

I. METHODS OF INCREASING FERMENTATION PRODUCT YIELD

[0016] The inventors have found that increased fermentation product yield, such as especially ethanol yield, is obtained when liquefying starch-containing material using an alpha-amylase in the presence of a thermostable phospholipase C (see Example).

[0017] Accordingly, in the first aspect the present disclosure relates to a method of increasing fermentation product yield during a fermentation product production process, wherein a phospholipase C is present and/or added during a liquefaction step of the fermentation product production process.

[0018] As used herein, the phrase "present and/or added during" a particular step of a fermentation product production process means that an amount of an enzyme (e.g., phospholipase C) is added before or during the particular step of the fermentation product production process.

[0019] In a preferred embodiment the fermentation product is ethanol and the method increases ethanol yield.

[0020] In a preferred embodiment the phospholipase C, e.g., one derived from a strain of Penicillium, for example Penicillium emersonii, is the mature part of the sequence shown as SEQ ID NO: 2, or a sequence having a sequence identity thereto of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%.

[0021] In an embodiment the phospholipase C shown in SEQ ID NO: 2 is present and/or added during liquefaction.

[0022] In a preferred embodiment the phospholipase C, e.g., one derived from a strain of Trichoderma, for example Trichoderma harzianum, is the mature part of the sequence shown as SEQ ID NO: 7, or a sequence having a sequence identity thereto of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the mature part of the sequence shown in SEQ ID NO: 8, or a sequence having a sequence identity thereto of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%

[0023] In an embodiment the phospholipase C shown in SEQ ID NO: 7 is present and/or added during liquefaction. In an embodiment the phospholipase C shown in SEQ ID NO: 8 is present and/or added during liquefaction.

[0024] In a preferred embodiment the phospholipase C, e.g., one derived from a strain of Rasamsonia, for example Rasamsonia eburnean, is the mature part of the sequence shown as SEQ ID NO: 9, or a sequence having a sequence identity thereto of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%.

[0025] In an embodiment the phospholipase C shown in SEQ ID NO: 9 is present and/or added during liquefaction.

[0026] The liquefaction is carried out by liquefying a starch-containing material at a temperature above the initial gelatinization temperature using an alpha-amylase, e.g., a bacterial alpha-amylase and the phospholipase C.

[0027] In an embodiment, the phospholipase C has a Melting Point (DSC) of at least about 80.degree. C.

[0028] Examples of suitable and preferred enzyme can be found below.

II. PROCESS OF PRODUCING FERMENTATION PRODUCTS

[0029] In another aspect the present disclosure relates to processes of producing fermentation products, comprising:

(a) liquefying a starch-containing material using an alpha-amylase in the presence of a phospholipase C; (b) saccharifying the liquified starch-containing material using a carbohydrate-source generating enzyme to form fermentable sugars; and (c) fermenting the fermentable sugars using a fermenting organism to product the fermentation product.

[0030] Liquefaction step (a), saccharification step (b) and fermentation step (c) are carried out sequentially, though saccharification step (b) and fermentation step (c) may be carried out simultaneously (SSF).

A. Liquefaction Step (a)

[0031] Generally the starch-containing material in step (a) may contain 20-55 wt.-% dry solids (DS), preferably 25-40 wt.-% dry solids, more preferably 30-35% dry solids.

[0032] The alpha-amylase and/or the phospholipase C may be added before and/or during liquefaction step (a).

[0033] In an embodiment the pH in step (a) is between 4-7, preferably between pH 4.5-6.

[0034] Step (a) may be carried out at as a liquefaction step at a temperature above the initial gelatinization temperature.

[0035] The term "initial gelatinization temperature" means the lowest temperature at which gelatinization of the starch commences. Starch heated in water begins to gelatinize between 50.degree. C. and 75.degree. C.; the exact temperature of gelatinization depends on the specific starch, and can readily be determined by the skilled artisan. Thus, the initial gelatinization temperature may vary according to the plant species, to the particular variety of the plant species as well as with the growth conditions. In the context of this disclosure the initial gelatinization temperature of a given starch-containing material is the temperature at which birefringence is lost in 5% of the starch granules using the method described by Gorinstein. S. and Lii. C, Starch/Starke, Vol. 44 (12) pp. 461-466 (1992).

[0036] In an embodiment step (a) is carried out at a temperature between 70 and 100.degree. C. In an embodiment step (a) is carried about at a temperature between 80-90.degree. C. In an embodiment step (a) is carried about at a temperature of about 82.degree. C. In an embodiment step (a) is carried about at a temperature of about 83.degree. C. In an embodiment step (a) is carried about at a temperature of about 84.degree. C. In an embodiment step (a) is carried about at a temperature of about 86.degree. C. In an embodiment step (a) is carried about at a temperature of about 87.degree. C. In an embodiment step (a) is carried about at a temperature of about 88.degree. C.

[0037] In an embodiment a jet-cooking step may be carried out before in step (a). Jet-cooking may be carried out at a temperature between 95-140.degree. C. for about 1-15 minutes, preferably for about 3-10 minutes, especially around about 5 minutes.

[0038] In an embodiment a process of the disclosure further comprises, before step (a), and optional jet-cooking step, the steps of:

i) reducing the particle size of the starch-containing material, preferably by dry milling; and ii) forming a slurry comprising the starch-containing material and water.

[0039] The starch-containing starting material, such as whole grains, may be reduced in particle size, e.g., by milling, in order to open up the structure, to increase the surface area and allowing for further processing. Generally there are two types of processes: wet and dry milling. In dry milling whole kernels are milled and used. Wet milling gives a good separation of germ and meal (starch granules and protein). Wet milling is often applied at locations where the starch hydrolysate is used in production of, e.g., syrups. Both dry and wet millings are well known in the art of starch processing. According to the present disclosure dry milling is preferred. In an embodiment the particle size is reduced to between 0.05 to 3.0 mm, preferably 0.1-0.5 mm, or so that at least 30%, preferably at least 50%, more preferably at least 70%, even more preferably at least 90% of the starch-containing material fit through a sieve with a 0.05 to 3.0 mm screen, preferably 0.1-0.5 mm screen. In another embodiment at least 50%, preferably at least 70%, more preferably at least 80%, especially at least 90% of the starch-containing material fit through a sieve with #6 screen.

[0040] The aqueous slurry may contain from 10-55 w/w-% dry solids (DS), preferably 25-45 w/w-% dry solids (DS), more preferably 30-40 w/w-% dry solids (DS) of starch-containing material.

[0041] The slurry may be heated to above the initial gelatinization temperature, preferably to between 70-95.degree. C., such as between 80-90.degree. C., between pH 5.0-7.0, preferably between 5.0 and 6.0, for 30 minutes to 5 hours, such as around 2 hours.

[0042] In an embodiment liquefaction step a) is carried out for 0.5-5 hours at a temperature from 70-95.degree. C. at a pH from 4-6.

[0043] In a preferred embodiment liquefaction step a) is carried out for 0.5-3 hours at a temperature from 80-90.degree. C. at a pH from 4-6.

[0044] The alpha-amylase and/or phospholipase C may initially be added to the aqueous slurry to initiate liquefaction (thinning). In an embodiment only a portion of the enzymes is added to the aqueous slurry, while the rest of the enzymes are added during liquefaction step a).

[0045] The aqueous slurry may in an embodiment be jet-cooked to further gelatinize the slurry before being subjected to liquefaction in step a). The jet-cooking may be carried out at a temperature between 95-160.degree. C., such as between 110-145.degree. C., preferably 120-140.degree. C., such as 125-135.degree. C., preferably around 130.degree. C. for about 1-15 minutes, preferably for about 3-10 minutes, especially around about 5 minutes.

[0046] The alpha-amylase used in step (a) may be any alpha-amylase, but is preferably a bacterial alpha-amylase. In a preferred embodiment the bacterial alpha-amylase is derived from the genus Bacillus. A preferred bacterial alpha-amylase may be derived from a strain of Bacillus stearothermophilus, and may be a variant of a Bacillus stearothermophilus alpha-amylase, such as the one shown as SEQ ID NO: 1. Bacillus stearothermophilus alpha-amylases are typically truncated naturally during production. In particular the alpha-amylase may be a truncated Bacillus stearothermophilus alpha-amylase having from 485-495 amino acids, such as one being around 491 amino acids long (SEQ ID NO: 1).

[0047] According to the present disclosure the Bacillus stearothermophilus alpha-amylase may be the one shown as SEQ ID NO: 1 or one having a sequence identity thereto of at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96.degree./h, at least 97%, at least 98%, at least 99%.

[0048] In an embodiment the bacterial alpha-amylase may be selected from the group of Bacillus stearothermophilus alpha-amylase variants comprising a deletion of one or two amino acids at any of positions R179, G180, I181 and/or G182, preferably the double deletion disclosed in WO 96/23873--see, e.g., page 20, lines 1-10 (hereby incorporated by reference), preferably corresponding to deletion of positions I181+G182 compared to the amino acid sequence of Bacillus stearothermophilus alpha-amylase set forth as SEQ ID NO: 3 disclosed in WO 99/19467 or SEQ ID NO: 1 herein or the deletion of amino acids R179+G180 using SEQ ID NO: 1 herein for numbering.

[0049] In a preferred embodiment the Bacillus stearothermophilus alpha-amylase variant comprises one of the following set of mutations:

[0050] R179*+G180*;

[0051] I181*+G182*;

[0052] I181*+G182*+N193F; preferably

[0053] I181*+G182*+N193F+E129V+K177L+R179E;

[0054] I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;

[0055] I181*+G182*+N193F+V59A Q89R+E129V+K177L+R179E+Q254S+M284V; and

[0056] I181*+G182*+N193F+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQ ID NO: 1 for numbering).

[0057] In an embodiment the Bacillus stearothermophilus alpha-amylase variant has a sequence identity to SEQ ID NO: 1 of at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100%.

[0058] In an embodiment the Bacillus stearothermophilus alpha-amylase variant has from 1-12 mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mutations, compared to the parent alpha-amylase, especially the alpha-amylase shown as SEQ ID NO: 1.

[0059] Commercially available bacterial alpha-amylase products and products containing alpha-amylases include TERMAMYL.TM. SC, LIQUOZYME.TM. SC, LIQUOZYME.TM. LpH, AVANTEC.TM., AVANTEC.TM. AMP, BAN (Novozymes A/S, Denmark) DEX-LO.TM., SPEZYME.TM. XTRA, SPEZYME.TM. AA, SPEZYME.TM. FRED-L, SPEZYME.TM. ALPHA, GC358.TM., SPEZYME.TM. RSL, SPEZYME.TM. HPA and SPEZYME.TM. DELTA AA (from DuPont, USA), FUELZYME.TM. (Verenium, USA).

[0060] A bacterial alpha-amylase may be added in step (a) in an amount well-known in the art.

[0061] In an embodiment the bacterial alpha-amylase, e.g., Bacillus alpha-amylase, such as especially Bacillus stearothermophilus alpha-amylase, or variant thereof, is dosed in liquefaction in a concentration between 0.01-10 KNU-A/g DS, e.g., between 0.02 and 5 KNU-A/g DS, such as 0.03 and 3 KNU-A, preferably 0.04 and 2 KNU-A/g DS, such as especially 0.01 and 2 KNU-A/g DS. In an embodiment the bacterial alpha-amylase, e.g., Bacillus alpha-amylase, such as especially Bacillus stearothermophilus alpha-amylases, or variant thereof, is dosed to liquefaction in a concentration of between 0.0001-1 mg EP (Enzyme Protein)/g DS, e.g., 0.0005-0.5 mg EP/g DS, such as 0.001-0.1 mg EP/g DS.

[0062] According to the present disclosure a phospholipase C, preferably a thermostable phospholliase C having a Melting Point (DSC) of at least about 80.degree. C., is present in and/or added to liquefaction step a) in combination with an alpha-amylase, such as a bacterial alpha-amylase (described above).

[0063] The thermostability of a phospholipase C may be determined as described in Example 2 of WO 2014/090161 (incorporated herein by reference).

[0064] In an embodiment the phospholipase C has a Melting Point (DSC) above 82.degree. C., such as above 84.degree. C., such as above 86.degree. C., such as above 88.degree. C., such as above 88.degree. C., such as above 90.degree. C., such as above 92.degree. C., such as above 94.degree. C., such as above 96.degree. C., such as above 98.degree. C., such as above 100.degree. C., such as between 80.degree. C. and 110.degree. C., such as between 82.degree. C. and 110.degree. C., such as between 87.degree. C. and 110.degree. C.

[0065] In a preferred embodiment the phospholipase C has at least 60%, such as at least 70%, such as at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 2 herein, preferably derived from a strain of the genus Penicillium, such as a strain of Penicillium emersonii.

[0066] In an embodiment the phospholipase C comprises or consists of the amino acid sequence of SEQ ID NO: 2, or an allelic variant thereof; or is a fragment thereof having phospholipase C activity. In another embodiment, the phospholipase C comprises or consists of the mature polypeptide of SEQ ID NO: 2, or a variant of the mature polypeptide of SEQ ID NO: 2 comprising a substitution, deletion, and/or insertion at one or more positions. In another embodiment, the phospholipase C comprises or consists of amino acids 1 to 594 of SEQ ID NO: 2.

[0067] In a preferred embodiment the phospholipase C has at least 60%, such as at least 70%, such as at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 7 herein, preferably derived from a strain of the genus Trichoderma, such as a strain of Trichoderma harzianum.

[0068] In an embodiment the phospholipase C comprises or consists of the amino acid sequence of SEQ ID NO: 7, or an allelic variant thereof; or is a fragment thereof having phospholipase C activity. In another embodiment, the phospholipase C comprises or consists of the mature polypeptide of SEQ ID NO: 7 (e.g., amino acid residues 19-643 of SEQ ID NO: 7, or a variant of the mature polypeptide of SEQ ID NO: 7 comprising a substitution, deletion, and/or insertion at one or more positions. In another embodiment, the phospholipase C comprises or consists of amino acids 1 to 594 of SEQ ID NO: 7.

[0069] In a preferred embodiment the phospholipase C has at least 60%, such as at least 70%, such as at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 8 herein, preferably derived from a strain of the genus Trichoderma, such as a strain of Trichoderma harzianum.

[0070] In an embodiment the phospholipase C comprises or consists of the amino acid sequence of SEQ ID NO: 8, or an allelic variant thereof; or is a fragment thereof having phospholipase C activity. In another embodiment, the phospholipase C comprises or consists of the mature polypeptide of SEQ ID NO: 8 (e.g., amino acid residues 19-645 of SEQ ID NO: 8, or a variant of the mature polypeptide of SEQ ID NO: 8 comprising a substitution, deletion, and/or insertion at one or more positions. In another embodiment, the phospholipase C comprises or consists of amino acids 1 to 594 of SEQ ID NO: 8.

[0071] In a preferred embodiment the phospholipase C has at least 60%, such as at least 70%, such as at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 9 herein, preferably derived from a strain of the genus Rasamsonia, such as a strain of Rasamsonia eburnea.

[0072] In an embodiment the phospholipase C comprises or consists of the amino acid sequence of SEQ ID NO: 9, or an allelic variant thereof; or is a fragment thereof having phospholipase C activity. In another embodiment, the phospholipase C comprises or consists of the mature polypeptide of SEQ ID NO: 9 (e.g., amino acid residues 19-611 of SEQ ID NO: 9, or a variant of the mature polypeptide of SEQ ID NO: 9 comprising a substitution, deletion, and/or inseration at one or more positions. In another embodiment, the phospholipase C comprises or consists of amino acids 1 to 594 of SEQ ID NO: 9.

[0073] 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.

[0074] The term "fragment" means a polypeptide having one or more (e.g., several) amino acids absent from the amino and/or carboxyl terminus of a mature polypeptide; wherein the fragment has phospholipase C activity.

[0075] The term "phospholipase C activity" means the activity that catalyzes the reaction: A phosphatidylcholine+H20=1,2-sn-diacylglycerol+choline phosphate. Phospholipase C activity may be determined using a phospholipase C activity assay (see, for example, the procedure described in Example 1 of WO 2014/090161, which is incorporated herein by reference. An enzyme having "phospholipase C activity" may belong to EC 3.1.4.3.

[0076] 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. In one embodiment, the mature polypeptide is amino acids 1 to 594 of SEQ ID NO: 2. Amino acids -16 to -1 of SEQ ID NO: 2 are a signal peptide. 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.

[0077] For purposes of the present disclosure, the 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 5.0.0 or later. The 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).

[0078] The term "variant" means a polypeptide having phospholipase C activity comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (e.g., several) positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position.

[0079] A phospholipase C may be added and/or present in step (a) in an amount effective to increase fermentation product yield, such as especially ethanol yield, during SSF steps (b) and (c) or fermentation step (c).

[0080] In an embodiment the phospholipase C, such as especially Penicillium emersonii phoshophlipase C, or variant thereof, is dosed in liquefaction in a concentration of about 0.1-50,000 .mu.g EP (Enzyme Protein)/g DS, such as 10,000 .mu.g EP (Enzyme Protein)/g DS, or especially such as 5-1000 .mu.g EP/g DS.

[0081] In an embodiment the phospholipase C, such as especially Trichoderma harzianum phoshophlipase C, or variant thereof, is dosed in liquefaction in a concentration of about 0.1-50,000 .mu.g EP (Enzyme Protein)/g DS, such as 10,000 .mu.g EP (Enzyme Protein)/g DS, or especially such as 5-1000 .mu.g EP/g DS.

[0082] In an embodiment the phospholipase C, such as especially Rasamsonia eburnea phoshophlipase C, or variant thereof, is dosed in liquefaction in a concentration of about 0.1-50,000 .mu.g EP (Enzyme Protein)/g DS, such as 10,000 .mu.g EP (Enzyme Protein)/g DS, or especially such as 5-1000 .mu.g EP/g DS.

[0083] Optionally, an endoglucanase (e.g., thermostable endoglucanase), hemicellulase (e.g., xylanase, preferably a thermostable xylanase), a protease, a carbohydrate-source generating enzyme, (e.g., glucoamylase, preferably a thermostable glucoamylase), a pullulanase, and/or a phytase may be present and/or added during liquefaction step (a). The enzymes may be added individually or as one or more blend compositions. In some embodiments, liquefaction step (a) is carried out in the absence of a protease.

[0084] In some embodiments, the glucoamylase comprises a variant of Penicillium oxalicum glucoamylase having the following mutations: K79V+P2N+P4S+P11F+T65A+Q327F (using SEQ ID NO: 3 herein for numbering).

B. Saccharification Step (b)

[0085] Liquefaction step (a) is followed by saccharification of dextrins in step (b).

[0086] In an embodiment a process of the disclosure may comprise a pre-saccharification step, i.e., after step (a), but before saccharification step (b), carried out for 40-90 minutes at a temperature between 30-65.degree. C.

[0087] According to the present disclosure saccharification step (b) may be carried out at a temperature from 20-75.degree. C., preferably from 40-70.degree. C., such as around 60.degree. C., and at a pH between 4 and 5.

[0088] In a preferred embodiment fermentation step (c) or simultaneous saccharification and fermentation (SSF) (i.e., combined steps (b) and (c)) may be carried out at a temperature between 20-60.degree. C., preferably between 25-40.degree. C., such as around 32.degree. C. In an embodiment fermentation step (c) or simultaneous saccharification and fermentation (SSF) are ongoing for 6 to 120 hours, in particular 24 to 96 hours.

[0089] According to the present disclosure a carbohydrate-source generating enzyme, preferably a glucoamylase, is present and/or added during saccharification step (b) and/or fermentation step (c) or simultaneous saccharification step (b) and fermentation step (c) (SSF).

[0090] The term "carbohydrate-source generating enzyme" includes any enzymes generating fermentable sugars. A carbohydrate-source generating enzyme is capable of producing one or more carbohydrates that can be used as an energy source by the fermenting organism(s) in question, for instance, when used in a process of the disclosure for producing ethanol. The generated carbohydrates may be converted directly or indirectly to the desired fermentation product, preferably ethanol. According to the disclosure a mixture of carbohydrate-source generating enzymes may be used.

[0091] Specific examples of carbohydrate-source generating enzyme activities include glucoamylase (being glucose generators), beta-amylase and maltogenic amylase (being maltose generators). A "maltogenic alpha-amylase" (glucan 1,4-alpha-maltohydrolase, E.C. 3.2.1.133) is able to hydrolyze amylose and amylopectin to maltose in the alpha-configuration. A maltogenic amylase from Bacillus stearothermophilus strain NCIB 11837 is commercially available from Novozymes A/S. Maltogenic alpha-amylases are described in U.S. Pat. Nos. 4,598,048, 4,604,355 and 6,162,628, which are hereby incorporated by reference. The maltogenic amylase may in a preferred embodiment be added in an amount of 0.05-5 mg total protein/gram DS or 0.05-5 MANU/g DS.

[0092] In a preferred embodiment the carbohydrate-source generating enzyme is a glucoamylase.

[0093] The process of the disclosure, including steps (b) and/or (c), may be carried out using any suitable glucoamylase. The glucoamylase may be of any origin, in particular of fungal origin.

[0094] Contemplated glucoamylases include those from the group consisting of Aspergillus glucoamylases, in particular A. niger G1 or G2 glucoamylase (Boel et al. (1984), EMBO J. 3 (5), p. 1097-1102), or variants thereof, such as those disclosed in WO 92/00381, WO 00/04136 and WO 01/04273 (from Novozymes, Denmark); the A. awamori glucoamylase disclosed in WO 84/02921, A. oryzae glucoamylase (AgriC. Biol. Chem. (1991), 55 (4), p. 941-949), or variants or fragments thereof. Other Aspergillus glucoamylase variants include variants with enhanced thermal stability: G137A and G139A (Chen et al. (1996), Prot. Eng. 9, 499-505); D257E and D293E/Q (Chen et al. (1995), Prot. Eng. 8, 575-582); N182 (Chen et al. (1994), Biochem. J. 301, 275-281); disulphide bonds, A246C (Fierobe et al. (1996), Biochemistry, 35, 8698-8704; and introduction of Pro residues in position A435 and S436 (Li et al. (1997), Protein Eng. 10, 1 199-1204.

[0095] Other glucoamylases contemplated include glucoamylase derived from a strain of Athelia, preferably a strain of Athelia rolfsii (previously denoted Corticium rolfsii) glucoamylase (see U.S. Pat. No. 4,727,026 and (Nagasaka, Y. et al. (1998) "Purification and properties of the raw-starch-degrading glucoamylases from Corticium rolfsii, Appl Microbial Biotechnol 50:323-330), Talaromyces glucoamylases, in particular derived from Talaromyces emersonii (WO 99/28448), Talaromyces leycettanus (U.S. Pat. No. Re. 32,153), Talaromyces duponti, Talaromyces thermophilus (U.S. Pat. No. 4,587,215). Also contemplated are Trichoderma reesei glucoamylases including the one disclosed as SEQ ID NO: 4 in WO 2006/060062 and glucoamylases being at least 80% or at least 90% identical thereto (hereby incorporated by reference).

[0096] In an embodiment the glucoamylase is derived from a strain of Aspergillus, preferably A, niger, A. awamori, or A. oryzae; or a strain of Trichoderma, preferably T. reesei; or a strain of Talaromyces, preferably T. emersonii.

[0097] In an embodiment the glucoamylase present and/or added during saccharification step (b) and/or fermentation step (c) is of fungal origin, such as from a strain of Pycnoporus, or a strain of Gloephyllum. In an embodiment the glucoamylase is derived from a strain of the genus Pycnoporus, in particular a strain of Pycnoporus sanguinous described in WO 2011/066576 (SEQ ID NOs 2, 4 or 6), such as the one shown as SEQ ID NO: 4 in WO 2011/066576.

[0098] In a preferred embodiment the glucoamylase is derived from a strain of the genus Gloeophyllum, such as a strain of Gloeophyllum sepiarium or Gloeophyllum trabeum, in particular a strain of Gloeophyllum as described in WO 2011/068803 (SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16). In a preferred embodiment the glucoamylase is the Gloeophyllum sepiarium shown in SEQ ID NO: 2 in WO 2011/068803.

[0099] Other contemplated glucoamylases include glucoamylase derived from a strain of Trametes, preferably a strain of Trametes cingulata disclosed as SEQ ID NO: 34 in WO 2006/069289 (which is hereby incorporated by reference).

[0100] Bacterial glucoamylases contemplated include glucoamylases from the genus Clostridium, in particular C. thermoamylolyticum (EP 135,138), and C. thermohydrosulfuricum (WO 86/01831).

[0101] Commercially available compositions comprising glucoamylase include AMG 200L; AMG 300 L; SAN.TM. SUPER, SAN.TM. EXTRA L, SPIRIZYME.TM. PLUS, SPIRIZYME.TM. FUEL, SPIRIZYME.TM. ULTRA, SPIRIZYME.TM. EXCEL, SPIRIZYME.TM. ACHIEVE, SPIRIZYME.TM. B4U and AMG.TM. E (from Novozymes A/S); OPTIDEX.TM. 300 (from Genencor Int.); AMIGASE.TM. and AMIGASE.TM. PLUS (from DSM); G-ZYME.TM. G900, G-ZYME.TM. and G990 ZR (from Genencor Int).

[0102] Glucoamylases may in an embodiment be added in an amount of 0.02-20 AGU/g DS, preferably 0.05-5 AGU/g DS (in whole stillage), especially between 0.1-2 AGU/g DS.

[0103] Glucoamylase may be added in an effective amount, preferably in the range from 0.001-1 mg enzyme protein per g DS, preferably 0.01-0.5 mg enzyme protein per g dry solid (DS).

[0104] Optionally an alpha-amylase (EC 3.2.1.1) may be added during saccharification step (b) and/or fermentation step (c). The alpha-amylase may be of any origin, but is typically of filamentous fungus origin. According to the disclosure an alpha-amylases adding during saccharification and/or fermentation is typically a fungal acid alpha-amylase.

[0105] The fungal acid alpha-amylases may be an acid fungal alpha-amylase derived from a strain of the genus Aspergillus, such as Aspergillus oryzae and Aspergillus niger.

[0106] A suitable fungal acid alpha-amylase is one derived from a strain Aspergillus niger. In a preferred embodiment the fungal acid alpha-amylase is the one from A. niger disclosed as "AMYA_ASPNG" in the Swiss-prot/TeEMBL database under the primary accession no. P56271 and described in more detail in WO 89/01969 (Example 3), The acid Aspergillus niger acid alpha-amylase is also shown as SEQ ID NO: 1 in WO 2004/080923 (Novozymes) which is hereby incorporated by reference. Also variants of said acid fungal amylase having at least 70% identity, such as at least 80% or even at least 90% identity, such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 1 in WO 2004/080923 are contemplated. A suitable commercially available acid fungal alpha-amylase derived from Aspergillus niger is SP288 (available from Novozymes A/S, Denmark).

[0107] The fungal acid alpha-amylase may also be a wild-type enzyme comprising a carbohydrate-binding module (CBM) and an alpha-amylase catalytic domain (i.e., a non-hybrid), or a variant thereof. In an embodiment the wild-type fungal acid alpha-amylase is derived from a strain of Aspergillus kawachii.

[0108] A specific example of a contemplated hybrid alpha-amylase includes the Rhizomucor pusillus alpha-amylase with Aspergillus niger glucoamylase linker and starch-binding domain (SBD) (which is disclosed in Table 5 as a combination of amino acid sequences SEQ ID NO: 20, SEQ ID NO: 72 and SEQ ID NO: 96 in U.S. application Ser. No. 11/316,535) (hereby incorporated by reference), In another embodiment the hybrid fungal acid alpha-amylase is a Meripilus giganteus alpha-amylase with Athelia rolfsii glucoamylase linker and SBD (SEQ ID NO: 102 in U.S. 60/638,614) (hereby incorporated by reference). Other specific examples of contemplated hybrid alpha-amylases include those disclosed in U.S. Patent Publication no. 2005/0054071, including those disclosed in Table 3 on page 15, such as Aspergillus niger alpha-amylase with Aspergillus kawachii linker and starch binding domain.

[0109] In a preferred embodiment the fungal acid alpha-amylase is one disclosed in WO 2013/006756 including the following variants: Rhizomucor pusillus alpha-amylase variant having an Aspergillus niger glucoamylase linker and starch-binding domain (SBD) which further comprises at least one of the following substitutions or combinations of substitutions: D165M; Y141W; Y141R; K136F; K192R; P224A; P224R; S123H+Y141W; G20S+Y141W; A76G+Y141W; G128D+Y141W; G128D+D143N; P219C+Y141W; N142D+D143N; Y141W+K192R; Y141W+D143N; Y141W+N383R; Y141W+P219C+A265C; Y141W+N142D+D143N; Y141W+K192R V410A; G128D+Y141W+D143N; Y141W+D143N+P219C; Y141W+D143N+K192R; G128D+D143N+K192R; Y141W+D143N+K192R+P219C; G128D+Y141W+D143N+K192R; or G128D+Y141W+D143N+K192R+P219C.

[0110] An acid alpha-amylase may according to the present disclosure be added in an amount of 0.1 to 10 AFAU/g DS, preferably 0.10 to 5 AFAU/g DS, especially 0.3 to 2 AFAU/g DS.

C. Fermenting Organisms

[0111] The term "fermenting organism" refers to any organism, including bacterial and fungal organisms, especially yeast, suitable for use in a fermentation process and capable of producing the desired fermentation product.

[0112] Examples of fermenting organisms used in fermentation step (c) or simultaneous saccharification and fermentation (i.e., SSF) for converting fermentable sugars in the fermentation medium into fermentation products, such as especially ethanol, include fungal organisms, such as especially yeast. Preferred yeast includes strains of Saccharomyces spp., in particular, Saccharomyces cerevisiae.

[0113] Suitable concentrations of the viable fermenting organism during fermentation, such as SSF, are well known in the art or can easily be determined by the skilled person in the art. In one embodiment the fermenting organism, such as ethanol fermenting yeast, (e.g., Saccharomyces cerevisiae) is added to the fermentation medium so that the viable fermenting organism, such as yeast, count per mL of fermentation medium is in the range from 10.sup.5 to 10.sup.12, preferably from 10.sup.7 to 10.sup.10, especially about 5.times.10.sup.7.

[0114] "Fermentation medium" refers to the environment in which fermentation is carried out. The fermentation medium includes the fermentation substrate, that is, the carbohydrate source that is metabolized by the fermenting organism. According to the present disclosure the fermentation medium may comprise nutrients and growth stimulator(s) for the fermenting organism(s). Nutrient and growth stimulators are widely used in the art of fermentation and include nitrogen sources, such as ammonia; urea, vitamins and minerals, or combinations thereof.

[0115] Examples of commercially available yeast includes, e.g., RED STAR.TM. and ETHANOL RED.TM. yeast (available from Fermentis/Lesaffre, USA), FALI (available from Fleischmann's Yeast, USA), SUPERSTART and THERMOSACC.TM. fresh yeast (available from Ethanol Technology, WI, USA), BIOFERM AFT and XR (available from NABC--North American Bioproducts Corporation, GA, USA), GERT STRAND (available from Gert Strand AB, Sweden), and FERMIOL (available from DSM Specialties).

D. Starch-Containing Materials

[0116] Any suitable starch-containing material may be used as starting material according to the present disclosure. Examples of starch-containing materials, suitable for use in a process of the disclosure, include whole grains, corn, wheat, barley, rye, milo, sago, cassava, tapioca, sorghum, rice, peas, beans, or sweet potatoes, or mixtures thereof or starches derived there from, or cereals. Contemplated are also waxy and non-waxy types of corn and barley.

[0117] In a preferred embodiment the starch-containing material, used for fermentation product production, such as especially ethanol production, is corn or wheat.

E. Fermentation Products

[0118] The term "fermentation product" means a product produced by a process including a fermentation step using a fermenting organism. Fermentation products contemplated according to the invention include alcohols (e.g., ethanol, methanol, butanol; polyols such as glycerol, sorbitol and inositol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, succinic acid, gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H2 and CO.sub.2); antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, B.sub.12, beta-carotene); and hormones. In a preferred embodiment the fermentation product is ethanol, e.g., fuel ethanol; drinking ethanol, i.e., potable neutral spirits; or industrial ethanol or products used in the consumable alcohol industry (e.g., beer and wine), dairy industry (e.g., fermented dairy products), leather industry and tobacco industry. Preferred beer types comprise ales, stouts, porters, lagers, bitters, malt liquors, happoushu, high-alcohol beer, low-alcohol beer, low-calorie beer or light beer. Preferably processes of the present disclosure are used for producing an alcohol, such as ethanol. The fermentation product, such as ethanol, obtained according to the present disclosure, may be used as fuel, which is typically blended with gasoline. However, in the case of ethanol it may also be used as potable ethanol.

F. Recovery

[0119] Subsequent to fermentation, or SSF, the fermentation product may be separated from the fermentation medium. The slurry may be distilled to extract the desired fermentation product (e.g., ethanol), Alternatively the desired fermentation product may be extracted from the fermentation medium by micro or membrane filtration techniques. The fermentation product may also be recovered by stripping or other method well known in the art.

III. USE OF PHOSPHOLIPASE C DURING LIQUEFACTION FOR INCREASING FERMENTATION PRODUCT YIELD

[0120] In yet another aspect, the present disclosure relates to the use of a phospholipase C during liquefaction in a fermentation product production process for increasing yield of a fermentation product (e.g., ethanol yield).

[0121] Any phospholipase C, for example a phospholipase C described above, can be used in a liquefaction step of an ethanol production process to increase ethanol yield. In preferred embodiments, the phospholipase C used in the liquefaction step has at least 60%, such as at least 70%, such as at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 2 herein, preferably derived from a strain of the genus Penicillium, such as a strain of Penicillium emersonii.

IV. EXAMPLES OF PREFERRED EMBODIMENTS OF THE DISCLOSURE

[0122] In a preferred embodiment the present disclosure relates to a process for producing ethanol from starch-containing material comprising the steps of:

(a) liquefying the starch-containing material at a pH in the range between 4.0-6.5 at a temperature in the range from 70-100.degree. C. using:

[0123] an alpha-amylase derived from Bacillus stearothermophilus;

[0124] a phospholipase C, preferably having a Melting Point (DSC) of at least about 80.degree. C.; (b) saccharifying using a glucoamylase enzyme; and (c) fermenting using a fermenting organism.

[0125] In a preferred embodiment the process of the disclosure comprises the steps of:

(a) liquefying the starch-containing material at a pH in the range between 4.5-6.2 at a temperature above the initial gelatinization temperature using:

[0126] an alpha-amylase, preferably derived from Bacillus stearothermophilus, having a T1/2 (min) at pH 4.5, 85.degree. C., 0.12 mM CaCl.sub.2 of at least 10;

[0127] a phospholipase C, preferably having a Melting Point (DSC) of at least about 80.degree. C.; (b) saccharifying using a glucoamylase enzyme; and (c) fermenting using a fermenting organism.

[0128] In a preferred embodiment the process of the disclosure comprises the steps of:

(a) liquefying the starch-containing material at a pH in the range between 4.0-6.5 at a temperature between 70-100.degree. C. using:

[0129] a bacterial alpha-amylase, preferably derived from Bacillus stearothermophilus, having a T1/2 (min) at pH 4.5, 85.degree. C., 0.12 mM CaCl.sub.2) of at least 10;

[0130] a phospholipase C, preferably having a Melting Point (DSC) of at least about 80.degree. C.; (b) saccharifying using a glucoamylase enzyme; and (c) fermenting using a fermenting organism.

[0131] In a preferred embodiment the process of the disclosure comprises the steps of:

(a) liquefying the starch-containing material at a pH in the range between 4.0-6.5 at a temperature above the initial gelatinization temperature using:

[0132] an alpha-amylase shown in SEQ ID NO: 1 having a double deletion in positions R179+G180 or I181+G182, and optional substitution N193F; and optionally further one of the following set of substitutions:

[0133] E129V+K177L+R179E;

[0134] V59A+089R+E129V+K177L+R179E+H208Y+K220P+N224 L+Q254S;

[0135] E129V+K177L+R179E+K220P+N224L+S242Q+Q254S;

[0136] V59A+Q89R+E129V+K177L+R179E+0254S+M284V (using SEQ ID NO: 1 herein for numbering);

[0137] a phospholipase C, preferably having a Melting Point (DSC) of at least about 80.degree. C., such as a phospholipase C having at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 2 herein; (b) saccharifying using a glucoamylase enzyme; (c) fermenting using a fermenting organism.

[0138] In a preferred embodiment the process of the disclosure comprises the steps of:

(a) liquefying the starch-containing material at a pH in the range between 4.0-6.5 at a temperature above the initial gelatinization temperature using:

[0139] an alpha-amylase shown in SEQ ID NO: 1 having a double deletion in positions R179+G180 or I181+G182, and optional substitution N193F; and optionally further one of the following set of substitutions:

[0140] E129V+K177L+R179E;

[0141] V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;

[0142] E129V+K177L+R179E+K220P+N224L+S242Q+Q254S;

[0143] V59A+Q89R+E129V+K177L+R179E+Q254S+M284V (using SEQ ID NO: 1 herein for numbering);

[0144] a phospholipase C, preferably having a Melting Point (DSC) of at least about 80.degree. C., such as a phospholipase C having at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 7 herein; (b) saccharifying using a glucoamylase enzyme; (c) fermenting using a fermenting organism.

[0145] In a preferred embodiment the process of the disclosure comprises the steps of:

(a) liquefying the starch-containing material at a pH in the range between 4.0-6.5 at a temperature above the initial gelatinization temperature using:

[0146] an alpha-amylase shown in SEQ ID NO: 1 having a double deletion in positions R179+G180 or I181+G182, and optional substitution N193F; and optionally further one of the following set of substitutions:

[0147] E129V+K177L+R179E;

[0148] V59A+089R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;

[0149] E129V+K177L+R179E+K220P+N224L+S242Q+Q254S;

[0150] V59A+Q89R+E129V+K177L+R179E+Q254S+M284V (using SEQ ID NO: 1 herein for numbering);

[0151] a phospholipase C, preferably having a Melting Point (DSC) of at least about 80.degree. C., such as a phospholipase C having at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 8 herein; (b) saccharifying using a glucoamylase enzyme; (c) fermenting using a fermenting organism.

[0152] In a preferred embodiment the process of the disclosure comprises the steps of:

(a) liquefying the starch-containing material at a pH in the range between 4.0-6.5 at a temperature above the initial gelatinization temperature using:

[0153] an alpha-amylase shown in SEQ ID NO: 1 having a double deletion in positions R179+G180 or I181+G182, and optional substitution N193F; and optionally further one of the following set of substitutions:

[0154] E129V+K177L+R179E;

[0155] V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;

[0156] E129V+K177L+R179E+K220P+N224L+S242Q+Q254S;

[0157] V59A+Q89R+E129V+K177L+R179E+Q254S+M284V (using SEQ ID NO: 1 herein for numbering);

[0158] a phospholipase C, preferably having a Melting Point (DSC) of at least about 80.degree. C., such as a phospholipase C having at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 9 herein; (b) saccharifying using a glucoamylase enzyme; (c) fermenting using a fermenting organism.

[0159] In a preferred embodiment the process of the disclosure comprises the steps of:

(a) liquefying the starch-containing material at a pH in the range between 4.0-6.5 at a temperature between 70-100.degree. C. using:

[0160] an alpha-amylase derived from Bacillus stearothermophilus having a double deletion in positions I181+G182, and optional substitution N193F; and optionally further one of the following set of substitutions:

[0161] V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S; or

[0162] V59A+Q89R+E129V+K177L+R179E+Q254S+M284V (using SEQ ID NO: 1 herein for numbering);

[0163] a phospholipase C, preferably having a Melting Point (DSC) of at least about 80.degree. C.; such as an phospholipase C having at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 2 herein; and

[0164] optionally a Penicillium oxalicum glucoamylase in SEQ ID NO: 3 herein, preferably having substitutions selected from the group of:

[0165] K79V; or

[0166] K79V+P11F+T65A+Q327F; or

[0167] K79V+P2N+P4S+P11F+T65A+Q327F (using SEQ ID NO: 3 herein for numbering); (b) saccharifying using a glucoamylase enzyme; (c) fermenting using a fermenting organism.

[0168] In a preferred embodiment the process of the disclosure comprises the steps of:

(a) liquefying the starch-containing material at a pH in the range between 4.0-6.5 at a temperature between 70-100.degree. C. using:

[0169] an alpha-amylase derived from Bacillus stearothermophilus having a double deletion in positions I181+G182, and optional substitution N193F; and optionally further one of the following set of substitutions:

[0170] V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S; or

[0171] V59A+Q89R+E129V+K177L+R179E+Q254S+M284V (using SEQ ID NO: 1 herein for numbering);

[0172] a phospholipase C, preferably having a Melting Point (DSC) of at least about 80.degree. C.; such as an phospholipase C having at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 7 herein; and

[0173] optionally a Penicillium oxalicum glucoamylase in SEQ ID NO: 3 herein, preferably having substitutions selected from the group of:

[0174] K79V; or

[0175] K79V+P11F+T65A+Q327F; or

[0176] K79V+P2N+P45+P11F+T65A+Q327F (using SEQ ID NO: 3 herein for numbering); (b) saccharifying using a glucoamylase enzyme; (c) fermenting using a fermenting organism.

[0177] In a preferred embodiment the process of the disclosure comprises the steps of:

(a) liquefying the starch-containing material at a pH in the range between 4.0-6.5 at a temperature between 70-100.degree. C. using:

[0178] an alpha-amylase derived from Bacillus stearothermophilus having a double deletion in positions I181+G182, and optional substitution N193F; and optionally further one of the following set of substitutions:

[0179] V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S; or

[0180] V59A+089R+E129V+K177L+R179E+0254S+M284V (using SEQ ID NO: 1 herein for numbering);

[0181] a phospholipase C, preferably having a Melting Point (DSC) of at least about 80.degree. C.; such as an phospholipase C having at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 8 herein; and

[0182] optionally a Penicillium oxalicum glucoamylase in SEQ ID NO: 3 herein, preferably having substitutions selected from the group of:

[0183] K79V; or

[0184] K79V+P11F+T65A+Q327F; or

[0185] K79V+P2N+P45+P11F+T65A+Q327F (using SEQ ID NO: 3 herein for numbering); (b) saccharifying using a glucoamylase enzyme; (c) fermenting using a fermenting organism.

[0186] In a preferred embodiment the process of the disclosure comprises the steps of:

(a) liquefying the starch-containing material at a pH in the range between 4.0-6.5 at a temperature between 70-100.degree. C. using:

[0187] an alpha-amylase derived from Bacillus stearothermophilus having a double deletion in positions I181+G182, and optional substitution N193F; and optionally further one of the following set of substitutions:

[0188] V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S; or

[0189] V59A+Q89R+E129V+K177L+R179E+Q254S+M284V (using SEQ ID NO: 1 herein for numbering):

[0190] a phospholipase C, preferably having a Melting Point (DSC) of at least about 80.degree. C.; such as an phospholipase C having at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 9 herein; and

[0191] optionally a Penicillium oxalicum glucoamylase in SEQ ID NO: 3 herein, preferably having substitutions selected from the group of:

[0192] K79V; or

[0193] K79V+P11F+T65A+Q327F; or

[0194] K79V+P2N+P4S+P11F+T65A+Q327F (using SEQ ID NO: 3 herein for numbering); (b) saccharifying using a glucoamylase enzyme; (c) fermenting using a fermenting organism.

[0195] In another preferred embodiment the disclosure relates to processes of producing ethanol, comprising:

(a) liquifying a starch-containing material using the alpha-amylase shown as SEQ ID NO: 1 or an alpha-amylase having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99% sequence identity to SEQ ID NO: 1; (b) saccharifying the liquified starch-containing material using a carbohydrate-source generating enzyme, in particular a glucoamylase, to form fermentable sugars; (c) fermenting the fermentable sugars into ethanol using a fermenting organism; wherein the phospholipase C shown as SEQ ID NO: 2 or a phospholipase C having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99% sequence identity to SEQ ID NO: 2 is present and/or added during step (a).

[0196] In another preferred embodiment the disclosure relates to processes of producing ethanol, comprising:

(a) liquifying a starch-containing material using the alpha-amylase shown as SEQ ID NO: 1 or an alpha-amylase having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99% sequence identity to SEQ ID NO: 1; (b) saccharifying the liquified starch-containing material using a carbohydrate-source generating enzyme, in particular a glucoamylase, to form fermentable sugars; (c) fermenting the fermentable sugars into ethanol using a fermenting organism; wherein the phospholipase C shown as SEQ ID NO: 7 or a phospholipase C having at least 60%, at least 70%, at least 80%; at least 90%, at least 95%, at least 97%; at least 99% sequence identity to SEQ ID NO: 7 is present and/or added during step (a).

[0197] In another preferred embodiment the disclosure relates to processes of producing ethanol, comprising:

(a) liquifying a starch-containing material using the alpha-amylase shown as SEQ ID NO: 1 or an alpha-amylase having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99% sequence identity to SEQ ID NO: 1; (b) saccharifying the liquified starch-containing material using a carbohydrate-source generating enzyme; in particular a glucoamylase, to form fermentable sugars; (c) fermenting the fermentable sugars into ethanol using a fermenting organism; wherein the phospholipase C shown as SEQ ID NO: 8 or a phospholipase C having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99% sequence identity to SEQ ID NO: 8 is present and/or added during step (a).

[0198] In another preferred embodiment the disclosure relates to processes of producing ethanol, comprising:

(a) liquifying a starch-containing material using the alpha-amylase shown as SEQ ID NO: 1 or an alpha-amylase having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99% sequence identity to SEQ ID NO: 1; (b) saccharifying the liquified starch-containing material using a carbohydrate-source generating enzyme, in particular a glucoamylase, to form fermentable sugars; (c) fermenting the fermentable sugars into ethanol using a fermenting organism; wherein the phospholipase C shown as SEQ ID NO: 9 or a phospholipase C having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99% sequence identity to SEQ ID NO: 9 is present and/or added during step (a).

[0199] In a preferred embodiment a cellulase or cellulolytic enzyme composition is present and/or added during fermentation or simultaneous saccharification and fermentation.

[0200] In a preferred embodiment a cellulase or cellulolytic enzyme composition derived from Trichoderma reesei is present and/or added during fermentation or simultaneous saccharification and fermentation (SSF).

[0201] In a preferred embodiment a cellulase or cellulolytic enzyme composition and a glucoamylase are present and/or added during fermentation or simultaneous saccharification and fermentation.

[0202] In an embodiment the cellulase or cellulolytic enzyme composition is derived from Trichoderma reesei, Humicola insolens, Chrysosporium lucknowense or Penicillium decumbens.

[0203] The disclosure described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the disclosure. Any equivalent embodiments are intended to be within the scope of this disclosure. Indeed, various modifications of the disclosure 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. Various references are cited herein, the disclosures of which are incorporated by reference in their entireties. The present disclosure is further described by the following examples which should not be construed as limiting the scope of the disclosure.

Materials & Methods

[0204] Alpha-Amylase 369 (AA369): Bacillus stearothermophilus alpha-amylase with the mutations: I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+Q254S+M284V truncated to 491 amino acids (SEQ ID NO: 1 herein).

[0205] Phospholipase C (PePLC): Penicillium emersonii phospholipase C (SEQ ID NO: 2 herein).

[0206] Glucoamylase SA (GSA): Blend comprising Talaromyces emersonii glucoamylase disclosed as SEQ ID NO: 34 in WO99/28448 or SEQ ID NO: 4 herein, Trametes cingulata glucoamylase disclosed as SEQ ID NO: 2 in WO 06/69289 or SEQ ID NO: 5 herein, and Rhizomucor pusillus alpha-amylase with Aspergillus niger glucoamylase linker and starch binding domain (SBD) disclosed in SEQ ID NO: 6 herein having the following substitutions G128D+D143N (activity ratio in AGU:AGU:FAU-F is about 20:5:1).

[0207] Yeast: ETHANOL RED.TM. available from Red Star/Lesaffre, USA.

Determination of Td by Differential Scanning Calorimetry.

[0208] The thermostability of the phospholipase C (PePLC) was determined at pH 4.0, pH 5.5 and pH 7.0 by Differential Scanning calorimetry (DSC) using a VP-Capillary Differential Scanning calorimeter (MicroCal Inc., Piscataway, N.J., USA) at a protein concentration of approximately 0.5 mg/ml. The thermal denaturation temperature, Td (.degree. C.), was taken as the top of denaturation peak (major endothermic peak) in thermograms (Cp vs. T) obtained after heating enzyme solutions in buffer at a constant programmed heating rate of 200 K/hr. Sample- and reference-solutions (approx. 0.2 ml) were loaded into the calorimeter (reference: buffer without enzyme) from storage conditions at 10.degree. C. and thermally pre-equilibrated for 20 minutes at 20.degree. C. prior to DSC scan from 20.degree. C. to 100.degree. C. Denaturation temperatures (Td) were determined at an accuracy of approximately +/-1.degree. C. Tds obtained under these conditions for PePLC were 90 (pH 4.0), 88 (pH 5.5), and 83 (pH 7.0).

[0209] The thermostability of Harzianum (U49A3) was determined by Differential Scanning calorimetry (DSC) using a VP-Capillary Differential Scanning calorimeter (MicroCal Inc., Piscataway, N.J., USA). The thermal denaturation temperature, Td (.degree. C.), was taken as the top of denaturation peak (major endothermic peak) in thermograms (Cp vs. T) obtained after heating enzyme solutions (approx, 0.5 mg/ml) in buffer (50 mM acetate buffer pH 5.0) at a constant programmed heating rate of 200 K/hr.

[0210] Sample- and reference-solutions (approx. 0.2 ml) were loaded into the calorimeter (reference: buffer without enzyme) from storage conditions at 10 deg C. and thermally pre-equilibrated for 20 minutes at 20.degree. C. prior to DSC scan from 20.degree. C. to 100.degree. C. Denaturation temperatures were determined at an accuracy of approximately +1-1.degree. C., Td obtained under these conditions for U49A3 was 79 deg C.

[0211] The thermostability of Rasamsonia (U4BCJ) was determined by Differential Scanning calorimetry (DSC) using a VP-Capillary Differential Scanning calorimeter (MicroCal Inc., Piscataway, N.J., USA). The thermal denaturation temperature, Td (.degree. C.), was taken as the top of denaturation peak (major endothermic peak) in thermograms (Cp vs. T) obtained after heating enzyme solutions (approx. 0.5 mg/ml) in buffer (50 mM acetate buffer pH 5.5) at a constant programmed heating rate of 200 K/hr.

[0212] Sample- and reference-solutions (approx. 0.2 ml) were loaded into the calorimeter (reference: buffer without enzyme) from storage conditions at 10 deg C. and thermally pre-equilibrated for 20 minutes at 20.degree. C. prior to DSC scan from 20.degree. C. to 100.degree. C. Denaturation temperatures were determined at an accuracy of approximately +/-1.degree. C. Td obtained under these conditions for U4BCJ was 82 deg C.

EXAMPLE

[0213] Use of Thermostable Phospholipase C in Liquefaction Increases Ethanol Yield Liquefactions were performed on a 100 g scale using ground corn and backset obtained from different industrial corn ethanol plants to be used for the experiment. The dry solids (% DS) of the corn flour was 85.70% and the dry solids of the backset was 8.67%, both determined by Mettler-Toldeo HB43 halogen moisture balance. For liquefaction of the corn flour, 34.89 g corn flour was weighed into 200 mL Lab-O-Mat canisters along with 30 g backset and 35.11 g tap water to target a % DS of 32.5%. The pH of the corn slurry was adjusted to 5.0 for liquefaction.

[0214] The alpha-amylase (AA369) was used for liquefaction. In addition, thermostable Phospholipase C (PLC) from P. emersonii was evaluated at two doses, 10 and 50 .mu.g EP/g DS. Each canister was dosed with the appropriate amount of diluted enzyme shown in Table 1 below. Alpha-amylase was dosed based on weight of ground corn and PLC was dosed based on total weight of corn slurry and % DS. After enzymes were added, the canisters were closed and sealed tightly, shaken thoroughly, and then placed in a Mathis Lab-O-Mat. The program used starts out at room temperature and ramps up 6.degree. C./min for 12 minutes until it reaches 80.degree. C. The temperature is then controlled at 85.degree. C. for 105 minutes resulting in a total run of 1 hour and 57 minutes. The canisters are rotated back and forth at 45 rpm during the program.

TABLE-US-00001 TABLE 1 Enzyme Dosage In Liquefaction Amylase PLC AA369 0.016% w/w corn 0 .mu.g EP/g DS AA369 0.016% w/w corn 10 .mu.g EP/g DS AA369 0.016% w/w corn 50 .mu.g EP/g DS

[0215] After the Lab-O-Mat incubation, the canisters were removed and cooled in a room temperature water bath for 15 minutes. Once canisters were cooled, a spatula was used to remove as much material as possible into the corresponding labelled beaker. Liquefact weights were obtained to prepare each mash to 200 ppm urea and 3 ppm penicillin, and adjusted to pH 5 using 40% H.sub.2SO.sub.4 if necessary. The % DS of the alpha-amylase-only liquefact was measured on the Mettler-Toldeo moisture balance at 33.90% DS. This % DS was used to calculate doses for all treatments, which were run in triplicates. To prepare for fermentation, approximately 5 grams of liquefact was aliquoted into the appropriate number of 15 mL Nunc flip top tubes. Exact weight of mash added was calculated by weighing the tubes before and after mash addition. Each tube was dosed with the appropriate amount of diluted Glucoamylase SA (GSA). The actual glucoamylase dosage was 0.6 AGU/g DS with the addition of DI water to normalize the % DS of all the tubes for direct comparison. Each tube was dosed with 100 .mu.l of Ethanol Red rehydrated yeast (rehydrated with tap water), vented by drilling a 1/64'' hole in the cap, and then vortexed. Tubes were placed in a 32.degree. C. waterbath for 52 hours but removed twice a day for vortexing.

[0216] After 52 hours of fermentation, samples were collected for HPLC analysis. This process began with stopping the enzyme and yeast reactions by adding 50 .mu.L of 40% H.sub.2SO.sub.4 (10 .mu.L/g corn mash) and vortexing to distribute the acid. Samples were then centrifuged at 1430.times.g for 10 minutes and the supernatant was filtered through a 0.45 .mu.m filter into a HPLC vial. Samples were stored at 4.degree. C. until analysis. The system used to determine ethanol and oligosaccharides concentration was the Agilent 1100/1200 HPLC system coupled with an RI detector. The separation column was a BioRad Aminex HPX-87H ion exclusion column (300 mm.times.7.8 mm).

RESULTS

[0217] The final ethanol and percentage of ethanol increase by addition of Phospholipase C is summarized in Table 2 below.

TABLE-US-00002 TABLE 2 Summarized Ethanol Yield and Precent Change Results Ethanol Ethanol Liquefaction Treatment (% w/v) Increase (%) AA369 only Control 12.13 -- AA369 plus 10 .mu.g PePLC 12.22 0.74% AA369 plus 50 .mu.g PePLC 12.31 1.48%

Sequence CWU 1

1

91515PRTBacillus stearothermophilus 1Ala Ala Pro Phe Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Leu 1 5 10 15 Pro Asp Asp Gly Thr Leu Trp Thr Lys Val Ala Asn Glu Ala Asn Asn 20 25 30 Leu Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr Lys 35 40 45 Gly Thr Ser Arg Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp 50 55 60 Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr 65 70 75 80 Lys Ala Gln Tyr Leu Gln Ala Ile Gln Ala Ala His Ala Ala Gly Met 85 90 95 Gln Val Tyr Ala Asp Val Val Phe Asp His Lys Gly Gly Ala Asp Gly 100 105 110 Thr Glu Trp Val Asp Ala Val Glu Val Asn Pro Ser Asp Arg Asn Gln 115 120 125 Glu Ile Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp Phe 130 135 140 Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr His 145 150 155 160 Phe Asp Gly Val Asp Trp Asp Glu Ser Arg Lys Leu Ser Arg Ile Tyr 165 170 175 Lys Phe Arg Gly Ile Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu 180 185 190 Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met Asp His 195 200 205 Pro Glu Val Val Thr Glu Leu Lys Asn Trp Gly Lys Trp Tyr Val Asn 210 215 220 Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys 225 230 235 240 Phe Ser Phe Phe Pro Asp Trp Leu Ser Tyr Val Arg Ser Gln Thr Gly 245 250 255 Lys Pro Leu Phe Thr Val Gly Glu Tyr Trp Ser Tyr Asp Ile Asn Lys 260 265 270 Leu His Asn Tyr Ile Thr Lys Thr Asn Gly Thr Met Ser Leu Phe Asp 275 280 285 Ala Pro Leu His Asn Lys Phe Tyr Thr Ala Ser Lys Ser Gly Gly Ala 290 295 300 Phe Asp Met Arg Thr Leu Met Thr Asn Thr Leu Met Lys Asp Gln Pro 305 310 315 320 Thr Leu Ala Val Thr Phe Val Asp Asn His Asp Thr Glu Pro Gly Gln 325 330 335 Ala Leu Gln Ser Trp Val Asp Pro Trp Phe Lys Pro Leu Ala Tyr Ala 340 345 350 Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp 355 360 365 Tyr Tyr Gly Ile Pro Gln Tyr Asn Ile Pro Ser Leu Lys Ser Lys Ile 370 375 380 Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln His 385 390 395 400 Asp Tyr Leu Asp His Ser Asp Ile Ile Gly Trp Thr Arg Glu Gly Val 405 410 415 Thr Glu Lys Pro Gly Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro 420 425 430 Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala Gly Lys Val 435 440 445 Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn Ser 450 455 460 Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Val Trp 465 470 475 480 Val Pro Arg Lys Thr Thr Val Ser Thr Ile Ala Arg Pro Ile Thr Thr 485 490 495 Arg Pro Trp Thr Gly Glu Phe Val Arg Trp Thr Glu Pro Arg Leu Val 500 505 510 Ala Trp Pro 515 2610PRTPenicillium emersoniiSIGNAL(1)..(16)mat_peptide(17)..(610) 2Met Arg Val Leu Ala Leu Ile Ala Ala Leu Ala Thr Val Ala Thr Ala -15 -10 -5 -1 Ser Ala Pro Tyr Asp Lys Arg Asp Leu Ala Gln Glu Ile Trp Asp Asp 1 5 10 15 Ile Lys Asn Ala Val Asp Cys Ala Gly Cys Gln Val Val Leu Thr Ala 20 25 30 Leu Lys Gly Val Ala Asp Leu Gly Thr Thr Ala Leu Val Asp Val Leu 35 40 45 Thr Glu Val Cys Asn Ile Ser Gly Lys Glu Asp Ser Asp Val Cys Ser 50 55 60 Gly Ile Ile Ser Arg Glu Gly Pro Val Leu Asp Tyr Val Leu Gln His 65 70 75 80 Leu Asp Ile Gly Ser His Thr Ser Gln Val Ile Cys Ala Ser Ala Phe 85 90 95 Gly Leu Cys Gln Tyr Pro Glu Val Arg Pro Tyr Asn Leu Thr Phe Pro 100 105 110 Lys Pro Lys Pro Asn Thr Thr Arg Pro Glu Pro Ser Gly Glu Ser Pro 115 120 125 Ile Gln Val Val His Phe Ser Asp Thr His Val Asp Leu Ser Tyr Glu 130 135 140 Thr Gly Ser Asn Tyr Asn Cys Thr Lys Pro Ile Cys Cys Arg Pro Tyr 145 150 155 160 Thr Ala Glu Asp Ala Pro Gly Asn Thr Thr Thr Pro Cys Gly Pro Tyr 165 170 175 Gly Asn Thr Lys Cys Asp Ala Pro Leu Ser Leu Glu Glu Ser Met Phe 180 185 190 Ala Ala Ile Lys Ala Leu Asn Pro Gln Pro Ala Phe Ser Ile Tyr Thr 195 200 205 Gly Asp Val Val Ala His Asp Ile Trp Leu Val Asp Gln Asn Glu Val 210 215 220 Ile Glu Asp Leu Asn Ala Thr Tyr Asp Arg Met Ala Gly Leu Gly Leu 225 230 235 240 Val Tyr Ala Ala Ile Gly Asn His Asp Thr Ala Pro Val Asn Asp Leu 245 250 255 Pro Thr Ser Asn Ile Pro Ser Glu Tyr Ser Ala Asn Trp Thr Tyr Glu 260 265 270 Ala Leu Ser Tyr Asp Phe Thr Met Leu Thr Gln Ser Ala Ser Ala Gln 275 280 285 Thr Ala Ala Asn Tyr Gly Ser Tyr Ser Ala Ile Tyr Pro Gly Ser Tyr 290 295 300 Gly Thr Asp Leu Arg Val Ile Ser Tyr Asn Ser Ile Phe Tyr Tyr Val 305 310 315 320 Asp Asn Phe Trp Ala Tyr Gln Asp Pro Met Glu Phe Asp Pro Asp Gly 325 330 335 Gln Leu Ala Trp Leu Ile Asn Glu Leu Gln Glu Ala Glu Thr Ala Gly 340 345 350 Gln Arg Val Trp Ile Ile Ala His Val Pro Thr Gly Thr Ser Asp His 355 360 365 Phe His Asp Tyr Ser His Tyr Phe Asp Gln Ile Val Gln Arg Tyr Glu 370 375 380 Ala Thr Ile Ala Ala Leu Phe Tyr Gly His Thr His Ile Asp Gln Phe 385 390 395 400 Gln Ile Ser Tyr Ser Asn Tyr Ser Asn Arg Ala Phe Asp Thr Ala Thr 405 410 415 Ala Ile Gly Tyr Ile Met Pro Ser Leu Thr Pro Thr Ser Gly Pro Pro 420 425 430 Thr Phe Arg Val Tyr Asp Val Asp Pro Lys Thr Phe Ala Val Leu Asp 435 440 445 Phe Thr Asn Tyr Ile Ala Asn Ile Ser Asp Pro Ala Phe Gln Ser Gly 450 455 460 Pro Ser Trp Gln Lys Tyr Tyr Ser Ala Lys Glu Thr Tyr Gly Ser Leu 465 470 475 480 Leu Ser Pro Pro Val Thr Asp Pro Thr Ala Glu Leu Thr Pro Ala Phe 485 490 495 Trp His Asn Val Thr Val Ala Phe Glu Gln Asp Asn Ala Thr Phe Gln 500 505 510 Glu Tyr Trp Ala Arg Gln Thr Arg Gly Tyr Asp Val Ser Ser Cys Thr 515 520 525 Gly Ser Cys Ile Thr Gln Ala Ile Cys Gly Leu Arg Ala Gly Asp Ala 530 535 540 Gln Tyr Asn Cys Val Thr Pro Thr Pro Gly Phe Asn Phe Ala Lys Arg 545 550 555 560 Asp Thr Ser Asn Pro Lys Gln Ala Leu Ser His Val Glu Lys Cys Glu 565 570 575 Gly Ser Gly Leu Leu Gly Leu Leu Arg Arg Met Val Ala Asp Ser Lys 580 585 590 Ser Ser 3595PRTPenicillium oxalicum 3Arg Pro Asp Pro Lys Gly Gly Asn Leu Thr Pro Phe Ile His Lys Glu 1 5 10 15 Gly Glu Arg Ser Leu Gln Gly Ile Leu Asp Asn Leu Gly Gly Arg Gly 20 25 30 Lys Lys Thr Pro Gly Thr Ala Ala Gly Leu Phe Ile Ala Ser Pro Asn 35 40 45 Thr Glu Asn Pro Asn Tyr Tyr Tyr Thr Trp Thr Arg Asp Ser Ala Leu 50 55 60 Thr Ala Lys Cys Leu Ile Asp Leu Phe Glu Asp Ser Arg Ala Lys Phe 65 70 75 80 Pro Ile Asp Arg Lys Tyr Leu Glu Thr Gly Ile Arg Asp Tyr Lys Ser 85 90 95 Ser Gln Ala Ile Leu Gln Ser Val Ser Asn Pro Ser Gly Thr Leu Lys 100 105 110 Asp Gly Ser Gly Leu Gly Glu Pro Lys Phe Glu Ile Asp Leu Asn Pro 115 120 125 Phe Ser Gly Ala Trp Gly Arg Pro Gln Arg Asp Gly Pro Ala Leu Arg 130 135 140 Ala Thr Ala Met Ile Thr Tyr Ala Asn Tyr Leu Ile Ser His Gly Gln 145 150 155 160 Lys Ser Asp Val Ser Gln Val Met Trp Pro Ile Ile Ala Asn Asp Leu 165 170 175 Ala Tyr Val Gly Gln Tyr Trp Asn Asn Thr Gly Phe Asp Leu Trp Glu 180 185 190 Glu Val Asp Gly Ser Ser Phe Phe Thr Ile Ala Val Gln His Arg Ala 195 200 205 Leu Val Glu Gly Ser Gln Leu Ala Lys Lys Leu Gly Lys Ser Cys Asp 210 215 220 Ala Cys Asp Ser Gln Pro Pro Gln Ile Leu Cys Phe Leu Gln Ser Phe 225 230 235 240 Trp Asn Gly Lys Tyr Ile Thr Ser Asn Ile Asn Thr Gln Ala Ser Arg 245 250 255 Ser Gly Ile Asp Leu Asp Ser Val Leu Gly Ser Ile His Thr Phe Asp 260 265 270 Pro Glu Ala Ala Cys Asp Asp Ala Thr Phe Gln Pro Cys Ser Ala Arg 275 280 285 Ala Leu Ala Asn His Lys Val Tyr Val Asp Ser Phe Arg Ser Ile Tyr 290 295 300 Lys Ile Asn Ala Gly Leu Ala Glu Gly Ser Ala Ala Asn Val Gly Arg 305 310 315 320 Tyr Pro Glu Asp Val Tyr Gln Gly Gly Asn Pro Trp Tyr Leu Ala Thr 325 330 335 Leu Gly Ala Ser Glu Leu Leu Tyr Asp Ala Leu Tyr Gln Trp Asp Arg 340 345 350 Leu Gly Lys Leu Glu Val Ser Glu Thr Ser Leu Ser Phe Phe Lys Asp 355 360 365 Phe Asp Ala Thr Val Lys Ile Gly Ser Tyr Ser Arg Asn Ser Lys Thr 370 375 380 Tyr Lys Lys Leu Thr Gln Ser Ile Lys Ser Tyr Ala Asp Gly Phe Ile 385 390 395 400 Gln Leu Val Gln Gln Tyr Thr Pro Ser Asn Gly Ser Leu Ala Glu Gln 405 410 415 Tyr Asp Arg Asn Thr Ala Ala Pro Leu Ser Ala Asn Asp Leu Thr Trp 420 425 430 Ser Phe Ala Ser Phe Leu Thr Ala Thr Gln Arg Arg Asp Ala Val Val 435 440 445 Pro Pro Ser Trp Gly Ala Lys Ser Ala Asn Lys Val Pro Thr Thr Cys 450 455 460 Ser Ala Ser Pro Val Val Gly Thr Tyr Lys Ala Pro Thr Ala Thr Phe 465 470 475 480 Ser Ser Lys Thr Lys Cys Val Pro Ala Lys Asp Ile Val Pro Ile Thr 485 490 495 Phe Tyr Leu Ile Glu Asn Thr Tyr Tyr Gly Glu Asn Val Phe Met Ser 500 505 510 Gly Asn Ile Thr Ala Leu Gly Asn Trp Asp Ala Lys Lys Gly Phe Pro 515 520 525 Leu Thr Ala Asn Leu Tyr Thr Gln Asp Gln Asn Leu Trp Phe Ala Ser 530 535 540 Val Glu Phe Ile Pro Ala Gly Thr Pro Phe Glu Tyr Lys Tyr Tyr Lys 545 550 555 560 Val Glu Pro Asn Gly Asp Ile Thr Trp Glu Lys Gly Pro Asn Arg Val 565 570 575 Phe Val Ala Pro Thr Gly Cys Pro Val Gln Pro His Ser Asn Asp Val 580 585 590 Trp Gln Phe 595 4618PRTTalaromyces emersonii 4Met Ala Ser Leu Val Ala Gly Ala Leu Cys Ile Leu Gly Leu Thr Pro 1 5 10 15 Ala Ala Phe Ala Arg Ala Pro Val Ala Ala Arg Ala Thr Gly Ser Leu 20 25 30 Asp Ser Phe Leu Ala Thr Glu Thr Pro Ile Ala Leu Gln Gly Val Leu 35 40 45 Asn Asn Ile Gly Pro Asn Gly Ala Asp Val Ala Gly Ala Ser Ala Gly 50 55 60 Ile Val Val Ala Ser Pro Ser Arg Ser Asp Pro Asn Tyr Phe Tyr Ser 65 70 75 80 Trp Thr Arg Asp Ala Ala Leu Thr Ala Lys Tyr Leu Val Asp Ala Phe 85 90 95 Ile Ala Gly Asn Lys Asp Leu Glu Gln Thr Ile Gln Gln Tyr Ile Ser 100 105 110 Ala Gln Ala Lys Val Gln Thr Ile Ser Asn Pro Ser Gly Asp Leu Ser 115 120 125 Thr Gly Gly Leu Gly Glu Pro Lys Phe Asn Val Asn Glu Thr Ala Phe 130 135 140 Thr Gly Pro Trp Gly Arg Pro Gln Arg Asp Gly Pro Ala Leu Arg Ala 145 150 155 160 Thr Ala Leu Ile Ala Tyr Ala Asn Tyr Leu Ile Asp Asn Gly Glu Ala 165 170 175 Ser Thr Ala Asp Glu Ile Ile Trp Pro Ile Val Gln Asn Asp Leu Ser 180 185 190 Tyr Ile Thr Gln Tyr Trp Asn Ser Ser Thr Phe Asp Leu Trp Glu Glu 195 200 205 Val Glu Gly Ser Ser Phe Phe Thr Thr Ala Val Gln His Arg Ala Leu 210 215 220 Val Glu Gly Asn Ala Leu Ala Thr Arg Leu Asn His Thr Cys Ser Asn 225 230 235 240 Cys Val Ser Gln Ala Pro Gln Val Leu Cys Phe Leu Gln Ser Tyr Trp 245 250 255 Thr Gly Ser Tyr Val Leu Ala Asn Phe Gly Gly Ser Gly Arg Ser Gly 260 265 270 Lys Asp Val Asn Ser Ile Leu Gly Ser Ile His Thr Phe Asp Pro Ala 275 280 285 Gly Gly Cys Asp Asp Ser Thr Phe Gln Pro Cys Ser Ala Arg Ala Leu 290 295 300 Ala Asn His Lys Val Val Thr Asp Ser Phe Arg Ser Ile Tyr Ala Ile 305 310 315 320 Asn Ser Gly Ile Ala Glu Gly Ser Ala Val Ala Val Gly Arg Tyr Pro 325 330 335 Glu Asp Val Tyr Gln Gly Gly Asn Pro Trp Tyr Leu Ala Thr Ala Ala 340 345 350 Ala Ala Glu Gln Leu Tyr Asp Ala Ile Tyr Gln Trp Lys Lys Ile Gly 355 360 365 Ser Ile Ser Ile Thr Asp Val Ser Leu Pro Phe Phe Gln Asp Ile Tyr 370 375 380 Pro Ser Ala Ala Val Gly Thr Tyr Asn Ser Gly Ser Thr Thr Phe Asn 385 390 395 400 Asp Ile Ile Ser Ala Val Gln Thr Tyr Gly Asp Gly Tyr Leu Ser Ile 405 410 415 Val Glu Lys Tyr Thr Pro Ser Asp Gly Ser Leu Thr Glu Gln Phe Ser 420 425 430 Arg Thr Asp Gly Thr Pro Leu Ser Ala Ser Ala Leu Thr Trp Ser Tyr 435 440 445 Ala Ser Leu Leu Thr Ala Ser Ala Arg Arg Gln Ser Val Val Pro Ala 450 455 460 Ser Trp Gly Glu Ser Ser Ala Ser Ser Val Pro Ala Val Cys Ser Ala 465 470 475 480 Thr Ser Ala Thr Gly Pro Tyr Ser Thr Ala Thr Asn Thr Val Trp Pro 485 490 495 Ser Ser Gly Ser Gly Ser Ser Thr Thr Thr Ser Ser Ala Pro Cys Thr 500 505 510 Thr Pro Thr Ser Val Ala Val Thr Phe Asp Glu Ile Val Ser Thr Ser 515 520 525 Tyr Gly Glu Thr Ile Tyr Leu Ala Gly Ser Ile Pro Glu Leu Gly Asn 530 535 540 Trp Ser Thr Ala Ser Ala Ile Pro Leu Arg Ala Asp Ala Tyr Thr Asn 545 550

555 560 Ser Asn Pro Leu Trp Tyr Val Thr Val Asn Leu Pro Pro Gly Thr Ser 565 570 575 Phe Glu Tyr Lys Phe Phe Lys Asn Gln Thr Asp Gly Thr Ile Val Trp 580 585 590 Glu Asp Asp Pro Asn Arg Ser Tyr Thr Val Pro Ala Tyr Cys Gly Gln 595 600 605 Thr Thr Ala Ile Leu Asp Asp Ser Trp Gln 610 615 5574PRTTrametes cingulata 5Met Arg Phe Thr Leu Leu Thr Ser Leu Leu Gly Leu Ala Leu Gly Ala 1 5 10 15 Phe Ala Gln Ser Ser Ala Ala Asp Ala Tyr Val Ala Ser Glu Ser Pro 20 25 30 Ile Ala Lys Ala Gly Val Leu Ala Asn Ile Gly Pro Ser Gly Ser Lys 35 40 45 Ser Asn Gly Ala Lys Ala Gly Ile Val Ile Ala Ser Pro Ser Thr Ser 50 55 60 Asn Pro Asn Tyr Leu Tyr Thr Trp Thr Arg Asp Ser Ser Leu Val Phe 65 70 75 80 Lys Ala Leu Ile Asp Gln Phe Thr Thr Gly Glu Asp Thr Ser Leu Arg 85 90 95 Thr Leu Ile Asp Glu Phe Thr Ser Ala Glu Ala Ile Leu Gln Gln Val 100 105 110 Pro Asn Pro Ser Gly Thr Val Ser Thr Gly Gly Leu Gly Glu Pro Lys 115 120 125 Phe Asn Ile Asp Glu Thr Ala Phe Thr Asp Ala Trp Gly Arg Pro Gln 130 135 140 Arg Asp Gly Pro Ala Leu Arg Ala Thr Ala Ile Ile Thr Tyr Ala Asn 145 150 155 160 Trp Leu Leu Asp Asn Lys Asn Thr Thr Tyr Val Thr Asn Thr Leu Trp 165 170 175 Pro Ile Ile Lys Leu Asp Leu Asp Tyr Val Ala Ser Asn Trp Asn Gln 180 185 190 Ser Thr Phe Asp Leu Trp Glu Glu Ile Asn Ser Ser Ser Phe Phe Thr 195 200 205 Thr Ala Val Gln His Arg Ala Leu Arg Glu Gly Ala Thr Phe Ala Asn 210 215 220 Arg Ile Gly Gln Thr Ser Val Val Ser Gly Tyr Thr Thr Gln Ala Asn 225 230 235 240 Asn Leu Leu Cys Phe Leu Gln Ser Tyr Trp Asn Pro Thr Gly Gly Tyr 245 250 255 Ile Thr Ala Asn Thr Gly Gly Gly Arg Ser Gly Lys Asp Ala Asn Thr 260 265 270 Val Leu Thr Ser Ile His Thr Phe Asp Pro Ala Ala Gly Cys Asp Ala 275 280 285 Val Thr Phe Gln Pro Cys Ser Asp Lys Ala Leu Ser Asn Leu Lys Val 290 295 300 Tyr Val Asp Ala Phe Arg Ser Ile Tyr Ser Ile Asn Ser Gly Ile Ala 305 310 315 320 Ser Asn Ala Ala Val Ala Thr Gly Arg Tyr Pro Glu Asp Ser Tyr Met 325 330 335 Gly Gly Asn Pro Trp Tyr Leu Thr Thr Ser Ala Val Ala Glu Gln Leu 340 345 350 Tyr Asp Ala Leu Ile Val Trp Asn Lys Leu Gly Ala Leu Asn Val Thr 355 360 365 Ser Thr Ser Leu Pro Phe Phe Gln Gln Phe Ser Ser Gly Val Thr Val 370 375 380 Gly Thr Tyr Ala Ser Ser Ser Ser Thr Phe Lys Thr Leu Thr Ser Ala 385 390 395 400 Ile Lys Thr Phe Ala Asp Gly Phe Leu Ala Val Asn Ala Lys Tyr Thr 405 410 415 Pro Ser Asn Gly Gly Leu Ala Glu Gln Tyr Ser Arg Ser Asn Gly Ser 420 425 430 Pro Val Ser Ala Val Asp Leu Thr Trp Ser Tyr Ala Ala Ala Leu Thr 435 440 445 Ser Phe Ala Ala Arg Ser Gly Lys Thr Tyr Ala Ser Trp Gly Ala Ala 450 455 460 Gly Leu Thr Val Pro Thr Thr Cys Ser Gly Ser Gly Gly Ala Gly Thr 465 470 475 480 Val Ala Val Thr Phe Asn Val Gln Ala Thr Thr Val Phe Gly Glu Asn 485 490 495 Ile Tyr Ile Thr Gly Ser Val Pro Ala Leu Gln Asn Trp Ser Pro Asp 500 505 510 Asn Ala Leu Ile Leu Ser Ala Ala Asn Tyr Pro Thr Trp Ser Ile Thr 515 520 525 Val Asn Leu Pro Ala Ser Thr Thr Ile Glu Tyr Lys Tyr Ile Arg Lys 530 535 540 Phe Asn Gly Ala Val Thr Trp Glu Ser Asp Pro Asn Asn Ser Ile Thr 545 550 555 560 Thr Pro Ala Ser Gly Thr Phe Thr Gln Asn Asp Thr Trp Arg 565 570 6583PRTRhizomucor pusillus 6Ala Thr Ser Asp Asp Trp Lys Gly Lys Ala Ile Tyr Gln Leu Leu Thr 1 5 10 15 Asp Arg Phe Gly Arg Ala Asp Asp Ser Thr Ser Asn Cys Ser Asn Leu 20 25 30 Ser Asn Tyr Cys Gly Gly Thr Tyr Glu Gly Ile Thr Lys His Leu Asp 35 40 45 Tyr Ile Ser Gly Met Gly Phe Asp Ala Ile Trp Ile Ser Pro Ile Pro 50 55 60 Lys Asn Ser Asp Gly Gly Tyr His Gly Tyr Trp Ala Thr Asp Phe Tyr 65 70 75 80 Gln Leu Asn Ser Asn Phe Gly Asp Glu Ser Gln Leu Lys Ala Leu Ile 85 90 95 Gln Ala Ala His Glu Arg Asp Met Tyr Val Met Leu Asp Val Val Ala 100 105 110 Asn His Ala Gly Pro Thr Ser Asn Gly Tyr Ser Gly Tyr Thr Phe Gly 115 120 125 Asp Ala Ser Leu Tyr His Pro Lys Cys Thr Ile Asp Tyr Asn Asp Gln 130 135 140 Thr Ser Ile Glu Gln Cys Trp Val Ala Asp Glu Leu Pro Asp Ile Asp 145 150 155 160 Thr Glu Asn Ser Asp Asn Val Ala Ile Leu Asn Asp Ile Val Ser Gly 165 170 175 Trp Val Gly Asn Tyr Ser Phe Asp Gly Ile Arg Ile Asp Thr Val Lys 180 185 190 His Ile Arg Lys Asp Phe Trp Thr Gly Tyr Ala Glu Ala Ala Gly Val 195 200 205 Phe Ala Thr Gly Glu Val Phe Asn Gly Asp Pro Ala Tyr Val Gly Pro 210 215 220 Tyr Gln Lys Tyr Leu Pro Ser Leu Ile Asn Tyr Pro Met Tyr Tyr Ala 225 230 235 240 Leu Asn Asp Val Phe Val Ser Lys Ser Lys Gly Phe Ser Arg Ile Ser 245 250 255 Glu Met Leu Gly Ser Asn Arg Asn Ala Phe Glu Asp Thr Ser Val Leu 260 265 270 Thr Thr Phe Val Asp Asn His Asp Asn Pro Arg Phe Leu Asn Ser Gln 275 280 285 Ser Asp Lys Ala Leu Phe Lys Asn Ala Leu Thr Tyr Val Leu Leu Gly 290 295 300 Glu Gly Ile Pro Ile Val Tyr Tyr Gly Ser Glu Gln Gly Phe Ser Gly 305 310 315 320 Gly Ala Asp Pro Ala Asn Arg Glu Val Leu Trp Thr Thr Asn Tyr Asp 325 330 335 Thr Ser Ser Asp Leu Tyr Gln Phe Ile Lys Thr Val Asn Ser Val Arg 340 345 350 Met Lys Ser Asn Lys Ala Val Tyr Met Asp Ile Tyr Val Gly Asp Asn 355 360 365 Ala Tyr Ala Phe Lys His Gly Asp Ala Leu Val Val Leu Asn Asn Tyr 370 375 380 Gly Ser Gly Ser Thr Asn Gln Val Ser Phe Ser Val Ser Gly Lys Phe 385 390 395 400 Asp Ser Gly Ala Ser Leu Met Asp Ile Val Ser Asn Ile Thr Thr Thr 405 410 415 Val Ser Ser Asp Gly Thr Val Thr Phe Asn Leu Lys Asp Gly Leu Pro 420 425 430 Ala Ile Phe Thr Ser Ala Thr Gly Gly Thr Thr Thr Thr Ala Thr Pro 435 440 445 Thr Gly Ser Gly Ser Val Thr Ser Thr Ser Lys Thr Thr Ala Thr Ala 450 455 460 Ser Lys Thr Ser Thr Ser Thr Ser Ser Thr Ser Cys Thr Thr Pro Thr 465 470 475 480 Ala Val Ala Val Thr Phe Asp Leu Thr Ala Thr Thr Thr Tyr Gly Glu 485 490 495 Asn Ile Tyr Leu Val Gly Ser Ile Ser Gln Leu Gly Asp Trp Glu Thr 500 505 510 Ser Asp Gly Ile Ala Leu Ser Ala Asp Lys Tyr Thr Ser Ser Asp Pro 515 520 525 Leu Trp Tyr Val Thr Val Thr Leu Pro Ala Gly Glu Ser Phe Glu Tyr 530 535 540 Lys Phe Ile Arg Ile Glu Ser Asp Asp Ser Val Glu Trp Glu Ser Asp 545 550 555 560 Pro Asn Arg Glu Tyr Thr Val Pro Gln Ala Cys Gly Thr Ser Thr Ala 565 570 575 Thr Val Thr Asp Thr Trp Arg 580 7643PRTTrichoderma harzianum 7Met Arg Pro Ser Ser Thr Leu Ser Leu Leu Ala Leu Gly Ser Val Ala 1 5 10 15 Gln Ala Ala Val Ser Leu Glu Asp Ser Ser Leu Ser Pro Arg Glu Ile 20 25 30 Glu Asn Ile Glu Arg Ala Ile Glu Ala Arg Ser Leu Ala Asp Asp Ile 35 40 45 Trp Asn Asp Ile Lys Asn Ala Ala Thr Cys Thr Ala Cys Gln Gly Ile 50 55 60 Leu Val Leu Leu Lys Gly Ile Ala Val Phe Gly Asp Gly Ala Phe Val 65 70 75 80 Ser Val Ala Thr Glu Leu Cys Lys Leu Ala Lys Val Glu Asp Asp Asp 85 90 95 Val Cys Glu Gly Thr Val Gly Leu Glu Gly Pro Ile Ile Ala Asp Ala 100 105 110 Ile Arg Asn Met Asp Leu Gly Ser Asp Thr Ser Lys Leu Phe Cys Gly 115 120 125 Ser Phe Leu Gly Leu Cys Pro Glu Pro Ser Val Pro Gln Trp Lys Ile 130 135 140 Pro Phe Pro Ser Pro Lys Pro Gln Thr Gly Arg Pro Ala Pro Ser Gly 145 150 155 160 Lys Thr Pro Leu Lys Val Val Gln Tyr Ser Asp Ile His Ile Asp Pro 165 170 175 Leu Tyr Val Ser Gly Ser Ser Ser Asn Cys Thr Lys Pro Ile Cys Cys 180 185 190 Arg Pro Tyr Thr Ala Ala Asp Glu Pro Gly Lys Ser Thr Ser Pro Ala 195 200 205 Gly Pro Asn Gly Asp His Lys Cys Asp Thr Pro Val Gly Leu Glu Ile 210 215 220 Ser Met Tyr Gln Ala Ile Lys Asn Ile Val Pro Asp Ala Ala Leu Thr 225 230 235 240 Leu Phe Thr Gly Asp Ile Val Asp His Ala Ile Trp Asn Thr Ser Gln 245 250 255 Pro Tyr Asn Gln Lys Gln Ile Ser Asp Ala Tyr Thr Tyr Met Ser Gln 260 265 270 Tyr Leu Gly Ile Val Tyr Gly Thr Ala Gly Asn His Glu Ser Asn Pro 275 280 285 Thr Asn Ala Phe Pro Pro Arg Ser Ile Ser Asn Ser Ser Gln Trp Val 290 295 300 Tyr Asp Ala Leu Ser Asp Gln Trp Thr Arg Trp Val Gly Ala Ser Ala 305 310 315 320 Glu Ser Glu Ile Glu Ser Ile Gly Ala Tyr Ser Thr Lys Tyr Pro Asn 325 330 335 Gly Asn Leu Arg Val Ile Ser Leu Asn Thr Asn Phe Tyr Tyr Arg Met 340 345 350 Asn Phe Trp Leu Tyr Gln Glu Asp Ile Glu Gln Asp Pro Asp Gly Gln 355 360 365 Ile Gln Trp Leu Val Asn Glu Leu Asp Ala Ala Glu Lys Ala Gly Glu 370 375 380 Arg Val Tyr Ile Ile Gly His Met Pro Leu Gly Glu Gly Asp Ala Phe 385 390 395 400 His Ala Gly Ser Asn Tyr Leu Asp Gln Val Val Asn Arg Tyr Ser Ser 405 410 415 Thr Ile Ala Ala Met Phe Phe Gly His Thr His Val Asp His Phe Glu 420 425 430 Val Ser Tyr Ser Asp Tyr Asn Ser Arg Asp Ala Ser His Ala Val Met 435 440 445 Ala Ser Tyr Ile Cys Pro Ser Leu Thr Pro Thr Ser Gly Met Pro Ser 450 455 460 Phe Arg Val Tyr Asp Val Asp Pro Glu Thr Phe Ala Val Leu Asp Thr 465 470 475 480 Thr Thr Tyr Ile Ala Asp Met Thr Asn Ala Asp Phe Gln Thr Thr Gly 485 490 495 Pro Val Trp Thr Lys Leu Tyr Ser Ala Lys Glu Thr Tyr Gly Ser Lys 500 505 510 Leu Asn Pro Pro Val Thr Asp Ala Ser Val Glu Leu Thr Pro Ala Phe 515 520 525 Trp His Asn Val Thr Ala Leu Phe Glu Ser Asn Ser Asp Val Phe Asn 530 535 540 Glu Tyr Ile Ser Leu Lys Ser Arg Gly Trp Asn Val Ala Ser Cys Thr 545 550 555 560 Gly Asp Cys Lys Thr Gln Glu Leu Cys Gln Leu Arg Ala Gly Arg Ser 565 570 575 Glu Asn Asn Cys Val Val Pro Ser Ile Gly Leu His Leu Asn Lys Arg 580 585 590 Ser Asp Glu Leu His Gly His Ser His His Arg Ser His Asp His Gln 595 600 605 Glu Cys Gly Met Ser Ala Gly Met Lys Thr Leu Gly Ser Leu Ala Val 610 615 620 Arg Lys Asp Leu Leu Asp Glu Leu Glu Ser Arg Ala Asn Glu Leu Lys 625 630 635 640 Ala Lys Ala 8645PRTTrichoderma harzianum 8Met Arg Pro Ser Ser Thr Leu Ser Leu Leu Ala Leu Gly Ser Val Ala 1 5 10 15 Gln Ala Ala Val Ser Leu Glu Asn Ser Ser Leu Pro Pro Arg Asp Ile 20 25 30 Glu Asn Ile Glu Arg Ala Ile Glu Ala Arg Ser Leu Ala Asp Asp Ile 35 40 45 Trp Asn Asp Ile Lys Asn Ala Ala Thr Cys Thr Ala Cys Gln Gly Ile 50 55 60 Leu Val Leu Leu Lys Gly Ile Ala Val Phe Gly Asp Gly Ala Phe Val 65 70 75 80 Ser Val Ala Thr Glu Leu Cys Lys Leu Ala Lys Val Glu Asp Asp Asp 85 90 95 Val Cys Glu Gly Thr Val Gly Leu Glu Gly Pro Ile Ile Ala Asp Ala 100 105 110 Ile Arg Asn Met Asp Leu Gly Ser Asp Thr Ser Lys Leu Phe Cys Gly 115 120 125 Ser Phe Leu Gly Leu Cys Pro Glu Pro Ser Val Pro Gln Trp Lys Ile 130 135 140 Pro Phe Pro Ser Pro Lys Pro Gln Thr Gly Arg Pro Ala Pro Ser Gly 145 150 155 160 Lys Thr Pro Leu Lys Val Val Gln Tyr Ser Asp Ile His Ile Asp Pro 165 170 175 Leu Tyr Val Ser Gly Ser Ser Ser Asn Cys Thr Lys Pro Ile Cys Cys 180 185 190 Arg Pro Tyr Thr Ala Ala Asp Glu Pro Gly Lys Ser Thr Ser Pro Ala 195 200 205 Gly Pro Asn Gly Asp His Lys Cys Asp Thr Pro Val Gly Leu Glu Ile 210 215 220 Ser Met Tyr Gln Ala Ile Lys Asn Ile Val Pro Asp Ala Ala Leu Thr 225 230 235 240 Leu Phe Thr Gly Asp Ile Val Asp His Ala Ile Trp Asn Thr Ser Gln 245 250 255 Pro Tyr Asn Gln Lys Gln Ile Ser Asp Ala Tyr Thr Tyr Met Ser Gln 260 265 270 Tyr Leu Gly Ile Val Tyr Gly Thr Ala Gly Asn His Glu Ser Asn Pro 275 280 285 Thr Asn Ala Phe Pro Pro Arg Ser Ile Ser Asn Ser Ser Gln Trp Val 290 295 300 Tyr Asp Ala Leu Ser Asp Gln Trp Thr Arg Trp Val Gly Ala Ser Ala 305 310 315 320 Glu Ser Glu Ile Glu Ser Ile Gly Ala Tyr Ser Thr Lys Tyr Pro Asn 325 330 335 Gly Asn Leu Arg Val Ile Ser Leu Asn Thr Asn Phe Tyr Tyr Arg Met 340 345 350 Asn Phe Trp Leu Tyr Gln Glu Gly Ile Glu Gln Asp Pro Asp Gly Gln 355 360 365 Ile Gln Trp Leu Val Ser Glu Leu Asp Ala Ala Glu Lys Ala Gly Glu 370 375 380 Arg Val Tyr Ile Ile Gly His Met Pro Leu Gly Glu Gly Asp Ala Phe 385 390 395 400 His Ala Gly Ser Asn Tyr Leu Asp Gln Val Val Asn Arg Tyr Ser Ser 405 410 415 Thr Ile Ala Ala Met Phe Phe Gly His Thr His Val Asp His Phe Glu 420 425

430 Val Ser Tyr Ser Asp Tyr Asn Ser Arg Asp Ala Ser His Ala Val Met 435 440 445 Ala Ser Tyr Ile Cys Pro Ser Leu Thr Pro Thr Ser Gly Met Pro Ser 450 455 460 Phe Arg Val Tyr Asp Val Asp Pro Glu Thr Phe Ala Val Leu Asp Thr 465 470 475 480 Thr Thr Tyr Ile Ala Asp Met Thr Asn Ala Asp Phe Gln Thr Thr Gly 485 490 495 Pro Val Trp Thr Lys Leu Tyr Ser Ala Lys Glu Thr Tyr Gly Ser Lys 500 505 510 Leu Asn Pro Pro Val Thr Asp Ala Ser Val Glu Leu Thr Pro Ala Phe 515 520 525 Trp His Asn Val Thr Ala Leu Phe Asp Ser Asn Ser Asp Val Phe Asn 530 535 540 Glu Tyr Ile Ser Leu Lys Ser Arg Gly Trp Asn Val Ala Ser Cys Thr 545 550 555 560 Gly Asp Cys Lys Thr Gln Glu Leu Cys Gln Leu Arg Ala Gly Arg Ser 565 570 575 Glu Asn Asn Cys Val Val Pro Ser Ile Gly Leu His Leu Asn Lys Arg 580 585 590 Ser Asp Glu Leu His Glu His Asp His Ser His His Gly Ser His Asp 595 600 605 His Gln Glu Cys Gly Met Ser Ala Gly Met Lys Thr Leu Gly Ser Leu 610 615 620 Ala Val Arg Lys Asp Leu Leu Asp Glu Leu Glu Thr Arg Ala Asn Glu 625 630 635 640 Leu Lys Ala Lys Ala 645 9611PRTRasamsonia eburnea 9Met Arg Ala Phe Leu Ile Thr Ala Leu Ala Ser Leu Ala Thr Ala Ala 1 5 10 15 Gly Ala Thr Tyr Asp Lys Arg Gly Leu Ala Gln Asp Ile Trp Asn Asp 20 25 30 Ile Lys Asn Ala Val Asp Cys Ala Gly Cys Gln Gly Ile Leu Thr Ala 35 40 45 Leu Lys Gly Leu Ser Tyr Leu Gly Thr Thr Ala Phe Val Asp Val Leu 50 55 60 Thr Glu Val Cys Asp Ile Ser Gly Val Glu Asp Ser Asp Val Cys Ser 65 70 75 80 Gly Ile Ile Ser Ser Glu Gly Pro Ala Leu Val Tyr Ile Leu Lys His 85 90 95 Leu Asp Ile Gly Ser His Thr Ser Gln Val Ile Cys Ala Ser Val Phe 100 105 110 Gly Leu Cys Gln Tyr Pro Ala Val Arg Ala Tyr Asn Leu Thr Phe Pro 115 120 125 Ser Pro Lys Pro Asp Lys Thr Cys Pro Glu Pro Ser Gly Glu Ser Pro 130 135 140 Val Gln Ile Val His Phe Ser Asp Thr His Ala Asp Leu Ser Tyr Glu 145 150 155 160 Thr Gly Ser Asn Tyr Asn Cys Thr Lys Pro Ile Cys Cys Arg Ser Tyr 165 170 175 Thr Ala Glu Asp Ala Pro Gly Asn Thr Thr Thr Pro Cys Gly Pro Tyr 180 185 190 Gly Asn Pro Lys Cys Asp Ala Pro Met Ser Leu Glu Glu Ser Met Phe 195 200 205 Ala Ala Ile Lys Ala Leu Ser Pro Gln Pro Ala Phe Ser Ile Tyr Thr 210 215 220 Gly Asp Val Val Ala His Asp Ile Trp Leu Val Asp Gln Asn Glu Val 225 230 235 240 Val Glu Asp Leu Asn Ala Thr Tyr Asp Arg Met Ala Gly Leu Gly Leu 245 250 255 Val Tyr Ala Ala Ile Gly Asn His Asp Thr Ala Pro Val Asn Asn Leu 260 265 270 Pro Thr Ser Asn Ile Pro Ser Gln Tyr Ser Ala Asn Trp Thr Tyr Glu 275 280 285 Ala Leu Glu Tyr His Phe Ser Leu Leu Thr Lys Ser Ala Ser Ala Gln 290 295 300 Thr Ala Glu Asn Tyr Gly Ser Tyr Ser Ser Val Tyr Arg Gly Arg Tyr 305 310 315 320 Gly Thr Asp Leu Arg Val Ile Ser Tyr Asn Ser Ile Phe Tyr Tyr Ile 325 330 335 Ala Asp Phe Trp Ala Tyr Gln Asp Pro Met Leu Tyr Asp Pro Asp Gly 340 345 350 Gln Leu Ala Trp Leu Ile Asn Glu Leu Gln Glu Ala Glu Thr Ala Gly 355 360 365 Gln Arg Val Trp Leu Ile Ala His Val Pro Ser Gly Thr Ala Asp His 370 375 380 Phe His Asp Tyr Ser His Tyr Phe Asp Gln Ile Val Gln Arg Tyr Glu 385 390 395 400 Ala Thr Ile Ala Ala Leu Phe Tyr Gly His Thr His Ile Asp Gln Phe 405 410 415 Gln Ile Ser Tyr Ser Asp Tyr Ser Asn Arg Ala Phe Asp Thr Ala Thr 420 425 430 Ala Ile Gly Tyr Ile Met Pro Ser Met Thr Pro Thr Ser Gly Pro Pro 435 440 445 Thr Phe Arg Val Tyr Asp Val Asp Pro Lys Thr Phe Ala Val Leu Asp 450 455 460 Phe Thr Asn Tyr Ile Ala Asn Ile Ser Asp Pro Ala Tyr Gln Ser Gly 465 470 475 480 Pro Thr Trp Gln Lys Tyr Tyr Ser Ala Lys Glu Ala Tyr Gly Ser Leu 485 490 495 Leu Ser Pro Pro Val Thr Asp Ala Thr Ala Glu Leu Thr Pro Ala Phe 500 505 510 Trp His Asn Val Thr Val Ala Phe Glu Asn Asp Asp Thr Ala Phe Gln 515 520 525 Glu Tyr Trp Ala Arg Gln Thr Arg Gly Tyr Ala Val Ser Ser Cys Thr 530 535 540 Gly Asp Cys Ile Thr Gln Ala Ile Cys Gly Leu Arg Ala Gly Glu Ser 545 550 555 560 Gln His Asn Cys Val Thr Pro Thr Pro Gly Phe Asn Phe Ala Lys Arg 565 570 575 Asp Val Ser Thr Asp Gly Gln Ala Leu Pro His Ile Glu Lys Cys Glu 580 585 590 Gly Ser Gly Leu Met Ala Leu Leu Ala Lys Met Val Ala Ser Asn Arg 595 600 605 Gln Ser Ser 610

Claims

1. A process for increasing fermentation product yield during a fermentation product production process, wherein a phospholipase C is present and/or added during a liquefaction step of the fermentation product production process.

2. A process for producing a fermentation product, comprising the steps of: (a) liquefying a starch-containing material using an alpha-amylase in the presence of a phospholipase C; (b) saccharifying the liquified starch-containing material using a carbohydrate-source generating enzyme to form fermentable sugars; and (c) fermenting the fermentable sugars using a fermenting organism to product the fermentation product.

3. The process of claim 1, wherein the phospholipase C is a thermostable phospholipase C having a Melting Point (DSC) of at least about 80.degree. C.

4. The process of claim 1, wherein the phospholipase C has: (i) at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity to the mature part of the polypeptide of SEQ ID NO: 2; (ii) at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity to the mature part of the polypeptide of SEQ ID NO: 7; (iii) at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, most preferably at least 94%, and at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity to the mature part of the polypeptide of SEQ ID NO: 8; or (iv) at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity to the mature part of the polypeptide of SEQ ID NO: 9.

5. The process of claim 1, wherein an alpha-amylase; and a phospholipase C having a Melting Point (DSC) above 80.degree. C.; are present and/or added in liquefaction step (a).

6. The process of claim 1, further comprises, prior to the liquefaction step a), the steps of: i) reducing the particle size of the starch-containing material; and ii) forming a slurry comprising the starch-containing material and water.

7. The process of claim 1, wherein at least 50% of the starch-containing material fit through a sieve with #6 screen.

8. The process of claim 1, wherein the pH during liquefaction is between 4.0-6.5.

9. The process of claim 1, wherein the temperature during liquefaction is in the range from 70-100.degree. C.

10. The process of claim 1, wherein a jet-cooking step is carried out before liquefaction in step a).

11. The process of claim 10, wherein the jet-cooking is carried out at a temperature between 95-160.degree. C. for about 1-15 minutes.

12. The process of claim 1, wherein saccharification and fermentation is carried out sequentially or simultaneously.

13. The process of claim 1, wherein saccharification is carried out at a temperature from 20-75.degree. C., and at a pH between 4 and 5.

14. The process of claim 1 wherein fermentation or simultaneous saccharification and fermentation (SSF) is carried out at a temperature from 25.degree. C. to 40.degree. C. for 6 to 120 hours.

15. The process of claim 1, wherein fermentation product is recovered after fermentation.

16. The process of claim 1, wherein the fermentation product is an alcohol.

17. The process of claim 1 wherein the starch-containing starting material is whole grains.

18. The process of claim 1, wherein the starch-containing material is derived from corn, wheat, barley, rye, milo, sago, cassava, manioc, tapioca, sorghum, rice or potatoes.

19. The process of claim 1, wherein the fermenting organism is yeast.

20. The process of claim 1, wherein the alpha-amylase is a bacterial alpha-amylase.

21. The process of claim 20, wherein the alpha-amylase is a Bacillus stearothermophilus alpha-amylase having the amino acid sequence of SEQ ID NO: 1 or having a sequence identity to SEQ ID NO: 1 of at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, and is truncated to have from 485 to 495 amino acids.

22. The process of claim 20, wherein the Bacillus stearothermophilus alpha-amylase has one or more of the following sets of mutations: I181*+G182*; I181*+G182*+N193F; I181*+G182*+E129V+K177L+R179E; I181*+G182*+N193F+E129V+K177L+R179E; I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S I181*+G182*+N193F+V59A Q89R+E129V+K177L+R179E+Q254S+M284V; and I181*+G182*+N193F+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQ ID NO: 1 for numbering).

23. The process of claim 20, wherein the Bacillus stearothermophilus alpha-amylase variant has a sequence identity to SEQ ID NO: 1 of at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%.

24. (canceled)

25. (canceled)

26. The process of claim 19, wherein the fermenting organism is a strain of Saccharomyces.

27. The process of claim 19, wherein the fermenting organism is a strain of Saccharomyces cerevisiae.