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Title:
ALPHA-AMYLASE, COMPOSITION AND METHOD
Document Type and Number:
WIPO Patent Application WO/2019/099235
Kind Code:
A1
Abstract:
The present disclosure relates to polypeptides having alpha-amylase activity and compositions comprising such polypeptides. Moreover, the disclosure also relates to methods of recombinantly producing such polypeptides or such compositions, as well as methods of using or applying the polypeptides or compositions thus produced in industrial settings.

Inventors:
TANG, Zhongmei (925 Page Mill Road, Palo Alto, California, 94304, US)
NI, Kefeng (Building 10, Lane 280Linhong Road, Shanghai 5, 200335, CN)
QIAN, Zhen (925 Page Mill Road, Palo Alto, California, 94304, US)
WU, Qihui (Room 601, No. 1. Lane 715Songhong Road,Changning District, Shanghai 0, 200000, CN)
ZHANG, Keya (Room 301, Building 20No. 239 Xiehe Road, Shanghai 5, 200335, CN)
ZOU, Zhengzheng (925 Page Mill Road, Palo Alto, California, 94304, US)
Application Number:
US2018/059321
Publication Date:
May 23, 2019
Filing Date:
November 06, 2018
Export Citation:
Click for automatic bibliography generation   Help
Assignee:
DANISCO US INC (925 Page Mill Road, Palo Alto, California, 94304, US)
International Classes:
C12N9/30; C11D3/386
Domestic Patent References:
WO2008080093A22008-07-03
WO2014099415A12014-06-26
WO2010091221A12010-08-12
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WO2011153516A22011-12-08
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WO1984002921A21984-08-02
WO1999028448A11999-06-10
WO1986001831A11986-03-27
WO2015017256A12015-02-05
WO2005001036A22005-01-06
Foreign References:
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US20110020899A12011-01-27
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US8945889B22015-02-03
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LIU ET AL.: "Improved heterologous gene expression in Trickoderma reesei by cellobiohydrolase I gene (cbhl) promoter optimization", ACTA BIOCHIM. BIOPHYS. SIN (SHANGHAI, vol. 40, no. 2, 2008, pages 158 - 65
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Attorney, Agent or Firm:
TODD, Stephen (925 Page Mill Road, Palo Alto, California, 94304, US)
Download PDF:
Claims:
CLAIMS

What is claimed is:

1. A polypeptide having alpha-amylase activity, selected from the group consisting of:

(a) a polypeptide comprising an amino acid sequence having preferably at least

91% identity to the polypeptide of SEQ ID NO: 3;

(h) a polypeptide comprising an amino acid sequence having preferably at least

91% identity to a catalytic domain of SEQ ID NO: 3;

(c) a polypeptide comprising an amino acid sequence having preferably at least

91% identity to a linker and a catalytic domain of SEQ ID NO: 3;

(d) a polypeptide encoded by a polynucleotide that hybridizes under preferably at least low stringency conditions, more preferably at least medium stringency conditions, even more preferably at least medium-high stringency conditions, most preferably at least high stringency, and even most preferably at least very high stringency conditions with

(i) the mature polypeptide coding sequence of SEQ ID NO: 1,

(ii) the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 1, or

(iii) a full-length complementar' strand of (i) or (ii);

(e) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having preferably at least 91% identity to the polypeptide coding sequence of SEQ ID NO: 3;

CO a variant comprising a substitution, deletion, and/or insertion of one or more (e.g., several) ammo acids of the polypeptide of SEQ ID NO: 3;

(g) a mature polypeptide produced by the processing of the polypeptide of SEQ ID NO: 2 by a signal peptidase or post translational modification during secretion from an expression host; and

(h) a fragment of a polypeptide of (a), (b), (c), (d), (e), (f) or (g) that has alpha-amylase activity.

2. A polynucleotide comprising a nucleotide sequence that encodes the polypeptide of claim 1

3. A vector comprising the polynucleotide of claim 2 operably linked to one or more control sequences that control the production of the polypeptide in an expression host.

4. A recombinant host cell comprising the polynucleotide of claim 2.

5. The host cell of claim 4, which is an ethanologenic microorganisms.

6. The host cell of claims 4 or 5, which further expresses and secretes one or more additional enzymes selected from the group comprising a protease, hemicellulase, cellulase, peroxidase, lipolytic enzyme, xylanase, lipase, phospholipase, esterase, perhydrolase, cutinase, pectinase, pectate lyase, mannanase, keratinase, reductase, oxidase, phenoloxidase, lipoxygenase, ligninase, glucoamylase, pullulanase, phytase, tannase, pentosanase, malanase, heta-glucanase, arabinosidase, hyaluronidase, chondroitinase, laccase, transferase, or a combination thereof.

7. A composition comprising the polypeptide of claim 1.

8 The composition of claim 7, further comprising a protease, hemicellulase, cellulase, peroxidase, lipolytic enzyme, xy!anase, lipase, phospholipase, esterase, perhydrolase, cutinase, pectmase, pectate lyase, maiinanase, keratinase, reductase, oxidase, phenoloxidase, lipoxygenase, ligninase, glucoamylase, pullulanase, phytase, tannase, pentosanase, malanase, heta-glucanase, arabinosidase, hyaluronidase, chondroitinase, laccase, transferase, or a combination thereof.

9 A method of producing a polypeptide having alpha-amylase activity, comprising;

(a) cultivating the host cell of claim 4 under conditions conducive for production of the polypeptide; and

(b) recovering the polypeptide.

10. A method for treating a starch-containing material with the polypeptide having alpha- amylase activity' of claim 1.

11. A method for saccharifying a starch substrate, comprising

a) contacting the starch substrate with the polypeptide having alpha-amylase activity of claim 1 ; and

b) saccharifying the starch substrate to produce the saccharides comprising glucose.

12. The method of claim 11, wherein saccharifying the starch substrate results in a high glucose syrup.

13. The method of claim 11 or 12, wherein the high glucose syrup comprises an amount of glucose selected from the list consisting of at least 95.5% glucose, at least 95.6% glucose, at least 95 7% glucose, at least 95 8% glucose, at least 95.9% glucose, at least 96% glucose, at least 96.1 % glucose, at least 96.2% glucose, at least 96.3% glucose, at least 96.4% glucose, at least 96.5% glucose and at least 97% glucose.

14. The method of any one of claims 10-12, further comprising fermenting the high glucose syrup to an end product.

15. The method of claim 14, wherein saccharifying and fermenting are carried out as a simultaneous saccharification and fermentation (SSF) process.

16. The method of claim 14 or 15, wherein the end product is alcohol, for example, ethanol.

17. The method of claim 14 or 15, wherein the end product is a biochemical selected from the group consisting of an ammo acid, an organic acid, citric acid, lactic acid, succinic acid, monosodium glutamate, gluconic acid, sodium gluconate, calcium gluconate, potassium gluconate, glucono delta-lactone, sodium erythorbate, omega 3 fatty acid, butanol, lysine, itaconic acid, 1 ,3-propanediol, biodiesel, and isoprene.

18. The method of any one of claims 1 1-17, wherein the starch substrate is about, 5% to 99%, 15% to 50% or 40-99% dry solid (DS).

19. The method of any one of claims 11-18, wherein the starch substrate is selected from wheat, barley, com, rye, nee, sorghum, bran, cassava, milo, millet, potato, sweet potato, tapioca, and any combination thereof.

20. The method of any one of claims 1 1-19, wherein the starch substrate comprises liquefied starch, gelatinized starch, or granular starch.

21. The method of any one of claims 11-20, further comprising adding a hexokinase, a xylanase, a glucose isomerase, a xylose isomerase, a phosphatase, a phytase, a pullulanase, a beta-amylase, a giucoamy!ase, a protease, a cel!ulase, a hemicellulase, a lipase, a cutinase, a trehalase, an isoamylase, a redox enzyme, an esterase, a transferase, a pectinase, a hydrolase, an aipha-giucosidase, a beta-glucosidase, or a combination thereof to the starch substrate.

22. A method of applying method of any one of claims 11 -21 for production of saccharides.

23. A saccharide produced by method of claim 22.

24. A method for saccharifying and fermenting a starch substrate to produce an end product, comprising

a) contacting the starch substrate with the polypeptide having alpha-amylase activity of claim 1 ;

b) saccharifying the starch substrate to produce the saccharides comprising glucose; and c) contacting the saccharides with a fermenting organism to produce an end product

25. The method of claim 24, wherein fermenting are carried out as a simultaneous saccharification and fermentation (SSF) process.

26. The method of claim 24 or 25, wherein the end product is alcohol, for example, ethanol.

27. The method of claim 24 or 25, wherein the end product is a biochemical selected from the group consisting of an amino acid, an organic acid, citric acid, lactic acid, succinic acid, monosodium glutamate, gluconic acid, sodium gluconate, calcium gluconate, potassium gluconate, glucono delta-lactone, sodium erythorbate, omega 3 fatty acid, butanol, lysine, itaconic acid, 1 ,3-propanediol, biodiesel, and isoprene.

Description:
ALPHA-AMYLASE, COMPOSITION AND METHOD

FIELD OF THE INVENTION

[001] The present disclosure relates to polypeptides having alpha-amylase activity and compositions comprising such polypeptides. The present disclosure further relates to polynucleotides encoding such polypeptides, engineered nucleic acid constructs, vectors and host cells comprising genes encoding such polypeptides, which may also enable the production of such polypeptides. Moreover, the disclosure relates to methods of recombinantly producing such polypeptides or such compositions, as well as methods of using or applying the polypeptides or compositions thus produced in industrial settings, for example, for starch treatment, such as liquefaction, saccharification and/or fermentation, or beverage preparation.

BACKGROUND

[002] Starch consists of a mixture of amylose (15-30% w/w) and amyiopectin (70-85% w/w). Amylose consists of linear chains of alpha- 1 ,4-linked glucose units having a molecular weight (MW) from about 60,000 to about 800,000 Amyiopectin is a branched polymer containing alpha- 1,6 branch points every 24-30 glucose units; its MW may be as high as 100 million.

[003] Alpha-amylases hydrolyze starch, glycogen, and related polysaccharides by cleaving internal alpha-1 , 4-glucosidic bonds at random. Alpha-amylase enzymes have been used for a variety of different purposes, such as, starch liquefaction, saccharification, fermentation, brewing, baking, textile desizing, textile washing, starch modification in the paper and pulp industry and digestability increasing in animal feed.

[004] Depending on the industrial applications, alpha-amylases suitable for these industrial processes can be diverse. There is as such always a need in the art for alternative alpha- amylases with improved or different properties such as pH optimum, temperature optimum, substrate specificities, and/or thermostability.

[QQ5] It is an object of the present disclosure to provide certain polypeptides having alpha- amylase activity , polynucleotides encoding the polypeptides, nucleic acid constructs that ca be used to produce such polypeptides, compositions comprising thereof, as well as methods of making and using such polypeptides. SUMMARY

[006] The present polypeptides, compositions and methods of using or applying the polypeptides or compositions. Aspects and embodiments of the polypeptides, compositions and methods are described in the following, independently -numbered paragraphs.

1. In one aspect, a polypeptide having alpha-amylase activity , selected from the group consisting of:

(a) a polypeptide comprising an amino acid sequence having preferably at least

90% identity to the polypeptide of SEQ ID NO: 3;

(b) a polypeptide comprising an amino acid sequence having preferably at least

90% identity' to a catalytic domain of SEQ ID NO: 3;

(c) a polypeptide comprising an amino acid sequence having preferably at least

90% identity to a linker and a catalytic domain of SEQ ID NO: 3;

(d) a polypeptide encoded by a polynucleotide that hybridizes under preferably at least low stringency conditions, more preferably at least medium stringency conditions, even more preferably at least medium-high stringency conditions, most preferably at least high stringency, and even most preferably at least very high stringency conditions with

(i) the mature polypeptide coding sequence of SEQ ID NO: 1,

(ii) the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 1, or

(iii) a full-length complementary strand of (i) or (ii);

(e) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having preferably at least 90% identity to the polypeptide coding sequence of SEQ ID NO: 3;

(f) a variant comprising a substitution, deletion, and/or insertion of one or more (e.g., several) ammo acids of the poly peptide of SEQ ID NO: 3;

(g) a mature polypeptide produced by the processing of the polypeptide of SEQ ID NO: 2 by a signal peptidase or post translational modification during secretion from an expression host; and

(h) a fragment of a polypeptide of (a), (b), (c), (d), (e), (Q or (g) that has alpha-amylase activity.

2. In another aspect, a polynucleotide comprising a nucleotide sequence that encodes the polypeptide of paragraph 1.

3. In another aspect, a vector comprising the polynucleotide of paragraph 2 operably linked to one or more control sequences that control the production of the polypeptide in an expression host.

D 4. In another aspect, a recombinant host cell comprising the polynucleotide of paragraph 2.

5. In some embodiments of the host cell of paragraph 4, which is an ethanologenic microorganisms.

6. In some embodiments of the host cell of paragraphs 4 or 5, which further expresses and secretes one or more additional enzymes selected from the group comprising a protease, hemicellulase, cellulase, peroxidase, lipolytic enzyme, xylanase, lipase, phospholipase, esterase, perhydrolase, cutinase, pectinase, pectate lyase, mannanase, keratinase, reductase, oxidase, phenoloxidase, lipoxygenase, iigninase, glucoamylase, pullulanase, phytase, tannase, pentosanase, malanase, beta-glucanase, arahinosidase, hyaluronidase, chondroitmase, laccase, transferase, or a combination thereof.

7. In another aspect, a composition comprising the polypeptide of paragraph 1.

8. In some embodiments of the composition of paragraph 7, further comprising a protease, hemicellulase, cellulase, peroxidase, lipolytic enzyme, xylanase, lipase, phospholipase, esterase, perhydrolase, cutinase, pectinase, pectate lyase, mannanase, keratinase, reductase, oxidase, phenoloxidase, lipoxygenase, Iigninase, glucoamylase, pullulanase, phytase, tannase, pentosanase, malanase, beta-glucanase, arahinosidase, hyaluronidase, chondroitmase, laccase, transferase, or a combination thereof.

9. In another aspect, a method of producing a polypeptide having alpha-amylase activity, comprising:

(a) cultivating the host cell of paragraph 4 under conditions conducive for production of the polypeptide; and

(b) recovering the polypeptide.

10. In another aspect, a method for treating a starch-containing material with the polypeptide having alpha-amylase activity of paragraph 1.

11. In another aspect, a method for saccharifying a starch substrate, comprising

contacting the starch substrate with the polypeptide having alpha-amylase activity 7 of paragraph 1; and

saccharifying the starch substrate to produce the saccharides comprising glucose.

12. In some embodiments of the method of paragraph 11, wherein saccharifying the starch substrate results in a high glucose syrup. 13. In some embodiments of the method of paragraph 11 or 12, wherein the high glucose syrup comprises an amount of glucose selected from the list consisting of at least 95.5% glucose, at least 95.6% glucose, at least 95.7% glucose, at least 95.8% glucose, at least 95.9% glucose, at least 96% glucose, at least 96.1 % glucose, at least 96.2% glucose, at least 96.3% glucose, at least 96.4% glucose, at least 96.5% glucose and at least 97% glucose.

14. In some embodiments of the method of any one of paragraphs 10-12, further comprising fermenting the high glucose syrup to an end product.

15. In some embodiments of the method of paragraph 14, wherein saccharifying and fermenting are carried out as a simultaneous saccharification and fermentation (SSF) process.

16. In some embodiments of the method of paragraph 14 or 15, wherein the end product is alcohol, for example, ethanol.

17. In some embodiments of the method of paragraph 14 or 15, wherein the end product is a biochemical selected from the group consisting of an amino acid, an organic acid, citric acid, lactic acid, succinic acid, monosodium glutamate, gluconic acid, sodium gluconate, calcium gluconate, potassium gluconate, giucono delta-lactone, sodium erythorbate, omega 3 fatty acid, butanol, lysine, itaeonic acid, 1 ,3-propanediol, biodiesel, and isoprene.

18. In some embodiments of the method of any one of paragraphs 11-17, wherein the starch substrate is about, 5% to 99%, 15% to 50% or 40-99% dry solid (DS).

19. In some embodiments of the method of any one of paragraphs 11-18, wherein the starch substrate is selected from wheat, barley, com, rye, rice, sorghum, bran, cassava, mho, millet, potato, sweet potato, tapioca, and any combination thereof.

20. In some embodiments of the method of any one of paragraphs 11-19, wherein the starch substrate comprises liquefied starch, gelatinized starch, or granular starch.

21. In some embodiments of the method of any one of paragraphs 1 1-20, further comprising adding a hexokinase, a xylanase, a glucose isomerase, a xylose isomerase, a phosphatase, a phytase, a pullulanase, a beta-amylase, a glucoamylase, a protease, a cellulase, a hemicei!ulase, a lipase, a cutinase, a trehalase, an isoamylase, a redox enzyme, an esterase, a transferase, a pectinase, a hydrolase, an a!pha-g!ueosidase, a beta-glucosidase, or a combination thereof to the starch substrate.

22. In another aspect, a method of applying method of any one of paragraphs 11-21 for production of saccharides. 23. In another aspect, a saccharide produced by method of paragraph 22.

24. In another aspect, a method for saccharify ing and fermenting a starch substrate to produce an end product, comprising

contacting the starch substrate with the polypeptide having alpha-amylase activity of paragraph 1;

saccharify ing the starch substrate to produce the saccharides comprising glucose; and contacting the saccharides with a fermenting organism to produce an end product.

25. In some embodiments of the method of paragraph 24, wherein fermenting are carried out as a simultaneous saccharification and fermentation (SSF) process.

26. In some embodiments of the method of paragraph 24 or 25, wherein the end product is alcohol, for example, ethanol.

27. In some embodiments of the method of paragraph 24 or 25, wherein the end product is a biochemical selected from the group consisting of an amino acid, an organic acid, citric acid, lactic acid, succinic acid, monosodium glutamate, gluconic acid, sodium gluconate, calcium gluconate, potassium gluconate, glucono delta-lactone, sodium erythorbate, omega 3 fatty' acid, butanol, lysine, itaconic acid, 1 , 3-propanediol, biodiesel, and isoprene.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts the pZKY258 expression vector harboring the synthetic gene of Asp Amy 14 alpha amylase

Figure 2 shows the dose response curves of starch solubilization activity for AspAmy 14 and AcAA alpha amylases. Panel A shows results at pH 3 7 and Panel B show results at pH 4.5

Figure 3 (Panels A, B and C) shows a MUSCLE multiple protein sequence alignment for AspAmy 14 and various homologous fungal alpha amylase described in Example 11.

DETAILED DESCRIPTION

[007] Described are polypeptides from Aspergillus having alpha-amylase activity and compositions comprising such polypeptides. The present disclosure further relates to polynucleotides encoding such polypeptides, engineered nucleic acid constructs, vectors and host cells comprising genes encoding such polypeptides, which may also enable the production of such polypeptides. Moreover, the disclosure relates to methods of recombinantly producing such polypeptides or such compositions, as well as methods of using or applying the polypeptides or compositions thus produced in industrial settings, for example, for starch liquefaction, saccharification, fermentation and food or beverage preparation. These and other aspects of the compositions and methods are described in detail, below.

[008] Prior to describing the various aspects and embodiments of the present compositions and methods, the following definitions and abbreviations are described.

[009] In accordance with this detailed description, the following abbreviations and definitions apply. Note that the singular forms“a,”“an,” and“the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to“an enzyme” includes a plurality of such enzymes, and reference to“the dosage” includes reference to one or more dosages and equivalents thereof known to those skilled in the art, and so forth.

[0010] The present document is organized into a number of sections for ease of reading; however, the reader will appreciate that statements made in one section may apply to other sections. In this manner, the headings used for different sections of the disclosure should not be construed as limiting.

[0011] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary' skill in the art. The following terms are provided below'.

1.1. Abbreviations and Acronyms

[0012] The following abbreviations/acronyms have the following meanings unless otherwise specified:

ABTS 2,2~azino~his-3-ethylbenzothiazolme-6-sulfome acid

cDNA complementary DNA

deoxyribonucleic acid

DPn degree of saccharide polymerization having n subunits ds or DS dry solids

GA glucoamylase

GAU/g ds giucoamylase activity unit/gram dry solids

IRS insoluble residual starch

kDa kiloDalton

MW molecular weight NCBI National Center for Biotechnology Information

PAHBAH p-hydroxy benzoic acid hydrazide

PEG poly ethyl eneg!y col

pi isoelectric point

PI performance index

ppm parts per million, e.g., pg protein per gram dry solid

RNA ribonucleic acid

SDS-PAGE sodium dodecy! sulfate polyacrylamide gel electrophoresis

SSF simultaneous saccharification and fermentation

SSU/g solid soluble starch unit/gram dry solids

sp. species

TrGA Trichoderma reesei glucoamylase

w/v weight/volume

w/w weight' weight

v/v volume/volume

wt% weight percent

°c degrees Centigrade

H 2 0 water

dl liO or DI deionized water

dIH 2 0 deionized water, Milli-Q filtration

g or gm grams

micrograms

mg milligrams

kg kilograms

mΐ. and mΐ microliters

mL and ml milliliters

M molar

mM millimoiar

micromolar

units

sec seconds

nnn(s) minute/minutes

hr(s) hour/hours

DO dissolved oxygen

ETOH ethanol eq. equivalents

N normal

PDB Protein Database

CAZy Carbohydrate- Active Enzymes database

Tris-HCl

HEPES 4-(2-hy droxy ethyl)- 1 -piperazineethanesulfonic acid

mS/cm milli-Siemens/cm

CV column volumes

1.2. Definitions

[0013] The terms“amylase” or“amylolytic enzyme” refer to an enzyme that is, among other things, capable of catalyzing the degradation of starch. Alpha-amylases are hydrolases that cleave the alpha-D-(l- 4) O-glycosidic linkages in starch. Generally, alpha-amylases (EC 3.2.1.1; alpha-D-( 1 -®4)-glucan glucanohydrolase) are defined as endo-acting enzymes cleaving alpha-D-(l 4) O-glycosidic linkages within the starch molecule in a random fashion yielding polysaccharides containing three or more (l-4)-alpha-linked D-glucose units. In contrast, the exo-acting amylolytic enzymes, such as beta-amylases (EC 3.2.1.2; alpha-D- (]— ->4)-giucan maltohydrolase) and some product-specific amylases like maltogemc alpha- amylase (EC 3.2.1.133) cleave the polysaccharide molecule from the non-reducing end of the substrate. Beta-amylases, alpha-glucosidases (EC 3.2.1.20; alpha-D-glucoside glucohydrolase), glucoamylase (EC 3.2.1.3; a!pha-D-(l- 4)~glucan glucohydrolase), and product-specific amylases like the maltotetraosidases (EC 3.2.1.60) and the maltohexaosidases (EC 3.2.1.98) can produce malto-oligosaccharides of a specific length or enriched syrups of specifi c m al tool! gos acch ari des .

[0014] The term“starch” refers to any material comprised of the complex polysaccharide carbohydrates of plants, comprised of amylose and amylopectin with the formula (C6H10O5)x, wherein X can be any number. The term includes plant-based materials such as grains, cereal, grasses, tubers and roots, and more specifically materials obtained from wheat, barley, com, rye, rice, sorghum, brans, cassava, millet, miio, potato, sweet potato, and tapioca. The term “starch” includes granular starch. The term“granular starch” refers to raw, i.e., uncooked starch, e.g., starch that has not been subject to ge!atinization or that has been subjected to temperatures at or below' the gelatinization temperature of the starch. [0015] The terms,“wild-type,”“parental,” or“reference,” with respect to a polypeptide, refer to a naturally-occurring polypeptide that does not include a man-made substitution, insertion, or deletion at one or more ammo acid positions. Similarly, the terms“wild-type,”“parental,” or“reference,” with respect to a polynucleotide, refer to a naturally-occurring polynucleotide that does not include a man-made nucleoside change. However, note that a polynucleotide encoding a wild-type, parental, or reference polypeptide is not limited to a naturally-occurring polynucleotide, and encompasses any polynucleotide encoding the wild-type, parental, or reference polypeptide.

[0016] Reference to the wild-type polypeptide is understood to include the mature form of the polypeptide. The term "mature polypeptide" is defined herein as a polypeptide in its final form following translation and any post-translational modifications, such as N-termmal processing, C-terminal truncation, glycosylation, phosphorylation, etc. Polypeptides, which are to be secreted, are translocated into the endoplasmic reticulum (ER). This is facilitated by a short hydrophobic N-terminal signal peptide, which allows for co- or post-translational translocation from the cytosol to the ER lumen and typically consists of 13 to 36 mostly hydrophobic ammo acids (pre-sequence) (Ng et al., 1996; Zimmermann et al., 2011). After cleavage of the signal peptide by an ER-resident signal peptidase, and correct folding by chaperones and foldases, the proteins are then transported to the Golgi network. Subsequently, proteins are delivered to their final cellular location, which may be the ER, Golgi, secretory vesicles, peroxisomes, endosomes, vacuole, cell wall or the cell exterior (recently reviewed by De!ic et al., 2013). Previous research has shown that the folding, secretion and processing ability of the cell is different for each protein and that the N-terminal amino acids may influence cleavage of the secretion leader (Wang et ah, 2014). Some proteins are modified posttranslationally, for example, by cleavage from a protein precursor, and therefore may have different amino acids at their N-terminus. The exact N-terminal sequence is also liable to exhibit a distinctive pattern depending on the experimental conditions.

[0017] The term“variant,” with respect to a polypeptide, refers to a polypeptide that differs from a specified wild-type, parental, or reference polypeptide in that it includes one or more naturally-occurring or man-made substitutions, insertions, or deletions of an amino acid. Similarly, the term“variant,” with respect to a polynucleotide, refers to a polynucleotide that differs in nucleotide sequence from a specified wild-type, parental, or reference polynucleotide. The identity of the wild-type, parental, or reference polypeptide or polynucleotide will be apparent from context. [0018] In the case of the present alpha-amylases,“activity” refers to alpha-amylase activity', which can he measured as described, herein.

[0019] The term“recombinant,” when used in reference to a subject cell, nucleic acid, protein or vector, indicates that the subject has been modified from its native state. Thus, for example, recombinant cells express genes that are not found within the native (non- recombinant) form of the cell, or express native genes at different levels or under different conditions than found in nature. Recombinant nucleic acids differ from a native sequence by one or more nucleotides and/or are operably linked to heterologous sequences, e.g , a heterologous promoter in an expression vector. Recombinant proteins may differ from a native sequence by one or more amino acids and/or are fused with heterologous sequences. A vector comprising a nucleic acid encoding an amylase is a recombinant vector.

[0020] The term“purified” refers to material (e.g., an isolated polypeptide or polynucleotide) that is in a relatively pure state, e.g., at least about 90% pure, at least about 95% pure, at least about 98% pure, or even at least about 99% pure.

[0021] The term“enriched” refers to material (e.g., an isolated polypeptide or polynucleotide) that is about 50% pure, at least about 60% pure, at least about 70% pure, or even at least about 70% pure.

[QQ22] The terms“thermostable” and“thermostability,” with reference to an enzyme, refer to the ability of the enzyme to retain activity after exposure to an elevated temperature. The thermostability' of an enzyme, such as an amylase enzy me, is measured by its half-life (ti /i ) given in minutes, hours, or days, during which half the enzyme activity is lost under defined conditions. The half-life may be calculated by measuring residual alpha-amylase activity for example following exposure to (i.e., challenge by) an elevated temperature.

[0023] A“pH range,” with reference to an enzyme, refers to the range of pH values under which the enzyme exhibits catalytic activity.

[0024] The terms“pH stable” and“pH stability,” with reference to an enzyme, relate to the ability' of the enzyme to retain activity over a w de range of pH values for a predetermined period of time (e.g., 15 min., 30 min., 1 hour).

[0025] The term “amino acid sequence” is synonymous with the terms “polypeptide,” “protein,” and“peptide,” and are used interchangeably. Where such amino acid sequences exhibit activity', they may be referred to as an“enzyme.” The conventional one-letter or three-letter codes for ammo acid residues are used, with amino acid sequences being presented in the standard amino-to-carboxy terminal orientation (i.e., N C).

[0026] The term “nucleic acid” encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids may be single stranded or double stranded, and may be chemically modified. The terms“nucleic acid” and“polynucleotide” are used interchangeably. Because the genetic code is degenerate, more than one codon may ¬ be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences that encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in 5'-to-3' orientation.

[0027] The term“hybridization” refers to the process by which one strand of nucleic acid forms a duplex with, i.e., base pairs with, a complementary strand, as occurs during blot hybridization techniques and PCR techniques. Stringent hybridization conditions are exemplified by hybridization under the following conditions: 65°C and 0.1X SSC (where IX SSC = 0.15 M NaCl, 0 015 M Na 3 citrate, pH 7 0). Hybridized, duplex nucleic acids are characterized by a melting temperature (T m ), where one half of the hybridized nucleic acids are unpaired with the complementary strand. Mismatched nucleotides within the duplex lower the T m .

[QQ28] A“synthetic” molecule is produced by in vitro chemical or enzymatic synthesis rather than by an organism.

[0029] The term“introduced” in the context of inserting a nucleic acid sequence into a cell, means“transfection”,“transformation” or“transduction,” as known in the art.

[0030] A“host strain” or“host cell” is an organism into which an expression vector, phage, virus, or other DNA construct, including a polynucleotide encoding a polypeptide of interest (e.g. , an amylase) has been introduced. Exemplary host strains are microorganism cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing the polypeptide of interest and/or fermenting saccharides. The term“host cell” includes protoplasts created from cells.

[0031] The term“heterologous” with reference to a polynucleotide or protein refers to a polynucleotide or protein that does not naturally occur in a host cell.

[0032] The term“endogenous” with reference to a polynucleotide or protein refers to a polynucleotide or protein that occurs naturally in the host cell.

[0033] The term“expression” refers to the process by which a polypeptide is produced based on a nucleic acid sequence. The process includes both transcription and translation. [0034] A“selecti ve marker” or“selectable marker” refers to a gene capable of being expressed in a host to facilitate selection of host cells carrying the gene. Examples of selectable markers include but are not limited to antimicrobials (e.g., hygromycin, bleomycin, or chloramphenicol) and/or genes that confer a metabolic advantage, such as a nutritional advantage on the host cell.

[0035] The term“vector” refers to a polynucleotide sequence designed to introduce nucleic acids into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes and the like.

[QQ36] An “expression vector” refers to a DNA construct comprising a DNA sequence encoding a polypeptide of interest, which coding sequence is operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, enhancers and sequences which control termination of transcription and translation.

[0037] The term“operably linked” means that specified components are in a relationship (including but not limited to juxtaposition) permitting them to function in an intended manner. For example, a regulatory sequence is operably linked to a coding sequence such that expression of the coding sequence is under control of the regulatory sequences.

[QQ38] A“signal sequence” is a sequence of amino acids attached to the N-terminal portion of a protein, which facilitates the secretion of the protein outside the cell. The mature form of an extracellular protein lacks the signal sequence, which is cleaved off during the secretion process.

[0039]“Biologically active” refer to a sequence having a specified biological activity, such an enzymatic activity.

[0040] The term“specific activity” refers to the number of moles of substrate that can be converted to product by an enzyme or enzyme preparation per unit time under specific conditions. Specific activity is generally expressed as units (U)/mg of protein.

[0041] “A cultured cell material comprising an amylase” or similar language, refers to a cell lysate or supernatant (including media) that includes an amylase as a component. The cell material may be from a heterologous host that is grown in culture for the purpose of producing the amylase.

[0042]“Percent sequence identity” means that a particular sequence has at least a certain percentage of amino acid residues identical to those in a specified reference sequence, when aligned using the CLUSTAL W algorithm with default parameters. See Thompson et al.

(1994) Nucleic Acids Res. 22:4673-4680. Default parameters for the CLUSTAL W algorithm are:

Gap opening penalty: 10.0

Gap extension penalty: 0.05

Protein weight matrix: BLOSUM series

DNA weight matrix: TUB

Delay divergent sequences %: 40

Gap separation distance: 8

DNA transitions weight: 0 50

List hydrophilic residues: GPSNDQEKR

Use negative matrix: OFF

Toggle Residue specific penalties: ON

Toggle hydrophilic penalties: ON

Toggle end gap separation penalty OFF.

[QQ43] Deletions are counted as non-identical residues, compared to a reference sequence. Deletions occurring at either termini are included. For example, a variant 500-amino acid residue polypeptide with a deletion of five amino acid residues from the C-terminus would have a percent sequence identity of 99% (495/500 identical residues x 100) relative to the parent polypeptide. Such a variant would be encompassed by the language,“a variant having at least 99% sequence identity to the parent/’

[0044]“Fused” polypeptide sequences are connected, i.e., operably linked, via a peptide bond between two subject polypeptide sequences.

[0045] The term“degree of polymerization” (DP) refers to the number (n) of anhydro- glucopyranose units in a given saccharide. Examples of DPI are the monosaccharides glucose and fructose. Examples of DP2 are the disaccharides maltose and sucrose. The term“DE,” or“dextrose equivalent,” is defined as the percentage of reducing sugar, i.e. , D- glucose, as a fraction of total carbohydrate in a syrup.

[0046] The term“dry solids content” (ds) refers to the total solids of a slurry in a dry weight percent basis. The term“slurry” refers to an aqueous mixture containing insoluble solids.

[0047] The phrase“simultaneous saccharification and fermentation (SSF)” refers to a process in the production of biochemicals in which a microbial organism, such as an ethanologenic microorganism, and at least one enzyme, such as an amylase, are present during the same process step SSF includes the contemporaneous hydrolysis of starch substrates (granular, liquefied, or solubilized) to saccharides, including glucose, and the fermentation of the saccharides into alcohol or other biochemical or biomaterial m the same reactor vessel.

|0048] An “ethanologenic microorganism” refers to a microorganism with the ability to convert a sugar or other carbohydrates to ethanol

[0049] The term“biochemicals” refers to a metabolite of a microorganism, such as citric acid, lactic acid, succinic acid, monosodium glutamate, gluconic acid, sodium gluconate, calcium gluconate, potassium gluconate, glucono delta-lactone, sodium erythorbate, omega 3 fatty acid, butanol, iso-butanol, an amino acid, lysine, itaconic acid, other organic acids, 1 ,3- propanediol, vitamins, or isoprene or other biomaterial.

[0050] The term “fermented beverage” refers to any beverage produced by a method comprising a fermentation process, such as a microbial fermentation, e.g., a bacterial and/or fungal fermentation. “Beer” is an example of such a fermented beverage, and the term“beer” is meant to comprise any fermented wort produced by fermentation/brewing of a starch- containing plant material. Often, beer is produced exclusively from malt or adjunct, or any combination of malt and adjunct.

[0051] The term“about” refers to ± 15% to the referenced value.

2, Polypeptides having alpha-amylase activity used in this invention

[0052] In a first aspect, the present invention relates to polypeptides comprising an amino acid sequence having preferably at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and even at least 99%, amino acid sequence identity to the polypeptide of SEQ ID NO: 3, and having alpha-amylase activity .

[0053] In some embodiments, the polypeptides of the present invention are the homologous polypeptides comprising amino acid sequences differ by ten amino acids, preferably by nine amino acids, preferably by eight amino acids, preferably by seven amino acids, preferably by six amino acids, preferably by five ammo acids, more preferably by four amino acids, even more preferably by three amino acids, most preferably by two amino acids, and even most preferably by one amino acid from the polypeptide of SEQ ID NO: 3.

[0054] In some embodiments, the polypeptides of the present invention are the variants of polypeptide of SEQ ID NO: 3, or a fragment thereof having alpha-amylase activity. [0055] In some embodiments, the polypeptides of the present invention are the catalytic regions comprising the amino acids 22 to 499 of SEQ ID NO: 2, predicted by Clustalx htps : // www. ncbi nlm. nih . go v/pubmed/ 17846036.

[0056] In some embodiments, the polypeptides of the present invention are the catalytic regions and linker regions comprising the amino acids 22 to 550 of SEQ ID NO: 2, predicted by Clustalx htps://www.ncbi . nlm. nih. gov/p ubmed/l 7846036.

[0057] In a second aspect, the present alpha-amylases comprise conservative substitution of one or several amino acid residues relative to the amino acid sequence of SEQ ID NO: 3. Exemplary conservative amino acid substitutions are listed in the Table I. Some conservative mutations can be produced by genetic manipulation, while others are produced by introducing synthetic amino acids into a polypeptide by other means.

Table 1. Conservative amino acid substitutions

[0058] In some embodiments, the present alpha-amylase comprises a deletion, substitution, insertion, or addition of one or a few amino acid residues relative to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the present alpha-amylases are derived from the amino acid sequence of SEQ ID NO: 3 by conservative substitution of one or several ammo acid residues. In some embodiments, the present alpha-amylases are derived from the amino acid sequence of SEQ ID NO: 3 by deletion, substitution, insertion, or addition of one or a few ammo acid residues relative to the amino acid sequence of SEQ ID NO: 3. In all cases, the expression "one or a few amino acid residues" refers to 10 or less, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acid residues.

[0059] In another embodiment, the present invention also relates to carbohydrate binding domain variants of SEQ ID NO: 3 comprising a substitution, deletion, and/or insertion at one or more (e.g., several) positions.

[0060] The present amylases may be "precursor," "immature," or "full-length," in which case they include a signal sequence, or "mature," in which case they lack a signal sequence. Mature forms of the polypeptides are generally the most useful. Unless otherwise noted, the ammo acid residue numbering used herein refers to the mature forms of the respecti v e amylase polypeptides. The present amylase polypeptides may also be truncated to remove the N or C- termini, so long as the resulting polypeptides retain amylase activity .

[0061] Alternatively, the ammo acid changes are of such a nature that the physico chemical properties of the polypeptides are altered. For example, amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like.

[QQ62] Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error- prone PCR, phage display (e.g., Lowman et a!., 1991, Biochem. 30: 10832-10837; U. S. Patent No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

[0063] Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host ceils (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagemzed DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual ammo acid residues a polypeptide of interest, and can be applied to polypeptides of unknown structure.

[0064] The present amylase may he a "chimeric" or "hybrid" polypeptide, in that it includes at least a portion from a first amylase, and at least a portion from a second amylase, g!ucoamy!ase, beta-amylase, alpha-giucosidase or other starch degrading enzymes, or even other glycosyl hydrolases, such as, without limitation, cellu!ases, hemicellu!ases, etc. (including such chimeric amylases that have recently been "rediscovered" as domain-swap amylases). The present amylases may further include heterologous signal sequence, an epitope to allow tracking or purification, or the like. Exemplary heterologous signal sequences are from B. licheniformis amylase (LAT), B. subtiiis (AmyE or AprE), and Streptomyces CelA.

3, Production of alpha-Amylases

[0065] The present alpha-amylases can be produced in host cells, for example, by secretion or intracellular expression. A cultured cell material (e.g. , a whole-cell broth) comprising an alpha- amylase can be obtained following secretion of the alpha-amylase into the cell medium. Optionally, the alpha-amylase can be isolated from the host cells, or even isolated from the cell broth, depending on the desired purity' of the final alpha-amylase. A gene encoding an alpha- amylase can be cloned and expressed according to methods well known in the art. Suitable host cells include bacterial, fungal (including yeast and filamentous fungi), and plant cells (including algae). Particularly useful host cells include Aspergillus niger, Aspergillus oryzae, Trichoderma reesi or Myceliopthora thermophila. Other host cells include bacterial cells, e.g. , Bacillus subtiiis or B. licheniformis, as well as Streptomyces.

[0066] Additionally, the host may express one or more accessory' enzymes, proteins, peptides. These may benefit liquefaction, saccharification, fermentation, SSF, and downstream processes. Furthermore, the host cell may produce ethanol and other biochemicals or biomaterials in addition to enzymes used to digest the various feedstock(s). Such host cells may be useful for fermentation or simultaneous saccharification and fermentation processes to reduce or eliminate the need to add enzymes. 3.1. Vectors

[0067] A DNA construct comprising a nucleic acid encoding alpha-amylases can be constructed to be expressed in a host cell. Because of the well-known degeneracy in the genetic code, different polynucleotides that encode an identical ammo acid sequence can be designed and made with routine skill. It is also well-known in the art to optimize codon use for a particular host cell. Nucleic acids encoding alpha-amylases can be incorporated into a vector. Vectors can be transferred to a host cell using well-known transformation techniques, such as those disclosed below.

[0068] The vector may be any vector that can be transformed into and replicated within a host cell. For example, a vector comprising a nucleic acid encoding an alpha-amylase can be transformed and replicated in a bacterial host cell. A vector comprising a nucleic acid encoding an alpha-amylase can also be transformed and conveniently integrated into the chromosome (in one or more copies) of a bacterial host cell, and the integration is generally considered to be an advantage, as the DNA sequence is more likely to be stably maintained in the cell. A representative useful vector is p2JM103BBI (Vogtentanz, Protein Expr Purif, 55:40- 52, 2007), which can be modified with routine skill and integrated into the chromosome of host cell such that they comprise other DNA fragments to improve the expression alpha-amylases of the invention.

[0069] Host cells that serve as expression hosts can include filamentous fungi, for example. The Fungal Genetics Stock Center (FGSC) Catalogue of Strains lists suitable vectors for expression in fungal host cells. See FGSC, Catalogue of Strains, University of Missouri, at www. fgsc.net (last modified January 17, 2007). A representative useful vector is pTrexSgM (see, Published US Patent Application 20130323798) and pTTT (see, Published US Patent Application 20110020899), which can be inserted into genome of host. The vectors pTrexSgM and pTTT can both be modified with routine skill such that they comprise and express a polynucleoti de encoding an alpha-amylase polypeptide of the inventi on.

[0070] A nucleic acid encoding an alpha-amylase can be operably linked to a suitable promoter, which allows transcription in the host ceil. The promoter may be any DNA sequence that shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Exemplary promoters for directing the transcription of the DNA sequence encoding an alpha-amylase, especially in a bacterial host, are the promoters derived from the lac operon of E. coli, the Streptomyces coelicolor agarase gene dagA or celA, the Bacillus licheniformis alpha-amylase gene (arnyL), the Bacillus stear other mophilus maltogenic amylase gene (amyM), the Bacillus amyloUquefaciem alpha-amylase gene (amyQ), the Bacillus subtilis xylA and xylB genes etc. For transcription in a fungal host, examples of useful promoters are those derived from the gene encoding Aspergillus oryzae TAKA amylase, Khizomucor miehei aspartic proteinase, Aspergillus mger neutral alpha-amylase, A. niger acid stable alpha-amylase, A. niger glucoamylase, Khizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase, or A. nidulans acetamidase. When a gene encoding an amylase is expressed in a bacterial species such as E. colt, a suitable promoter can be selected, for example, from a bacteriophage promoter including a T7 promoter and a phage lambda promoter. Examples of suitable promoters for the expression in a yeast species include but are not limited to the Gal 1 and Gal 10 promoters of Saccharomyces cerevisiae and the Pichia pastoris AOXl or AOX2 promoters. Examples of suitable promoter for the expression m filamentous fungi include but are not limited to cbhl, an endogenous, inducible promoter from 71 reesei. See Liu et al. (2008)“Improved heterologous gene expression in Trichoderma reesei by cellobiohydrolase I gene (cbhl) promoter optimization,” Acta Biochim. Biophys. Sin (Shanghai) 40(2): 158-65.

[0071] The coding sequence can be operably linked to a signal sequence. The DNA encoding the signal sequence may be the DNA sequence naturally associated with the amylase gene to he expressed or from a different genus or species. A DNA construct or vector comprising a signal sequence and a promoter sequence can be introduced into a fungal host cell and can be derived from the same source. For example, the signal sequence is the cbhl signal sequence that is operably linked to a cbhl promoter.

[0072] An expression vector may also comprise a suitable transcription terminator and, in eukaryotes, polyadenylation sequences operably linked to the DNA sequence encoding an alpha-amylase. Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.

[0073] The vector may further comprise a DNA sequence enabling the vector to replicate m the host cell. Examples of such sequences are the origins of replication of plasmids pUC19, pAC. ' YC ! 77. pUBl lO, pf.194. pAMBl , and pU702.

[0074] The vector may also comprise a selectable marker, e.g., a product of a gene complements a defect in the isolated host cell, such as the dal genes from B. subtilis or B. lichemformis, or a gene that confers antibiotic resistance such as, e.g. , ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Furthermore, the vector may comprise Aspergillus selection markers such as amdS , argB, niaD and xxsC, a marker giving rise to hygrornycin resistance, or the selection may be accomplished by co-transformation, such as known in the art. See e.g., International PCT Application WO 91/17243.

[0075] Intracellular expression may be advantageous in some respects, e.g., when using certain bacteria or fungi as host cells to produce large amounts of amylase for subsequent enrichment or purification. Extracellular secretion of amylase into the culture medium can also be used to make a cultured cell material comprising the isolated amylase.

[QQ76] The procedures used to ligate the DNA construct encoding an amylase, the promoter, terminator and other elements, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art. (see, e.g.. Sambrook et al. , MOLECULAR CLONING: A LABORATORY MANUAL, 2 nd ed., Cold Spring Harbor, 1989, and 3 rd ed., 2001).

|0077] An isolated cell, either comprising a DNA construct or an expression vector, is advantageously used as a host cell in the recombinant production of an amylase. The cell may be transformed with the DNA construct encoding the enzyme, conveniently by integrating the DNA construct (in one or more copies) in the host chromosome. This integration is generally considered to be an advantage, as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g., by homologous or heterologous recombination. Alternatively, the cell may be transformed with an expression vector as described above in connection with the different types of host cells.

[0078] Examples of suitable bacterial host organisms are Gram positive bacterial species such as Bacillaceae including Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis , Geobacillus (formerly Bacillus) stearothermophilus, Bacillus alkalophilus, Bacillus amyloUquefaciem, Bacillus coagulans, Bacillus laulus, Bacillus megaterium, and Bacillus thuringiensis; Streptomyces species such as Streptomyces murinus : lactic acid bacterial species including Lactococcus sp. such as Laciococcus lacks; Lactobacillus sp. including Lactobacillus reuteri; Leuconostoc sp.; Pediococcus sp.; and Streptococcus sp. Alternatively, strains of a Gram negative bacterial species belonging to Enterobacteriaceae including E. edit, or to Pseudomonadaceae can be selected as the host organism. [0079] A suitable yeast host organism can be selected from the biotechnologically relevant yeasts species such as but not limited to yeast species such as Pichia sp., Hansenula sp., or Kluyveromyces, Yarrowinia, Schizosaccharomyces species or a species of Saccharomyces, including Saccharomyces cerevisiae or a species belonging to Schizosaccharomyces such as, for example, S. pombe species. A strain of the methylotrophic yeast species, Pichia pastoris, can be used as the host organism. Alternatively, the host organism can be a Hansenula species.

[0080] Suitable host organisms among filamentous fungi include species of Aspergillus, e.g., Aspergillus niger , Aspergillus oryzae, Aspergillus tubigensis, Aspergillus aw amor i , or Aspergillus nidulans. A suitable procedure for transformation of Aspergillus host cells includes, for example, that described in EP 238023. Alternatively, strains of a Fusarium species, e.g., Fusarium oxysporum or of a Rhizomucor species such as Rhizomucor miehei can be used as the host organism. Other suitable strains include Mycelioplhora, Thermornyces and Mucor species. In addition, Trichoderma sp. can be used as a host. A suitable procedure for transformation of Trichoderma host cells includes, for example, that described by Penttila et al. [Gene 61(1987) 155-164] An amylase expressed by a fungal host cell can be glycosylated, /.<?., will comprise a glycosyl moiety. The glycosylation pattern can be the same or different as present in the wild-type amylase. The type and/or degree of glycosylation may impart changes in enzymatic and/or biochemical properties.

[0081] It is advantageous to delete genes from expression hosts, where the gene deficiency can be cured by the transformed expression vector. Known methods may be used to obtain a fungal host cell having one or more inactivated genes. Gene inactivation may be accomplished by complete or partial deletion, by insertional inactivation or by any other means that renders a gene nonfunctional for its intended purpose, such that the gene is prevented from expression of a functional protein. Any gene from a Trichoderma sp. or other filamentous fungal host that has been cloned can be deleted, for example, cbhl, chh2, egll, and egl2 genes. Gene deletion may be accomplished by inserting a form of the desired gene to be inactivated into a plasmid by methods known in the art.

[0082] The suitable host cell may be the ethanologenic microbial cell, which may express one or more of the amylase homologs described herein, and/or other Bacillus amylases (including from B. hcheniformis , B. stearothermophilus , B. substihs, and other Bacillus species), and/or amylases from other sources. These may further express a homologous or heterologous starch degrading enzymes, such as glucoamylase, i.e., a glucoamylase that is not the same species as the host cell. Additionally, the host may express one or more accessory enzymes, proteins, and/or peptides. These may benefit pre treatment, liquefaction, saccharification, fermentation, SSF, stillage, condensed distillers soluble or syrup, etc processes. Furthermore, the host cell may produce ethanol and other bioehemicals or biomaterials in addition to enzymes used to digest the various feedstock(s). Such host cells may be useful for fermentation or simultaneous saccharification and fermentation processes to reduce or eliminate the need to add enzymes.

[0083] Introduction of a DNA construct or vector into a host cell includes techniques such as transformation; electroporation; nuclear microinjection; transduction; transfection, e.g., lipofection mediated and DEAE-Dextrin mediated transfection; incubation with calcium phosphate DNA precipitate; high velocity bombardment with DNA-coated microprojectiles; and protoplast fusion. General transformation techniques are known in the art. See e.g., Sambrook et al. (2001 ), supra. The expression of heterologous protein in Trichoderma is described, for example, in U.S. Patent No. 6,022,725. Reference is also made to Cao et al. (2000) Science 9:991-1001 for transformation of Aspergillus strains.

3.3. Expression ami Fermentation

[QQ84] A method of producing an amylase may comprise cultivating a host cell as described above under conditions conducive to the production of the enzyme and recovering the enzyme from the cells and/or culture medium.

[0085] The medium used to cultivate the cells may be any conventional medium suitable for growing the host cell and obtaining expression of a alpha-amylase polypeptide. Suitable media and media components are available from commercial suppliers or may be prepared according to published recipes (e.g., as described in catalogues of the American Type Culture Collection).

[0086] Any of the fermentation methods well known in the art can suitably used to ferment the transformed or the derivative fungal strain as described above. In some embodiments, fungal cells are grown under batch or continuous fermentation conditions.

3.4. Identification of Amylase Activity

[0087] To evaluate the expression of an amylase in a host cell, assays can measure the expressed protein, corresponding mRNA, or alpha- amylase activity. For example, suitable assays include Northern blotting, reverse transcriptase polymerase chain reaction, and in situ hybridization, using an appropriately labeled hybridizing probe. Suitable assays also include measuring amylase activity in a sample, for example, by assays directly measuring reducing sugars such as glucose in the culture media. For example, glucose concentration may be determined using glucose reagent kit No. 15-UV (Sigma Chemical Co.) or an instrument, such as Techmcon autoanalyzer. Alpha- Amylase activity also may be measured by any known method, such as the PAHBAH or ABTS assays, described below'.

3,5, Methods for Enriching and Purifying Alpha-Amylases

[0088] Separation and concentration techniques are known in the art and conventional methods can be used to prepare a concentrated solution or broth comprising an alpha-amylase poly peptide of the invention

[QQ89] After fermentation, a fermentation broth is obtained, the microbial cells and various suspended solids, including residual raw fermentation materials, are removed by conventional separation techniques in order to obtain an alpha-amylase solution. Filtration, centrifugation, microfiltration, rotary vacuum drum filtration, ultrafiltration, centrifugation followed by ultra filtration, extraction, or chromatography, or the like, are generally used.

[0090] It may at times be desirable to concentrate a solution or broth comprising an alpha- amylase polypeptide to optimize recovery'. Use of un-concentrated solutions or broth would typically increase incubation time in order to collect the enriched or purified enzyme precipitate.

4, Compositions and Methods for Starch Degradation

[0091] The present alpha-amylases are useful for a variety of industrial applications. For example, alpha-amylases are useful in a starch degradation processes, particularly in liquefaction of gelatinized starch, simultaneous liquefaction and saccharification, saccharification, fermentation, and/or simultaneous saccharification and fermentation (SSF).

[QQ92] The starch conversion process may be a precursor to, or simultaneous with, a fermentation process designed to produce alcohol for fuel or drinking (i.e., potable alcohol), or other biochemicals or biomaterials. One skilled in the art is aware of various fermentation conditions that may be used in the production of these end-products. These various uses of alpha-amylases are described in more detail below.

4.1. Preparation of Starch Substrates

[0093] Those of general skill in the art are well aware of available methods that may be used to prepare starch substrates for use in the processes disclosed herein. For example, a useful starch substrate may be obtained from tubers, roots, stems, legumes, cereals or whole grain. More specifically, the granular starch may be obtained from com, cobs, wheat, barley, rye, triiicale, rnilo, sago, millet, cassava, tapioca, sorghum, rice, peas, bean, banana, or potatoes. Com contains about 60-68% starch; barley contains about 55-65% starch; millet contains about 75-80% starch; wheat contains about 60-65% starch; and polished rice contains 70-72% starch. Specifically, contemplated starch substrates are com starch and wheat starch. The starch from a grain may be ground or whole and includes corn solids, such as kernels, bran and/or cobs. The starch may also be highly refined raw starch or feedstock from starch refinery processes. Various starches also are commercially available. For example, corn starch is available from Cerestar, Sigma, and Katayama Chemical Industr' Co. (Japan); wheat starch is available from Sigma; sweet potato starch is available from Wako Pure Chemical Industry' Co. (Japan); and potato starch is available from Nakaari Chemical Pharmaceutical Co. (Japan).

[0094] The starch substrate can be a crude starch from milled whole grain, which contains non-starch fractions, e.g., germ residues and fibers. Milling may comprise wet milling or dry milling or grinding. In wet milling, whole grain is soaked in water or dilute acid to separate the grain into its component parts, e.g., starch, protein, germ, oil, kernel fibers. Wet milling efficiently separates the germ and meal (i.e., starch granules and protein) and is especially suitable for production of syrups. Starch to be processed may be a highly refined starch quality, for example, at least 90%, at least 95%, at least 97%, or at least 99.5% pure. In dry' milling or grmding, whole kernels are ground into a fine powder and often processed without fractionating the grain into its component parts. In some cases, oils and/or fiber from the kernels are recovered. Dry ground grain thus will comprise significant amounts of non-starch carbohydrate compounds, in addition to starch. Dry grinding of the starch substrate can be used for production of ethanol and other biochemical and biomaterials.

4.2. Gelatinization and Liquefaction of Starch

[0095] As used herein, the term“liquefaction’' or“liquefy” means a process by which gelatinized starch is converted to less viscous liquid containing shorter chain soluble dextrins, liquefaction-inducing and/or saccharifying enzymes optionally may be added. In some embodiments, the starch substrate prepared as described above is slurried with water. The starch slurry' may contain starch as a weight percent of dry' solids of about 10-55%, about 20- 45%, about 30-45%, about 30-40%, or about 30-35%. Alpha- Amylase (EC 3.2.1.1) may be added to the slurry, with a metering pump, for example. Tire alpha- amylase typically used for this application is a thermal stable, bacterial alpha- amylase, such as a Geobacillus stearothermophiius alpha- amylase, CytophagajApha- amylase, etc, for example SPEZYME® RSL (DuPont product), SPEZYME® AA (DuPont product), SPEZYME® Fred (DuPont product), CLEARFLOW® AA (DuPont product), SPEZYME® Alpha PF (DuPont product), SPEZYME® Powerliq (DuPont product) can be used here. The alpha-amylase may be supplied, for example, at about 1500 units per kg dry matter of starch. To optimize alpha- amylase stability and activity, the pH of the slurry typically is adjusted to about pH 5.5- 6 5 or a pFI most suitable for the amylase to be added and about 1 rnM of calcium (about 40 ppm free calcium ions) can also be added. Various alpha-amylases may require different conditions. Liquefaction alpha-amylase remaining in the slurry following liquefaction may be deactivated via a number of methods, including lowering the pH in a subsequent reaction step or by removing calcium from the slurry' in cases where the enzyme is dependent upon calcium.

|0096] The slurry of starch plus the alpha- amylase may be pumped continuously through a jet cooker, which is steam heated to 80-1 10°C, depending upon the source of the starch containing feedstock. Ge!atinization occurs rapidly under these conditions, and the enzymatic activity, combined with the significant shear forces, begins the hydrolysis of the starch substrate. The residence time m the jet cooker is brief. The partially gelatinized starch may then he passed into a series of holding tubes maintained at 105-1 10°C and held for 5-8 min. to complete the ge!atinization process (“primary liquefaction”). Hydrolysis to the required DE is completed in holding tanks at 85-95°C or higher temperatures for about 1 to 2 hours (“secondary liquefaction”). These tanks may contain baffles to discourage back mixing. As used herein, the term“minutes of secondary' liquefaction” refers to the time that has elapsed from the start of secondary liquefaction to the time that the Dextrose Equivalent (DE) is measured. The slurry is then allowed to cool to room temperature. This cooling step can be 30 minutes to 180 minutes, e.g. 90 minutes to 120 minutes. The liquefied starch typically is in the form of a slurry' having a dry solids content (w/w) of about 10-50%; about 10-45%; about 15-40%; about 20-40%; about 25-40%; or about 25-35%.

|0097] Liquefaction with alpha-amylases advantageously can be conducted at low pH, eliminating the requirement to adjust the pH to about pFT 5.5-6.5. Alpha-amylases can be used for liquefaction at a pH range of 2 to 7, e.g. , pH 3.0 - 7 5, pH 4.0 - 6.0, or pH 4.5 - 5.8. Alpha-amylases can maintain liquefying activity at a temperature range of about 70°C - 140°C, e.g., 85°C, 90°C, or 95°C. For example, liquefaction can be conducted with 800 pg an amylase in a solution of 25% DS com starch for 10 min at pH 5.8 and 85°C, or pH 4.5 and 95°C. Liquefying activity can be assayed using any of a number of known viscosity assay methods in the art.

[0098] In particular embodiments using the present alpha-amylases, startch liquifacti on is performed at a temperature range of 90-115°C, for the purpose of producing high-purity glucose syrups, HFCS, maltodextnns, etc.

[0099] The liquefied starch may be saccharified into a syrup rich in lower DP (e.g., DP I + DP2) saccharides, using alpha-amylases, optionally in the presence of another enzyme(s). The exact composition of the products of saccharification depends on the combination of enzymes used, as well as the type of starch processed. Advantageously, the syrup obtainable using the provided alpha-amylases may contain a weight percent of DP2 of the total oligosaccharides in the saccharified starch exceeding 30%, e.g , 45% - 65% or 55% - 65%. The weight percent of (DPI + DP2) in the saccharified starch may exceed about 70%, e.g., 75% - 85% or 80% - 85%. The present amylases also produce a relatively high yield of glucose, e.g., DPI > 20%, in the syrup product.

[QQ100] Whereas liquefaction is generally run as a continuous process, saccharification is often conducted as a batch process. Saccharification conditions are dependent upon the nature of the hquefact and type of enzymes available. In some cases, a saccharification process may involve temperatures of about 60-65°C and a pH of about 4.0-4.5, e.g., pH 4.3. Saccharification may be performed, for example, at a temperature between about 40°C, about 50°C, or about 55°C to about 60°C or about 65°C, necessitating cooling of the Liquefact. The pH may also be adjusted as needed. Saccharification is normally conducted in stirred tanks, which may take several hours to fill or empty. Enzymes typically are added either at a fixed ratio to dried solids, as the tanks are filled, or added as a single dose at the commencement of the filling stage. A saccharification reaction to make a syrup typically is run over about 24-72 hours, for example, 24-48 hours. When a maximum or desired DE has been attained, the reaction is stopped by heating to 85 °C for 5 min., for example. Further incubation will result in a lower DE, eventually to about 90 DE, as accumulated glucose re-polymerizes to isomaltose and/or other reversion products via an enzymatic reversion reaction and/or with the approach of thermodynamic equilibrium. Preferred!y, saccharification optimally is conducted at a temperature range of about 30°C to about 75°C, e.g., 45°C - 75°C or 47°C - 75°C. The saccharifying may be conducted over a pH range of about pH 3 to about pH 7, e.g., pH 3.0 - pH 6 5, pH 3 5 - pH 5.5, pH 3.5, pH 3.8, or pH 4 5. [00101] An amylase may be added to the slurry in the form of a composition. Amylase can be added to a slurry' of a granular starch substrate. An amylase can be added as a whole broth, clarified, enriched, partially purified, or purified enzyme. The amylase also can be added as a whole broth product.

[00102] An amylase may be added to the slurry as an isolated enzyme solution. For example, an amylase can be added in the form of a cultured cell material produced by host cells expressing such amylase. An amylase may also be secreted by a host cell into the reaction medium during the fermentation or SSF process, such that the enzyme is provided continuously into the reaction. The host cell producing and secreting amylase may also express an additional enzyme, such as a glucoamylase. For example, U.S. Patent No. 5,422,267 discloses the use of a glucoamylase in yeast for production of alcoholic beverages. For example, a host ceil, e.g., Trichoderma reesei or Aspergillus niger, Myceliopthora thermophila or Yeast, may be engineered to co-express an amylase and a glucoamylase, e.g., Humicola GA, Trichoderma GA, or variants of these, during saccharification. The host cell can be genetically modified so as not to express its endogenous glucoamylase and/or other enzymes, proteins or other materials. The host cell can be engineered to express a broad spectrum of various saccharolytic enzymes. For example, the recombinant yeast host cell can comprise nucleic acids encoding a glucoamylase, an a!pha-g!ucosidase, beta-amylase, an enzyme that utilizes pentose sugar, an alpha- amylase, a pulluianase, an isoamylase, an isopullulanase, a phytase, a protease, and/or other enzymes. See, e.g. , WO 201 1/153516 A2,

[00103] The present alpha- amylases can also be used in a granular or raw starch hydrolysis (RSH) or granular starch hydrolysis (GSH) process for producing desired sugars and fermentation products. The term "granular starch" means raw uncooked starch, i. e., starch in its natural form found in cereal, tubers or grains. A "raw starch hydrolysis" process (RSH) differs from conventional starch treatment processes, including liquefying gelatinized starch at high temperature using typically a bactenal alpha-amylase, followed by simultaneous saccharification and fermentation carried out in the presence of a glucoamylase and a fermentation organism and possibly other enzymes. RSH process includes sequentially or simultaneously saccharifying and fermenting granular starch at or below the gelatinization temperature of the starch substrate typically in the presence of at least an amylase and/or glucoamylase. The gelatinization temperature may vary according to the plant species, to the particular variety of the plant species as well as with the growth conditions. [00104] A fungal alpha-amylase described herein expressed in bacterial, fungal, yeast or ethanologenic microbial cells can be used in raw starch hydrolysis process described herein.

[00105] In addition, alpha-amylase that is other than the alpha-amylase described in this invention, glucoamylase, hexokmase, xylanase, glucose isomerase, xylose isomerase, phosphatase, phytase, pullulanase, beta-amylase, protease, cellulase, hemicellulase, lipase, cutinase, isoamylase, redox enzyme, esterase, transferase, peciinase, a-glucosidase, beta- glucosidase, or a combination thereof can also be used in raw starch hydrolysis process described herein. The said enzymes can be co-expressed with alpha-amylase in this invention or directly added into the raw starch hy drolysis process.

[00106] The soluble starch hydrolysate, particularly a glucose rich syrup, can be fermented by contacting the starch hydrolysate with a fermenting organism typically at a temperature around 32°C, such as from 30°C to 35°C for alcohol-producing yeast. The temperature and pH of the fermentation will depend upon the fermenting organism. End of fermentation (EOF) products include metabolites, such as citric acid, lactic acid, succinic acid, monosodium glutamate, gluconic acid, sodium gluconate, calcium gluconate, potassium gluconate, itaconic acid and other carboxylic acids, glucono delta-lactone, sodium erythorbate, amino acids, lysine and other amino acids, vitamins, omega 3 fatty acid, butanol, isoprene, 1,3-propanediol, vitamins, and other biomaterials.

[00107] Ethanologenic microorganisms include yeast, such as Saccharomyces cerevisiae and bacteria, e.g., Zymomonas moolis, expressing alcohol dehydrogenase and pyruvate decarboxylase. The ethanologenic microorganism can express xylose reductase and xylitol dehydrogenase, which convert xylose to xylulose. Improved strains of ethanologenic microorganisms, which can withstand higher temperatures, for example, are known in the art and can be used. See Liu el al. (2011) Sheng Wu Gong Cheng Xue Bao 27: 1049-56. Commercial sources of yeast include ETHANOL RED ® (LeSaffre); THERMOS ACC © (Lallemand); RED STAR® (Red Star); FERMIOL® (DSM Specialties); and SUPERSTART® (Alltech). Microorganisms that produce other metabolites, such as citric acid and lactic acid, by fermentation are also known m the art. See, e.g., Papagianni (2007) Biotechnol. Adv. 25:244-63; John et al. (2009) Biotechnol. Adv. 27:145-52.

[00108] The saccharification and fermentation processes may be earned out as an SSF process. Fermentation may comprise subsequent enrichment, purification, and recovery ' of ethanol, for example. During the fermentation, the ethanol content of the broth or“beer” may reach about 8-18% v/v, e.g., 14-15% v/v. The broth may be distilled to produce enriched, e.g., 96% pure, solutions of ethanol. Further, CO2 generated by fermentation may be collected with a CO2 scrubber, compressed, and marketed for other uses, e.g. , carbonating beverage or dry ice production. Solid waste from the fermentation process may be used as protem-rich products, e.g., livestock feed.

[00109] As mentioned above, an SSF process can be conducted with fungal cells that express and secrete amylase continuously throughout SSF. The fungal cells expressing amylase also can be the fermenting microorganism, e.g., an ethanologenic microorganism. Ethanol production thus can be carried out using a fungal cell that expresses sufficient amylase so that less or no enzyme has to be added exogenously. The fungal host cell ca be from an appropriately engineered fungal strain. Fungal host cells that express and secrete other enzymes, in addition to amylase, also can be used. Such cells may express glucoamylase and/or a pu!lulanase, phytase, alpha-glucosidase, isoamylase, beta-amylase ce!lulase, xy!anase, other hemicellulases, protease, beta-glucosidase, pectinase, esterase, redox enzymes, transferase, or other enzymes.

4,6. Post fermentation and the products from post fermentation

[00110] Fermentation products, such as ethanol, are produced by first degrading starch- containing material into fermentable sugars by liquefaction and saccharification, or liquefaction followed by SSF, or saccharification followed by fermentation (raw starch process), and converting the sugars directly or indirectly into the desired fermentation product using a fermenting organism. Liquid fermentation products such as ethanol are recovered from the fermented mash (often referred to as "beer" or "beer mash"), e.g., by distillation, which separates the desired fermentation product from other liquids and/or solids. The remaining faction, referred to as "whole stillage", is separated into a solid and a liquid phase, e.g., by centrifugation. The solid phase is referred to as "wet cake" (or "wet grains" or "WDG") and the liquid phase (supernatant) is referred to as "thin stillage". Wet cake is dried to provide "Distillers Dried Grains" (DDG) used as nutrient in animal feed. Thin stillage is typically evaporated to provide condensate and syrup (or "thick stillage") or may alternatively be recycled directly to the slurry tank as "backset". Condensate may either be forwarded to a methanator before being discharged or may be recycled to the slurry tank. The syrup consisting mainly of limit dextrins and non-fermen table sugars may be blended into DDG or added to the wet cake before drying to produce DDGS (Distillers Dried Grain with Solubles). [00111] It is known to commercially use the various byproducts and residues derived from the fermentation processes like the ethanol production process. Distillers residues or byproducts, as well as by-products of cereal and other food industry' manufacturing, are known to have a certain value as sources of protein and energy for animal feed. Furthermore, the oil from the by-products like Whole Stillage, Wet Cake, Thin Stillage, DDG and/or DDGS can be recovered as a separate by-product for use in biodiesel production or other products

[00112] The by-products like DDG, DDGS or WDG comprises proteins, fibers, fat and unconverted starch. The Wet-Cake may be used in dairy feedlots. The dried DDGs may be used in livestock, e,g, dairy, beef and swine feeds and poultry feeds. While the protein content is high, the amino acid composition is not well suited for monogastric animals if used as animal feed Furthermore, the by-products contain significant levels of Crude Fibers (CF), which are structural carbohydrates consisting of cellulose, hemicellulose and indigestible materials like lignin. The proportion of cellulose and lignin in the crude fibers fraction also determines the digestibility of crude fibers and its solubility in the intestine. The soluble non- starch-polysaccharides (NSP) cannot be digested by monogastric animals like swine and poultry and can cause an increase in viscosity, due to their ability to bind water, which can result in moist, sticky droppings and wet litter. Another effect of NSP is the so-called "Nutrient Encapsulation". Essentially, the starch, protein, oil and other nutrients are encapsulated within the plant cell which is an impermeable barrier preventing full utilization of the nutrients within the cell .

[00113] Furthermore, the soluble NSP can cause an increase in viscosity during fermentation and can influence separation and drying conditions of fermentation by-products like DDGS in the production process.

[00114] Therefore, a number of specific processes or treatment methods have been used and are being investigated for improving the quality of the by-products from fermentation processes. For example, adding enzymes to the liquefaction, saccharification, fermentation or SSF, whole stillage, w'et-cake, and/or thin stillage, etc, m the ethanol production process has been used to improve the solid-liquid separation in the process, and/or to alter or improve the yield and/or quality of the by-products. In addition, the enzymes are also being used or investigated as a route to access residual starch, and in some cases, to access the cellulosic and/or hemicellulosic sugars associated with the corn fiber. These sugars can then be utilized by appropriate hosts to produce fermentation products, including ethanol. The present amylases may be used in these processes, as well as other starch degrading enzymes, such as alpha-amylase that is other than the alpha-amylase described in this invention, glucoamylase, hexokinase, xylanase, glucose isomerase, xylose isomerase, phosphatase, phytase, pullulanase, beta-amylase, protease, cellulase, hemicellulase, lipase, cutinase, isoamylase, redox enzyme, esterase, transferase, pectinase, a-glucosidase, beta-glucosidase, or a combination thereof, even hemicellulases, ce!lulases. The enzymes can be added at any step in the process.

5, Compositions Comprising Alpha-Amylases

[00115] In some embodiments, a polypeptide comprising an amino acid sequence that is at least about 90%, at least about 95%, identical to that of SEQ ID NO: 3 can be used in the enzyme composition.

[00116] Alpha-amylases (EC 3.2.1.1) may be combined with a glucoamylase (EC 3.2, 1.3), e.g., a Trichoderma glucoamylase or variant thereof. An exemplary glucoamylase is Trichoderma reesei glucoamylase (TrGA) and variants thereof that possess superior specific activity and thermal stability. See U.S. Published Applications Nos. 2006/0094080, 2007/0004018, and 2007/0015266 (Danisco US Inc.). Suitable variants of TrGA include those with glucoamylase activity and at least 80%, at least 90%, or at least 95% sequence identity to wild-type TrGA. Alpha-amylases advantageously increase the yield of glucose produced in a saccharification process catalyzed by TrGA.

[00117] Alternatively, the glucoamylase may be another glucoamylase derived from plants (including algae), fungi, or bacteria. For example, the g!ucoamylases may be Aspergillus niger Gl or G2 glucoamylase or its variants (e.g , Boel et al. (1984) EMBO J. 3: 1097-1102; WO 92/00381; WO 00/04136 (Novo Nordisk A/S)); and A. awamori glucoamylase (e.g., WO 84/02921 (Cetus Corp.)). Other contemplated Aspergillus glucoamylase include variants with enhanced thermal stability, e.g., 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 ); A246C (Fierobe et al. (1996) Biochemistry , 35: 8698-8704); and variants with Pro residues in positions A435 and S436 (Li et al. (1997) Protein Eng. 10:1199- 1204). Other contemplated glucoamylases include Talaromyces glucoamylases, in particular derived from T enter sonii (e.g., WO 99/28448 (Novo Nordisk A'S), T. leycettanus (e.g., U.S. Patent No. RE 32,153 (CPC Internationa], Inc.)), T. duponti, or T. thermophilus (e.g, U.S. Patent No. 4,587,215). Contemplated bacterial glucoamylases include glucoamylases from the genus Clostridium , in particular C. thermoamylolyticum (e.g., EP 135138 (CPC International, Inc.) and C. thermohydrosulfur icum (e.g, WO 86/01831 (Michigan Biotechnology Institute)). Suitable glucoamylases include the glucoamylases derived from Aspergillus oryzae , such as a glucoamylase shown in SEQ ID NO:2 in WO 00/04136 (Novo Nordisk A/S). Also suitable are commercial glucoamylases, such as AMG 200L; AMG 300 L; SAN™ SUPER and AMG™ E (Novozymes); OPTIDEX® 300 and OPTIDEX L-400 (Damsco US Inc.); AMIGASE™ and AM1GASE™ PLUS (DSM); G-ZYME® G900 (Enzyme Bio-Systems); and G-ZYME® G990 ZR (A. nige r glucoamylase with a low protease content). Still other suitable glucoamylases include Aspergillus furnigaius glucoamylase, Talar omyces glucoamylase, Thielavia glucoamylase, Trametes glucoamylase, Thermomyces glucoamylase, Athelia glucoamylase, Pycnoporus glucoamylase, Penici!lim glucoamylases or Humicola glucoamylase (e.g., HgGA). Glucoamylases typically are added in an amount of about 0 1 - 2 glucoamylase units (GAU)/g ds, e.g., about 0.16 GAU/g ds, 0.23 GAU/g ds, or 0.33 GAU/g ds.

[00118] Other suitable enzymes that ca be used with amylase include a phytase, protease, pullulanase, beta- amylase, isoamylase, a different alpha-amylase, aipha-giucosidase, ceilulase, xylanase, other hemicellulases, beta-glucosidase, transferase, pectinase, lipase, cutinase, esterase, redox enzymes, or a combination thereof. For example, a debranching enzyme, such as an isoamylase (EC 3.2.1.68), may be added in effective amounts well known to the person skilled in the art. A pullulanase (EC 3.2, 1.41 ), e.g., PROMOZYME®, is also suitable. Further suitable enzymes include proteases, such as fungal and bacterial proteases. Fungal proteases include those obtained fro Aspergillus , such as A. niger, A. aw amor i, A. oryzae ; Mucor ( e.g., M . miehei ); Rhizopus and Trichoderma.

[00119] Beta-Amylases (EC 3.2.1.2) are exo-acting maltogemc amylases, which catalyze the hydrolysis of 1,4-alpha-glucosidic linkages into amylopectin and related glucose polymers, thereby releasing maltose. Beta-Amylases have been isolated from various plants and microorganisms. See Fogarty et al. (1979) in PROGRESS IN INDUSTRIAL MICROBIOLOGY, Vol. 15, pp. 1 12-115. These beta- Amylases have optimum temperatures in the range from 40°C to 65°C and optimum pFi in the range from about 4.5 to about 7.0. Contemplated beta-amylases include, but are not limited to, beta- amylases from barley SPEZYME® BBA 1500, SPEZYME® DBA, OPT!MALT™ ME, OPTIMALT™ BBA (Damsco US Inc.); and NOVOZYM™ WBA (Novozymes A/S).

[00120] Compositions comprising the present amylases may be aqueous or non-aqueous formulations, granules, powders, gels, slurries, pastes, etc., which may further comprise any one or more of the additional enzymes listed, herein, along with buffers, salts, preservatives, water, co-solvents, surfactants, and the like. [00121] All references cited herein are herein incorporated by reference in their entirety for all purposes. In order to further illustrate the compositions and methods, and advantages thereof, the following specific examples are given with the understanding that they are illustrative rather than limiting.

[00122] The protein sequence of a fungal alpha-amylase designated AspAmyl4 was identified from an Aspergillus sp. strain based on sequence homology. A synthetic gene encoding AspAmy l4 was ordered as a codon-optimized gene for expression in Trichoderma reesei. The codon-optimized synthetic gene encoding AspAmyT4 is set forth as SEQ ID NO: 1 :

ATGAAGTGGACCGTCTCTCTCTTCCCTTTGCTGTCCTTGTTCGGTCAGACAGCCCAT

GCCCTCACCCCAGCACAATGGCGCAGCCAGTCAATC I ACTTCCT GAT GACCGACCG

CTTCGGTCGAACGGACAATTCTACAACTGCCGCCTGCAACACTGCTGACAGAGTTT

GTACTTCGATAACGGCACTCGGGTGCATGTACTGATGTGTGCAGGTATACTGCGGT

GGTAGCTGGCAGGGGATCATCAATCATGTATGAGTGGATTATGATGGATATTCTCT

GTTTGATACTAACGCCACCAGCTCGATTACATCCAAGGAATGGGATTCACTGCCAT

CTGGATCACCCCAGTCACAGAGCAGTTCTATGAAGACACCGGCGACGGCACCTCC

TACCATGGGTACTGGCAGCAGAACATGTAGGCATTCGTCCTCGTTTCGTGTTCGGT

GCTAATGCATGCAGCTACAATGTCAATTCCAACTACGGAACGGCGCAAGACCTCA

AGAATCTCGCCAGTGCGTTGCACGCGCGCGGCATGCACCTGATGGTCGATGTGGTT

GCCAACCACATGGTAAGCTGTCTCTTCATGGAAATATAATAGAAACGAACTGAAC

TGGCGTAGGGCTACGACGGAGCCGGAAACTCCGTCGACTACGGCGTTTTCGATCC

GTTTTCCTCTTCGAGCTACTTCCACCCATACTGTCTCATCTCCGACTACAACAACCA

GACCAACGTCGAAGACTGCTGGCTCGGAGATACCACTGTTTCGTTACCTGATCTTG

ACACGACAAGCACAGACGTACGAAATATCTGGTACGACTGGGTTGAGGAACTGGT

TGCCAACTATTCCAGTCAGTAGCCCGCATCATATGAGTAGGGGGCGTACTGACAG

CCATAGTCGATGGCCTGCGGGTCGACACGGTAAAACATGTTGAGAAGGACTTTTG

GCCCGGCTACAACAGCGCAGCAGGCGTCTACTGTGTCGGTGAGGTGTTCTCGGGC

GATCCGGCATACACATGTCCATACCAGAACTACATGGACGGTGTGCTCAACTACC

CAATGTGAACATGCCTACCTTCCAGAAAACCCCAGAGGCTGACACACCGCAGCTA CTACCAACTCCTCTATGCGTTCGAGTCAACCAGCGGCAGCATGAGCAACCTGTACA

ACATGATCAACTCGGTTGCCTCCGACTGCAAGGATCCCACCCTACTGGGCAACTTT

ATCGAGAACCACGACAACCCGCGCTTTGCTTCGTAAGTCTTTCTTCCTCTATTCGTG

CAGTCCATGCTAAATCCCGCAGCTACACGAGTGACTACTCGCAAGCGAAGAATGT

GATCTCGTTTATCTTCCTCACCGATGGCATCCCCATCGTCTACGCCGGACAGGAAC

AGCACTACAGCGGCGGCAGCGACCCAGCCAACCGCGAGGCCACCTGGCTATCCGC

ATACTCAACCGGCGCCACGCTGTACACCTGGATCGCGTCGACAAACAAGATCCGC

AAGCTGGCGATATCCAAGGACACGGGATACGTGGAGGCCAAGGTATGCGCACACC

CCCGGCTCTGTAGCTCACGCTAACGCGGACAGAACAACCCCTTCTACTACGACTCC

AATACGATCGCCATGCGCAAGGGAACCACCGCCGGTGCGCAGGTCATCACCGTCT

TGAGCAACAAGGGCGCGTCGGGTAGCTCCTACACCCTCTCCTTGAGCGGTACGGG

CTACGCCGCCGGCGCGACCCTGGTCGAGATGTACACCTGCACCACGGCCACTGTA

GACTCAAGCGGCAACCTCCCGGTTCTAATGACATCCGGTTTGCCCAGAGTGTTTCT

ACCGTCGTCTTGGGTAAGTGGCAGCGGTCTTTGCGGCTCCGCTGTCTCTACTACAC

TCACGACAGTTTCCACTACGCTCACGACAGTCGCCGCGACCACGACGTCGACCAC

GACATCGACCACGACATCGACCACGACATCGACCACGACATCGACCACGACATCG

ACCACGACATCGACCACGACATCGACAACATGCACGGCCGCCACAGCCCTTCCCA

TTCTCTTCGAGGAACTCGTCACGACAACCTACGGAGAGAACATCTTCCTGACCGGC

TCGATCAGCCAACTGGGCAGCTGGAACACCGCCTCGGCCGTTGCCTTGTCGGCGA

GTAAGTACACCGCTTCCAAGCCGGAATGGTACGTGACCGTGACCTTGCCCGTGGG

CACCACG FTCCAGT AC A AG GTTA FC AAGAAAGAGGCGGACGGGAGTGT GGCGTGG

GAGAGTGATCCGAACCGATCGTACACGGTTCCGAGTGGCTGTGCGGGTGCGACAG

T G AC GGTT GTT GAT ACTT GG AGGT G A

|00123] The amino acid sequence of the AspAmyM precursor protein is set forth as SEQ ID NO: 2. The native signal peptide is shown in italics and underline.

AiATF/ SZE ZZA FGGlAgALTPAOWRSOSrYFLMTDRFGRTDNSTTAACNTADRVYC

GGSWQGIINHLDYIQGMGFTAIWITPVTEQFYEDTGDGTSYHGYWQQNIYNVNSNYG

TAQDLKNLASALHARGMHLMVDVVANHMGYDGAGNSVDYGVFDPFSSSSYFHPYC

LI SDYNNQTNVEDCWLGDTTVSLPDLDTTSTDVRNIWYDWVEELV ANY SIDGLRVDT VKHVEKDFWPGYNSAAGVYCVGEVFSGDPAYTCPYQNYMDGVLNYPIYYQLLYAFE STSGSMSNLYNMINSVASDCKDPTLLGNFIENHDNPRFASYTSDYSQAKNVISFIFLTD

GIPIVYAGQEQHYSGGSDPANREATWLSAYSTGATLYTWIASTNKIRKLAISKDTGY V EAKNNPFYYDSNTIAMRKGTTAGAQVITVLSNKGASGSSYTLSLSGTGYAAGATLVE

MYTCTTATVDSSGNLPVLMTSGLPRVFLPSSAWSGSGLCGSAVSTTLTTVSTTLTTV A

ATTTSTTTSTTTSTTTSTTTSTTTSTTTSTTTSTTCTAATALPILFEELVTTTYGEN IFLTG

SISQLGSWNTASAVALSASKYTASKPEWYVTVTLPVGTTFQYKFIKKEADGSVAWES

D PNR S YTVP S GC AG ATVTV VDTWR

[00124] The amino acid sequence of the mature form of AspAmy i4 confirmed by LC MS/MS

is set forth as SEQ ID NO: 3;

LTPAQWRSQS1YFLMTDRFGRTDNSTTAACNTADRVYCGGSWQG11NHLDYIQGMGF

TAIWITPVTEQFYEDTGDGTSYHGYWQQNIYNVNSNYGTAQDLKNLASALHARGMH

LMVDVVANHMGYDGAGNSVDYGVFDPFSSSSYFHPYCLISDYNNQTNVEDCWLGDT

TVSLPDLDTTSTDVRNIWYDWVEELVANYSIDGLRVDTVKHVEKDFWPGYNSAAGV

YCVGEVFSGDPAYTCPYQNYMDGVLNYPIYYQLLYAFESTSGSMSNLYNMINSVASD

CKDPTLLGNFIENHDNPRFASYTSDYSQAKNVISFIFLTDGIPIVYAGQEQHYSGGS DPA

NREATWLSAYSTGATLYTWIASTNK1RKLA1SKDTGYVEAKNNPFYYDSNTIAMRKG T

TAGAQVITVLSNKGASGSSYTLSLSGTGYAAGATLVEMYTCTTATVDSSGNLPVLMT

SGLPRVFLPSSWVSGSGLCGSAVSTTLTTVSTTLTTVAATTTSTTTSTTTSTTTSTT TSTT

TSTTTSTTTSTTCTAATALPILFEELVTTTYGENIFLTGSISQLGSWNTASAVALSA SKYT

ASKPEWYVTVTLPVGTTFQYKFIKKEADGSVAWESDPNRSYTVPSGCAGATVTVVDT

Expression of Aspergillus sp, alpha-amylase (AspAmyl4)

[00125] The DNA sequence of Asp Amy 14 was optimized for expression of AspAmyl4 m Trichoderma reesei and inserted into the pGXT expression vector (the same as the pTTTpyr2 vector described in published PCT Application WO2015/017256), resulting in pZKY258 (Figure 1).

[00126] The plasmid pZKY258 was transformed into a suitable Trichoderma reesei strain (method described in published PCT application WO 05/001036) using protoplast transformation (Te’o et al. (2002) I. Microbiol. Methods 51:393-99). Transformants were selected on a solid medium containing acetamide as the sole source of nitrogen. After 5 days of growth on acetamide plates, transformants were collected and subjected to fermentation in 250 mL shake flasks in defined media containing a mixture of glucose and sophorose.

EXAMPLE 3

Purification of AspAmyl4

[00127] The crude sample of AspAmyl 4 from fermentation was concentrated and ammonium sulfate was added into the concentrated sample to the final concentration of 1 M The solution was then loaded onto a 20 mL HiPrepTM Phenyl FF 16/10 column pre- equilibrated with 20 mM sodium acetate (pH 5.0) supplemented with 1 M (NH LSCfi. Elution was performed using 6 column volumes of 0.75 Vi ammonium sulfate. Fractions were collected and run on SDS-PAGE. The fractions containing the target protein were pooled, concentrated, and exchanged with the buffer to 20 mM NaH 2 PO4 (pH 7.0). The solution was then loaded into a 20 ml HiPrepTM Q FF 16/10 column pre-equilibrated with 20 mM NaPlbPCL (pH 7.0). Elution was performed using 6 column volumes of 0.3 NaCl. Fractions were collected and run on SDS-PAGE. The fractions containing the target protein were pooled, concentrated, and exchanged with the buffer to 20 mM sodium acetate pPT 5.0 using an Arnicon Ultra- 15 device with 10 K MWCO The purified sample is around 90% pure and stored in 40% glycerol at -20 °C until usage.

EXAMPLE 4

Potato amylopectin-hydrolyzing activity of AspAmyl4

[00128] Alpha- amylase activity was determined using a colorimetric assay to monitor the release of reducing sugars from potato amylopectin. The activity is reported as equivalents of glucose released per minute. Substrate solutions were prepared by mixing 9 mL of 1% (w/w, in water) potato amylopectin (Sigma, Cat. No. 10118), 1 mL of 0.5 M buffer (pH 5.0 sodium acetate or pH 8.0 HEPES), and 40 mE of 0.5 M CaCh into a 15-mL conical tube. Stock solution of purified alpha-amylase sample was made by diluting original sample to 20 ppm in water. Serial dilutions of enzyme sample and glucose standard were prepared in water in non- binding microtiter plates (MTP, Coming 3641). Then 90 pL of substrate solution (preincubated at 50 °C for 5 min at 600 rpm) and 10 pL of the enzyme serial dilution were added and mixed into in non-bindmg microtiter plates (MTP, Coming 3641). All the incubations were carried out at 50 °C for 10 min at 600 rpm in a thermomixer (Eppendorf). After incubation, 50 pL of 0.5 N NaOH were added to each well to stop the reaction. Total reducing sugars present in each well were measured using a PAHBAH method: 80 pL of 0.5 N NaOH was aliquoted into a microtiter plate, followed by the addition of 20 pL of PAHBAH reagent [5% w/v 4- hydroxybenzoic acid hydrazide in 0.5 N HC1] and 10 pL of each reaction mixture. Plates were incubated at 95 °C for 5 min and cooled down at 4 °C for 5 sec. Samples (80 pL) were then transferred to polystyrene microtiter plates (Costar 9017) and absorbance was read at 410 nm. Resulting absorbance values w¾re plotted against enzyme concentration and linear regression w¾s used to determine the slope of the linear region of the plot. Using the method mentioned above, specific activity of AspAmyM w¾s determined and compared with a reference fungal alpha-amylase, AcAA (described in U.S. Patent No. 8,945,889). Results for both enzymes are shown in Table 2.

Specific Activity (U/rag) = Slope (enzyme) / slope (std) * 100

Define: 1 11 = 1 pmol glucose equivalent/min

Table 2. Specific activity of AspAmyl4 and AcAA on potato amylopectin

[QQ129] The effect of pH (from 3.0 to 10.0) on AspAmyM activity was monitored using the PAHBAH assay protocol as described in Example 4. Buffer working solutions consisted of the combination of glycine/sodium acetate/HEPES (250 mM), with pH varying from 3.0 to 10.0. Substrate solutions were prepared by mixing 896 pL of 1% (w/w, in water) potato amylopectin (Sigma, Cat. No. 10118), 100 pL of 250 mM buffer working solution (pH from 3 0 to 10.0), and 4 pL of 0 5 M CaCh. Enzyme working solution was prepared in water at a certain dose (showing signal within linear range as per dose response curve). All the incubations were carried out at 50 °C for 10 min following the same protocol as described above for specific activity of AspAm\T4. The absorbance from a control (water-only) was subtracted, and the resulting values were converted to percentages of relative activity, by defining the activity at the optimal pH as 100%. As shown in Table 3, AspAmyM showed a similar pH profile to AcAA. with an optimum pH at 4.0 The pH range, within which the enzyme retained greater than 70% of maximum activity, was from pH 3.3 to 6.4.

Table 3. pH profile of Asp Amy 14 and AcAA

[00130] The effect of temperature (from 40 to 90°C) on alpha-amylase activity was monitored using the PAHBAH assay protocol as described in Example 4. Substrate solutions were prepared by mixing 3.6 mL of 1% (w/w, m water) potato amylopectin (Sigma, Cat. No. 10118), 0.4 mL of 0.5 M pH 5.0 sodium acetate buffer, and 16 m L of 0.5 M CaCL into a 15- mL conical tube. Enzyme working solution was prepared in water at 2.5 ppm. Prior to the reaction, 90 pi. of substrate solution was added in PCR plates (Axygen, PCR-96-HS-C) and incubated in Peltier Thermal Cyclers (BioRad) at desired temperatures (i.e. 40 to 90 °C) for 5 min. Then 10 pL of diluted enzyme was added to the substrate to initiate the reaction. Following 10 min incubation in the PCR machines, reactions were quenched and measured using the same protocol as described above for specific activity of Asp Amy 14. The absorbance from a control (water-only) was subtracted, and the resulting values were converted to percentages of relative acti vity, by defining the acti vity at the optimal temperature as 100%. As shown in Table 4. Asp Amy 14 showed an optimum temperature at 70 °C and retained greater than 70% of maximum activity between 54 °C and 75°C, whereas AcAA exhibited an optimum temperature of 63 °C arid maintained greater than 70% of maximum activity between 49 °C and 71 °C.

Table 4. Temperature profile of AspAmyM and AcAA

[00131] Thermostability of alpha-amylase AspAmyl4 was determined by measuring the enzyme activity before and after enzyme samples pre-incubated at temperatures from 40 to 90 °C for 2 h. Enzyme was diluted in 50 rnM of sodium acetate buffer (pH 5.0) containing 2 mM of CaCb to 10 ppm and 50 pL was aliquot to PCR strip tubes. The tubes were transferred to PCR machines at the desired temperature from 40 to 90 °C. After 2 h-preineubation, the enzymes were diluted to 2.5 ppm in water and assayed for their residual activities using the amylopectin/PAHBAH method as described in Example 4 The residual activities were converted to percentages of relative activity, by defining the activity of the sample kept on ice as 100%. The thermostability' was defined as the temperature at winch the sample remained 50% of activity. As shown in Table 5, AspAmy14 retained greater than 60% of initial activity after 2 h incubation at 60°C, whereas AcAA only maintained 5% residual activity under the same incubation conditions. Thermostability of Asp Amy 14 and AcAA

[QQ132] SSF is typically conducted at pH 3.8-4.8, 32 °C for 55 hours, and the enzymes used m the process should be able to maintain its activity under this condition during the whole process. Thus, it is very' useful to know the low' pH stability of the enzymes. Hie pH stability is evaluated by measuring the residual enzyme activity after preincubating the enzyme at pH 3.7 and pH 4.5, respectively, for a determined length of time. Residual enzyme activity' is assayed using the amyiopectin/PAHBAH method as described in Example 4. Enzyme stock solutions were prepared by diluting the samples to 400 ppm in water and stored at 4 °C. At each time point, 97.5 pL of dilution buffer (50 mM sodium acetate buffer, pH 3.7 or 4.5 with 2 mM CaC ) was added to PCR strip tubes, followed by adding 2,5 pL of enzyme stock solution (400 ppm) and mixing well. After incubation at 32 °C for different lime points, the enzymes were further diluted to 2.5 ppm in water and assayed for their residual activities. The residual activity' was converted to percentage of relative activity', by defining the activity' without preincubation at pH 3.7 or 4 5 as 100%. As shown in Table 6, AspAmy M showed greater pH stability than AcAA. After 24 hours at pH 3.7, AspAmy · retained almost 100% original activity ' , while AcAA only retained 41%. At pH 4.5 and 48 hours incubation, AspAmyM retained almost 100% original activity', while AcAA retained 81%.

Table 6, pH stability' of Asp Amy M and AcAA

40

|00133] The goal of starch solubilization assay is to evaluate the enzyme capability of removing insoluble residual starch by measuring the remaining insoluble starch at the end of the assay. This is performed by determining the optical density (QD) of each well at 260 nm. The large starch granules will scatter the light passing through the well; therefore the higher concentration of insoluble starch, the higher the OD. The substrate for solubilization assay was a blend of amyloge! (70% amylose content Hylon VII) and insoluble com starch (Sigma- aldrich, Batch #: 129K0076) which mimics the real substrate used in SSF. It was prepared by repeat washing corn starch and amylogel with water for 10 times via successive centrifugation/decanting, and then suspended in 100 mM sodium acetate buffer (pH 3.7 and pH 4.5, respectively) at 30% (w/w). Equal proportions of corn starch and amylogel slum' were mixed and diluted 25-fold in 100 mM sodium acetate (pH 3.7 and pH 4.5, respectively), it was then autoclaved for 60 minutes at 121 °C with a stir bar. As the mixture cooled, it was stirred overnight on a stir plate to prevent gelling. After that, the substrate was stored at 4 °C and ready for use. The solubilization substrate (150 pL) was mixed well (stirring) while it w¾s added to the UV/'vis plate (MTP, Coming 3635). Wide bore tips were required for transferring substrate. The enzyme solution (10 pL) was added to each well with final concentration from 0 to 12.5 ppm. The plate was then incubated for 24 hours at 32 °C while shaking at 250 rpm. After 24 hours, the plate w¾s briefly mixed to make sure that the particles w¾re suspended. Then the plate was read at 260 nm. As shown in Figure 2, AspAmyM showed better performance compared to AcAA in terms of insoluble starch removal at both pH 3.7 and 4.5, especially at the lower enzyme dosages. [00134] Asp Amy 14 and AcAA were evaluated for their performance under conditions (at pH 4.4, 32□ C) meant to represent industrial simultaneous sacharifi cation and fermentation (SSF) conditions. Com liquefact (34.85% dry solids) was stored at -20 °C until thawed for use. H2SO4 was added to adjust the pH to 4.4 and solid urea was added to 600 ppm. Dry yeast (Ethanol Red, Lesaffre Advanced Fermentations, France, #42138) was hydrated by adding 1 g to 4 ml water and incubated for 10 min before resuspension and addition to the liquefact at a dilution of 1:200. The liquefact (0.4 ml) was added to each well of a 96- deepwell microtiter plate containing a protease (Fermgen, Dupont, 0.124 SAPU/'g dry' solids), a T. reesei giucoamylase variant (3.5 pg/g dry solid) and an alpha-amylase (0-36 pg/g dry solids). The plate was sealed to allow' gas to escape but not to enter the wells, and placed in a forced-air incubator at 32 DC shaking at 300 rpm.

[QQ135] The reaction was stopped at 47 or 69 hrs by addition of 0.4 ml. 0.02N H2SO4 with shaking, followed by centrifugation and collection of the supernatant which was filtered through 0.2 micron membranes. Ethanol content w¾s determined by HPLC using a Rezex RFQ-Fast Acid column (Phenomenex) with a 0.01N IT2SO4 mobile phase. The ethanol produced during fermentation is given in table 7.

[00136] SAPU: One spectrophotometric acid protease unit (SAPU) is the enzyme activity that will liberate lpmol of tyrosine per minute under the conditions specified (pH 3.0 and 37°C). The assay is based on the enzymatic hydrolysis of a casein substrate in which the solubilized casein filtrate is determined spectrophotometrically.

[00137] Under the conditions of this assays, AspAmyl4 performs better than AcAA under most concentrations, specially the higher concentrations.

Table 7, SSF performance of AspAmyM and AcAA

EXAMPLE 11

Protein sequence analyses of mature alpha-amylase AspAmyM

[00138] Related proteins were identified by a BLAST search (Altschul et al., Nucleic Acids Res, 25:3389-402, 1997) using the mature amino acid sequences for AspAmyM (SEQ ID NO: 3 ) against Public and Genome Quest Patent databases with search parameters set to default values and a subset are shown on Tables 8A and 8B. Percent identity (PID) for both search sets is defined as the number of identical residues divided by the number of aligned residues in the pairwise alignment. Value labeled“Sequence length” on tables corresponds to the length (in ammo acids) for the proteins referenced with the listed Accession numbers, while“Aligned length” refers to sequence used for alignment and PID calculation.

Table 8 A. List of sequences with percent identity to AspAmy M mature sequence identified from the NCBI non-redundant protein database

Table SB. List of sequences with percent identity to AspAmyM mature sequence identified from Genome Quest database

[00139] An alignment of the mature sequences of AspAmyM (SEQ ID NO:3); XP 001209405.1 (aa 21-607 of SEQ ID NO:4); EDP53736.1(aa 24-630 of SEQ ID NO:5); XP_001265628.1 (aa 24-632 of SEQ ID NO:6); OXN35790.1 (aa 30-631 of SEQ ID NO: 7 ): 0X803711 1 (aa 22-633 of SEQ ID NO: 8); US20150337277-0004 (aa 22-643 of SEQ ID NO:9); and US20150337277-0006 (aa 22-628 of SEQ ID NO: 10) was performed with default parameters using the MUSCLE program from Geneious software (Biomatters Ltd.) (Robert C. Edgar MUSCLE: multiple sequence alignment with high accuracy and high throughput Nucl. Acids Res. (2004) 32 (5): 1792-1797). The multiple sequence alignment of mature AspAmyM alpha amylase and various other homologous sequences is shown on Figure 3.