ENZYMES HAVING PULLULANASE ACTIVITY

Abstract

The present invention relates to thermostable pullulanases useful for industrial and scientific purposes. The present invention provides methods for producing the modified pullulanase, enzymatic compositions comprising the modified pullulanase, and methods for use of the enzymatic compositions.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. patent application Ser. No. 14/773,696 filed on Sep. 8, 2015, now pending; which is a National Stage of International Application PCT/US2014/022542, filed Mar. 14, 2014, which claims benefit of priority of U.S. Ser. No. 61/801,084, filed Mar. 15, 2013. All of the above-mentioned applications are hereby incorporated by reference into the subject application in their entireties for all purposes.

FIELD OF THE INVENTION

[0002] This invention relates generally to enzymes, polynucleotides encoding the enzymes, the use of such polynucleotides and polypeptides, and more specifically to enzymes having pullulanase activity.

SEQUENCE LISTING

[0003] This application is being filed electronically via the USPTO EFS-WEB server, as authorized and set forth in MPEP .sctn. 502.05 and this electronic filing includes an electronically submitted sequence listing; the entire content of this sequence listing is hereby incorporated by reference into the specification of this application. The sequence listing is identified on the electronically filed ASCII (.txt) text file as follows:

TABLE-US-00001 File Name Date of Creation Size SEQUENCE_LISTING_160884US03 09/08/2015 8,133 bytes

BACKGROUND

[0004] Pullulanase is a specific kind of glucanase, an amylolytic exoenzyme, that degrades pullulan. Type I pullulanases specifically attack alpha-1,6 linkages, while type II pullulanases are also able to hydrolyse alph-1,4 linkages.

[0005] Pullulanase (EC 3.2.1.41) is also known as pullulan-6-glucanohydrolase (Debranching enzyme). Its substrate, pullulan, is regarded as a chain of maltotriose units linked by alpha-1,6-glycosidic bonds. Pullulanase will hydrolytically cleave pullulan (alpha-glucan polysaccharides).

[0006] Pullulanases are used for several industrial and commercial applications, including, saccharification of starch, liquefaction of starch, production of high-maltose corn syrup, production of high-fructose corn syrup, starch processing, in detergents, production of cyclodextrins, and production of low-calorie beer, in the baking industry, as well as dental plaque control. (Siew Ling Hii, Joo Shun Tan, Tau Chuan Ling, and Arbakariya Bin Ariff, "Pullulanase: Role in Starch Hydrolysis and Potential Industrial Applications," Enzyme Research, vol. 2012, Article ID 921362, 14 pages, 2012.)

SUMMARY OF THE INVENTION

[0007] The invention provides an isolated nucleic acid having a sequence as set forth in SEQ ID No.: 1 and variants thereof having at least 50% sequence identity to SEQ ID No.: 1 and encoding polypeptides having pullulanase activity, or encoding polypeptides having pullulanase activity having at least 50% sequence identity to SEQ ID No.: 2.

[0008] One aspect of the invention is an isolated nucleic acid having a sequence as set forth in SEQ ID No.: 1, sequences substantially identical thereto, and sequences complementary thereto.

[0009] Another aspect of the invention is an isolated nucleic acid including at least 10 consecutive bases of a sequence as set forth in SEQ ID No: 1 nucleic acid sequences, sequences substantially identical thereto, and the sequences complementary thereto.

[0010] In yet another aspect, the invention provides an isolated nucleic acid encoding a polypeptide having a sequence as set forth in SEQ ID No.: 2 and variants thereof encoding a polypeptide having pullulanase activity and having at least 50% sequence identity to such sequences. Another aspect of the invention is an isolated nucleic acid encoding a polypeptide or a functional fragment thereof having a sequence as set forth in SEQ ID No.: 2, and sequences substantially identical thereto.

[0011] Another aspect of the invention is an isolated nucleic acid encoding a polypeptide having at least 10 consecutive amino acids of a sequence as set forth in SEQ ID No.: 2, and sequences substantially identical thereto.

[0012] In yet another aspect, the invention provides a purified polypeptide having a sequence as set forth in SEQ ID No.: 2, and sequences substantially identical thereto

[0013] Another aspect of the invention is a method of making a polypeptide having a sequence as set forth in SEQ ID No.: 2, and sequences substantially identical thereto. The method includes introducing a nucleic acid encoding the polypeptide into a host cell, wherein the nucleic acid is operably linked to a promoter, and culturing the host cell under conditions that allow expression of the nucleic acid.

[0014] Another aspect of the invention is a method of making a polypeptide having SEQ ID No.: 2, and sequences substantially identical thereto. The method includes introducing a nucleic acid encoding the polypeptide into a host cell, wherein the nucleic acid is operably linked to a promoter, and culturing the host cell under conditions that allow expression of the nucleic acid, thereby producing the polypeptide.

[0015] Another aspect of the invention is a method of generating a variant including obtaining a nucleic acid having a sequence as set forth in SEQ ID No.: 1 or sequences substantially identical thereto, sequences complementary to the sequences of SEQ ID No.: 1, fragments comprising at least 30 consecutive nucleotides of the foregoing sequences, and changing one or more nucleotides in the sequence to another nucleotide, deleting one or more nucleotides in the sequence, or adding one or more nucleotides to the sequence.

[0016] Another aspect of the invention is a computer readable medium having stored thereon a sequence as set forth in SEQ ID No.: 1 sequences, and sequences substantially identical thereto, or a polypeptide sequence as set forth in SEQ ID No.: 2 amino acid sequences, and sequences substantially identical thereto.

[0017] Another aspect of the invention is an assay for identifying fragments or variants of SEQ ID No.: 2 amino acid sequences, and sequences substantially identical thereto, which retain the enzymatic function of the polypeptides of SEQ ID No.: 2 amino acid sequences, and sequences substantially identical thereto. The assay includes contacting the polypeptide of SEQ ID No.: 2 amino acid sequences, sequences substantially identical thereto, or polypeptide fragment or variant with a substrate molecule under conditions which allow the polypeptide fragment or variant to function, and detecting either a decrease in the level of substrate or an increase in the level of the specific reaction product of the reaction between the polypeptide and substrate thereby identifying a fragment or variant of such sequences.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.

[0019] FIG. 1 is a chromatogram displaying the results of a digestion using the pullulanase of SEQ ID No.: 1 & 2 with a 1% corn starch substrate at 50 degrees Celcius, and as further described in Example 1.

[0020] FIG. 2 is a chromatogram displaying the results of a digestion using the pullulanase of SEQ ID No.: 1 & 2 with a 1% pullulan at 50 degrees Celcius, and as further described in Example 1.

[0021] FIG. 3 is a chromatogram displaying the results of a digestion using the pullulanase of SEQ ID No.: 1 & 2 with a 1% corn starch substrate at 75 degrees Celcius, and as further described in Example 1.

[0022] FIG. 4 is SEQ ID No.: 1, the deoxyribonucleic acid (DNA) sequence of the present invention.

[0023] FIG. 5 is SEQ ID No.: 2, the amino acid sequence of the present invention.

EXAMPLES

Example 1

[0024] The pullulanase of the present invention (as embodied by SEQ ID No.: 1 & 2) was tested against 1% solids using an aliquot from a trial cell lysate. The results shown in FIGS. 1, 2, and 3, show that the pullulanase of the present invention is able to cleave both alpha-1,6 and alpha-1,4 bonds. As displayed in FIG. 2, the pullulanase of the present invention (as embodied by SEQ ID No. 1 & 2) was assayed with 1% pullulan at 50 degrees Celsius. As displayed in FIG. 1, the pullulanase of the present invention (as embodied by SEQ ID NO 1 & 2) was assayed with 1% corn starch at 50 degrees Celcius. As displayed in FIG. 3, the pullulanase of the present invention (as embodied by SEQ ID No. 1 & 2) was assayed with 1% corn starch at 75 degrees Celsius. As indicated in FIGS. 1, 2, and 3, the enzyme is a type II pullulanase, as the reaction products (major peaks) are glucose, maltose, and maltotriose. Additionally, the peak 2 product was confirmed to be maltose and not isomaltose, while the peak 3 product was confirmed to be maltotriose and not panose.

Example 2

[0025] The pullulanase of the present invention (as embodied by SEQ ID No. 1 & 2) melting point or thermal denaturation was determined using differential scanning calorimetry. The Tm of the present invention (as embodied by SEQ ID No. 1 & 2) is 84 degrees Celsius.

DETAILED DESCRIPTION

[0026] The present invention relates to a pullulanase enzyme, polynucleotides encoding the enzymes, methods of making and using these polynucleotides and polypeptides. The invention is directed to novel polypeptides having pullulanase activity, nucleic acids encoding them. The polypeptides of the invention can be used in a variety of commercial, medical, and industrial contexts. The polypeptides of the invention can be used as, e.g., an additive for a detergent, for processing foods and for chemical synthesis utilizing a reverse reaction, saccharification of starch, liquefaction of starch, production of high-maltose corn syrup, production of high-fructose corn syrup, starch processing, ethanol production, production of cyclodextrins, and production of low-calorie beer, in the baking industry, as well as dental plaque control.

[0027] In one aspect of the invention the pullulanase is a type II pullulanase or is capable of cleaving both alpha-1,6 and alpha-1,4 bonds. In another aspect of the invention the pullulanase of the present invention is capable of cleaving both alpha-1,6 and alpha-1,4 of pullulan yielding glucose, maltose, and maltotriose. In a further embodiment of the present invention the pullulanase of the present invention is thermostable and or thermotolerant. In a further embodiment of the invention the pullulanase of the present invention is active at 75 degrees Celsius. In a further embodiment of the invention, the pullulanase of the invention is capable of saccharification of starch at higher temperatures then currently employed, thereby reducing processing times, and increasing yields due to lessening the rate of retrogradation of materials resulting in drops in temperature. In a further embodiment of the present invention the pullulanase is capable of enhancing ethanol or glucose production by digesting limit dextrans that an amylase cannot digest.

[0028] In a further embodiment of the present invention the pullulanase of the present invention is coupled with an amylase enzyme.

[0029] In one aspect, the nucleic acid encodes at least one polypeptide having pullulanase activity.

[0030] "Synthetic" nucleic acids (including oligonucleotides), polypeptides or proteins of the invention include those prepared by any chemical synthesis, e.g., as described, below.

[0031] The phrases "nucleic acid" or "nucleic acid sequence" includes oligonucleotides, nucleotides, polynucleotides, or to a fragment of any of these, to DNA or RNA (e.g., mRNA, rRNA, tRNA) of genomic, recombinant or synthetic origin which may be single-stranded or double-stranded and may represent a sense or antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material, natural or synthetic in origin, including, e.g., iRNA such as miRNA or siRNA, ribonucleoproteins (e.g., iRNPs). The term encompasses nucleic acids, i.e., oligonucleotides, containing known analogues of natural nucleotides. The term also encompasses nucleic-acid-like structures with synthetic backbones, see e.g., Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197; Strauss-Soukup (1997) Biochemistry 36:8692-8698; Samstag (1996) Antisense Nucleic Acid Drug Dev 6:153-156.

[0032] "Recombinant" polypeptides or proteins refer to polypeptides or proteins produced by recombinant DNA techniques; e.g., produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide or protein.

[0033] The term "gene" includes a nucleic acid sequence comprising a segment of DNA involved in producing a transcription product (e.g., a message), which in turn is translated to produce a polypeptide chain, or regulates gene transcription, reproduction or stability. Genes can include regions preceding and following the coding region, such as leader and trailer, promoters and enhancers, as well as, where applicable, intervening sequences (introns) between individual coding segments (exons).

[0034] The invention provides isolated and recombinant nucleic acids, including expression cassettes such as expression vectors encoding the polypeptides of the invention. The invention provides probes comprising or consisting of nucleic acids of the invention. The invention also includes methods for discovering new pullulanase sequences using the nucleic acids of the invention. The invention also includes methods for inhibiting the expression of pullulanase genes, transcripts and polypeptides using the nucleic acids of the invention.

[0035] The nucleic acids of the invention can be made, isolated and/or manipulated by, e.g., cloning and expression of cDNA libraries, amplification of message or genomic DNA by PCR, and the like. In practicing the methods of the invention, homologous genes can be modified by manipulating a template nucleic acid, as described herein. The invention can be practiced in conjunction with any method or protocol or device known in the art, which are well described in the scientific and patent literature.

Sequence CWU 1

1

211836DNAThermococcus celer 1atgaccgccc aacgaccccc gcccaccggc ccgattccgg tcgtcggccc cacccggccc 60caacccagcg gcgacctcaa cttcgagcac gaccccaagc tgcccaaatt cgtctcgctg 120gcggcggatg ggctctcggt gcgctttcag gccggggagg gctcggtgaa gtgggccatc 180gtccaggccg agggcaggaa ctaccccatg caccggcagc tctggcagga cggcagcgag 240atctggcggg tagcgcttcc ccccagcata aagaactacc gcatccgcct cgagaccgcc 300cagaataagc ccgccgtgtt cggccctttt gacgcgaatc atccgttggt gggcctggac 360tgggtaggga agagcatcgg ctaccaggtc ttccccgaac gcttctggaa tggggacaaa 420accaacgaca tgcgggcgct cgagaccgac gagtaccgct tcaacgagac ctggaaccag 480aacccccagg cgactaagcc ctacctatcg aactggaacg acccacccgg tgagctgcac 540tgctgccacc agtatttcgg tggcgaccta gccgggttcc tggagaagct cccccacctc 600gaggctttgg gggtcaggct cgtctacttc aacccgctct tcgactccgg ctccgcccac 660ggctacgaca cccacgacta ctacaaaatc tctcccaaat tcggcgacga ggctcttcta 720cggcaggtgc tggacgcagc ccacgcccag gggatgcggg tgatcttcga cttcgtaccc 780aaccacactg gcctgggctt cttcgccttc caggacgtgg tgaaaaacgg gcgggcctcc 840cagtactgga actggtacac gattaggcga tggccgttcc gtcccggcga cgcctcggcc 900tacgacacct tctcgggggt ggggagctta cccaagctca acaccgccaa ccccgaggtg 960aaaaagtatc tgctcgaggt ggccgagcac tggatgcgct tcggcttcga cggtttgcgc 1020gtcgactacc cacagggcat cgtgaaccgc gaggatttct accgcgacct gcgccgggtg 1080gtgaaggggg ttaaacccga tgcctacatc gtggccgaga tctgggcgcg ggaccccagc 1140tggctgcaag gcgaccaggc cgatagcctg atgaactacg ccatcgggcg ggacattgtg 1200ctgcgcttcg cccggggcgg cggggtggct ttgtacagtg gacggcgggc cttggccgat 1260ctagtccgca tctacaccga ttaccccgag gcggtggtgg gccaggggtg gaacctcatt 1320ggctcacacg atactcccag ggtgctcacc gatttgggtg ggggtgctct aggcgacacc 1380ccgtcgcccg agtcgctggc ccgactgcgc ctggccatgg gcttgctcta cgccctcccc 1440ggaatgccga ttttcttcca gggggatgag tgcggtttca ccggggaggc cgggcagtac 1500ccggtcaatg agctataccg ctacccaatc cagtgggacc ggtgcaaccc cgacgtgctg 1560gagttttacc agcgactcgg caaggttcgg gctggcctag ccgctctgca aggcccggcg 1620ttccgggcct acgcgggtga gggggcggtg ttggcttttc tgcggggcga gccaggtcag 1680gaggtggtgt tggcggcttt caacaagggc agcgaaccag ccaacctggc ccttcccggc 1740ggcacctggc gcgatgccct tagcggccag accttccaaa gccaagcaga actcccggcg 1800attggttttc gctatctcgt acgggctggg cagtag 18362611PRTThermococcus celer 2Met Thr Ala Gln Arg Pro Pro Pro Thr Gly Pro Ile Pro Val Val Gly1 5 10 15Pro Thr Arg Pro Gln Pro Ser Gly Asp Leu Asn Phe Glu His Asp Pro 20 25 30Lys Leu Pro Lys Phe Val Ser Leu Ala Ala Asp Gly Leu Ser Val Arg 35 40 45Phe Gln Ala Gly Glu Gly Ser Val Lys Trp Ala Ile Val Gln Ala Glu 50 55 60Gly Arg Asn Tyr Pro Met His Arg Gln Leu Trp Gln Asp Gly Ser Glu65 70 75 80Ile Trp Arg Val Ala Leu Pro Pro Ser Ile Lys Asn Tyr Arg Ile Arg 85 90 95Leu Glu Thr Ala Gln Asn Lys Pro Ala Val Phe Gly Pro Phe Asp Ala 100 105 110Asn His Pro Leu Val Gly Leu Asp Trp Val Gly Lys Ser Ile Gly Tyr 115 120 125Gln Val Phe Pro Glu Arg Phe Trp Asn Gly Asp Lys Thr Asn Asp Met 130 135 140Arg Ala Leu Glu Thr Asp Glu Tyr Arg Phe Asn Glu Thr Trp Asn Gln145 150 155 160Asn Pro Gln Ala Thr Lys Pro Tyr Leu Ser Asn Trp Asn Asp Pro Pro 165 170 175Gly Glu Leu His Cys Cys His Gln Tyr Phe Gly Gly Asp Leu Ala Gly 180 185 190Phe Leu Glu Lys Leu Pro His Leu Glu Ala Leu Gly Val Arg Leu Val 195 200 205Tyr Phe Asn Pro Leu Phe Asp Ser Gly Ser Ala His Gly Tyr Asp Thr 210 215 220His Asp Tyr Tyr Lys Ile Ser Pro Lys Phe Gly Asp Glu Ala Leu Leu225 230 235 240Arg Gln Val Leu Asp Ala Ala His Ala Gln Gly Met Arg Val Ile Phe 245 250 255Asp Phe Val Pro Asn His Thr Gly Leu Gly Phe Phe Ala Phe Gln Asp 260 265 270Val Val Lys Asn Gly Arg Ala Ser Gln Tyr Trp Asn Trp Tyr Thr Ile 275 280 285Arg Arg Trp Pro Phe Arg Pro Gly Asp Ala Ser Ala Tyr Asp Thr Phe 290 295 300Ser Gly Val Gly Ser Leu Pro Lys Leu Asn Thr Ala Asn Pro Glu Val305 310 315 320Lys Lys Tyr Leu Leu Glu Val Ala Glu His Trp Met Arg Phe Gly Phe 325 330 335Asp Gly Leu Arg Val Asp Tyr Pro Gln Gly Ile Val Asn Arg Glu Asp 340 345 350Phe Tyr Arg Asp Leu Arg Arg Val Val Lys Gly Val Lys Pro Asp Ala 355 360 365Tyr Ile Val Ala Glu Ile Trp Ala Arg Asp Pro Ser Trp Leu Gln Gly 370 375 380Asp Gln Ala Asp Ser Leu Met Asn Tyr Ala Ile Gly Arg Asp Ile Val385 390 395 400Leu Arg Phe Ala Arg Gly Gly Gly Val Ala Leu Tyr Ser Gly Arg Arg 405 410 415Ala Leu Ala Asp Leu Val Arg Ile Tyr Thr Asp Tyr Pro Glu Ala Val 420 425 430Val Gly Gln Gly Trp Asn Leu Ile Gly Ser His Asp Thr Pro Arg Val 435 440 445Leu Thr Asp Leu Gly Gly Gly Ala Leu Gly Asp Thr Pro Ser Pro Glu 450 455 460Ser Leu Ala Arg Leu Arg Leu Ala Met Gly Leu Leu Tyr Ala Leu Pro465 470 475 480Gly Met Pro Ile Phe Phe Gln Gly Asp Glu Cys Gly Phe Thr Gly Glu 485 490 495Ala Gly Gln Tyr Pro Val Asn Glu Leu Tyr Arg Tyr Pro Ile Gln Trp 500 505 510Asp Arg Cys Asn Pro Asp Val Leu Glu Phe Tyr Gln Arg Leu Gly Lys 515 520 525Val Arg Ala Gly Leu Ala Ala Leu Gln Gly Pro Ala Phe Arg Ala Tyr 530 535 540Ala Gly Glu Gly Ala Val Leu Ala Phe Leu Arg Gly Glu Pro Gly Gln545 550 555 560Glu Val Val Leu Ala Ala Phe Asn Lys Gly Ser Glu Pro Ala Asn Leu 565 570 575Ala Leu Pro Gly Gly Thr Trp Arg Asp Ala Leu Ser Gly Gln Thr Phe 580 585 590Gln Ser Gln Ala Glu Leu Pro Ala Ile Gly Phe Arg Tyr Leu Val Arg 595 600 605Ala Gly Gln 610

Claims

1. An isolated, synthetic, or recombinant nucleic acid encoding a polypeptide having pullulanase activity, selected from the group consisting of: (a) a nucleic acid encoding a polypeptide having pullunase activity comprising a nucleic acid sequence having at least 50% sequence identity to SEQ ID NO:1; (b) a nucleic acid encoding a polypeptide having pullulanase activity comprising a nucleic acid sequence having at least 50% sequence identity to SEQ ID NO:1, or a fragment thereof, wherein the fragment encodes a polypeptide having pullulanase activity; (c) a nucleic acid sequence encoding a polypeptide having pullulanase activity comprising an amino acid sequence having at least 50% sequence identity to SEQ ID NO:2; (d) a nucleic acid sequence encoding a polypeptide having pullulanase activity comprising an amino acid sequence having at least 50% sequence identity to SEQ ID NO:2, or a fragment thereof, wherein the fragment has pullulanase activity; (e) the nucleic acid of (a), (b), (c), or (d) encoding a polypeptide having pullulanase activity but lacking a signal sequence or a carbohydrate binding module; (f) the nucleic acid of (a), (b), (c), (d) or (e) encoding a polypeptide having pullulanase activity, and further comprising a heterologous sequence; (g) the nucleic acid sequence of (g), wherein the heterologous sequence comprises a sequence encoding a heterologous signal sequence, carbohydrate binding module, catalytic domain (CD), or a combination thereof, or the heterologous signal sequence, carbohydrate binding module, or catalytic domain (CD) is derived from another pullulanase enzyme, or a non-pullulanase enzyme; or (h) a nucleic acid sequence fully complementary to (a), (b), (c), (d), (e), (f), or (g).

2. The isolated, synthetic, or recombinant nucleic acid of claim 1, wherein the pullulanase activity comprises the cleavage of both alpha-1,6 and alpha-1,4 bonds.

3. The isolated, synthetic, or recombinant nucleic acid of claim 1, wherein the pullulanase activity comprises type II pullulanase activity.

4. The isolated, synthetic, or recombinant nucleic acid of claim 1, wherein the pullulanase activity is thermostable and or thermotolerant.

5. An isolated, synthetic, or recombinant polypeptide having pullulanase comprising (a) an amino acid sequence having at least 50% identity, or complete sequence identity to sequence of SEQ ID No. 2; (b) having the amino acid sequence encoded by the nucleic acid of claim 1; (c) the amino acid sequence of (a) or (b), and comprising at least one conservative amino acid residue conservative substitutions; (d) the amino acid sequence of (a) or (b) or (c) or a fragment thereof with pullulanase activity.

6. The isolated, synthetic, or recombinant polypeptide of claim 5, where the pullulanase activity is thermostable.

7. The isolated, synthetic, or recombinant polypeptide of claim 5 wherein the polypeptide retains an pullulanase activity under conditions comprising a temperature range of between about 37.degree. C. to about 84.degree. C., or between about 55.degree. C. to about 85.degree. C., or between about 70.degree. C. to about 84.degree. C.

8. The isolated, synthetic, or recombinant polypeptide of claim 5, where the pullulanase activity is thermotolerant.

9. The isolated, synthetic, or recombinant nucleic acid of claim 5, wherein the polypeptide retains an pullulanase activity after exposure to a temperature in the range from greater than 37.degree. C. to about 84.degree. C., from greater than 55.degree. C. to about 84.degree. C.

10. A method of hydrolyzing a starch linkage comprising contacting a substance containing the starch with a polypeptide of claim 5, and sequences substantially identical thereto.

11. A detergent additive comprising with a polypeptide of claim 5.

12. A method of producing ethanol comprising use of the polypeptide of claim 5.

13. The method of claim 12, wherein the polypeptide is capable of digesting limit dextrans.

14. The method of claim 10 or 11 or 12, further comprising addition of an pullulanase or a combination thereof.

15. A detergent composition comprising the polypeptide of claim 5.