BR112020009525A2 - alpha-amylase, composition and method - Google Patents

Abstract

The present description relates to polypeptides that have alpha-amylase activity and compositions that comprise such polypeptides. In addition, the description also refers to methods for producing recombinantly, such polypeptides or such compositions, as well as methods for using or applying the polypeptides or compositions so produced in industrial environments.

Description

Descriptive report of the invention patent for “ALPHA-AMYLASE, COMPOSITION AND METHODS”.

FIELD OF THE INVENTION

[0001] The present invention relates to polypeptides that have alpha-amylase activity and compositions that comprise such polypeptides. The present invention also relates to polynucleotides that encode such polypeptides, genetically modified nucleic acid constructs, vectors and host cells that comprise genes that encode such polypeptides, which may also enable the production of such polypeptides. In addition, the description refers to methods for recombinantly producing such polypeptides or such compositions, as well as methods for using or applying the polypeptides or compositions so produced in industrial environments, for example, for treating starch, such as liquefaction, saccharification and / or fermentation or preparation of drinks.

BACKGROUND

[0002] Starch consists of a mixture of amylose (15 to 30% w / w) and amylopectin (70 to 85% w / w). Amylose consists of linear chains of alpha-1,4-linked glucose units that have a molecular weight (MW) of about 60,000 to about 800,000. Amylopectin is a branched polymer containing alpha-1.6 branch points every 24 to 30 units of glucose; your PM can be as high as 100 million.

[0003] Alpha-amylases hydrolyze starch, glycogen and related polysaccharides by randomly cleaving internal alpha-1,4-glucoside bonds. Alpha-amylase enzymes have been used for a variety of different purposes, such as liquefaction of starch, saccharification, fermentation, brewing, baking, degumming of textile products, washing of textile products, modification of starch in the pulp and paper industry and increased digestibility in animal feed.

[0004] Depending on industrial applications, the alpha-amylases suitable for these industrial processes can be diverse. Thus, there is a need in the technique of alternative alpha-amylases with different or improved properties, such as ideal pH, ideal temperature, substrate specificities and / or thermostability.

[0005] It is an object of the present invention to provide certain polypeptides that have alpha-amylase activity, polynucleotides that encode polypeptides, nucleic acid constructs that can be used to produce such polypeptides, compositions that comprise them, as well as methods to produce and use such polypeptides.

SUMMARY

[0006] The present polypeptides, compositions and methods are for use or application of the polypeptides or compositions. Aspects and modalities of the polypeptides, compositions and methods are described in the paragraphs independently numbered below.

1. In one aspect, a polypeptide that has alpha-amylase activity selected from the group consisting of: (a) a polypeptide that comprises an amino acid sequence that preferably has at least 90% identity with the polypeptide of SEQ ID NO: 3; (b) a polypeptide comprising an amino acid sequence that preferably has at least 90% identity to a catalytic domain of SEQ ID NO: 3; (c) a polypeptide comprising an amino acid sequence that preferably has at least 90% identity with 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 to high stringency conditions, most preferably , at least high stringency and, even more 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 sequence of encoding mature polypeptide of SEQ ID NO: 1 or (iii) a full length complementary strand of (i) or (ii); (e) a polypeptide encoded by a polynucleotide that comprises a nucleotide sequence that preferably has 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 (for example, several) amino acids of the polypeptide of SEQ ID NO: 3; (g) a mature polypeptide produced by processing the polypeptide of SEQ ID NO: 2 by a post-translational signaling or modification peptidase during secretion from an expression host; and (h) a fragment of a polypeptide from (a), (b), (c), (d), (e), (f) or (g) that has alpha-amylase activity.

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

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

4. In another aspect, a recombinant host cell that comprises the polynucleotide of paragraph 2.

5. In some embodiments, the host cell in paragraph 4 which is an ethanological microorganism.

6. In some embodiments, 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, peridrolase, cutinase, pectinase, pectate lyase, mannanase, keratinase, reductase, oxidase, phenoloxidase, lipoxygenase, ligninase, glycoamylase, pululanase, phytase, tanase, pentosanase, malanase, beta-glucanase, lacrosone, hondalidase, hialasidase, hial or a combination of them.

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

8. In some embodiments, the composition of paragraph 7 which further comprises a protease, hemicellulase, cellulase, peroxidase, lipolytic enzyme, xylanase, lipase, phospholipase, esterase, peridrolase, cutinase, pectinase, lyase pectate, mannase, keratinase, reductase , oxidase, phenoloxidase, lipoxygenase, ligninase, glycoamylase, pullulanase, phytase, tanase, pentosanase, malanase, beta-glucanase, arabinosidase, hyaluronidase, chondroitinase, laccase, transferase or a combination thereof.

9. In another aspect, a method for producing a polypeptide that has alpha-amylase activity comprising: (a) cultivating the host cell of paragraph 4, under conditions conducive to the production of the polypeptide; and (b) recovering the polypeptide.

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

11. In another aspect, a method for saccharifying a starch substrate which comprises bringing the starch substrate into contact with the polypeptide that has the alpha-amylase activity of paragraph 1; and saccharify the starch substrate to produce the saccharides that comprise glucose.

12. In some embodiments of the method in paragraph 11, in which saccharifying the starch substrate results in a syrup with a high glucose content.

13. In some modalities of the method of paragraph 11 or 12, in which the syrup with a high glucose content 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, the method of any of paragraphs 10 to 12 which further comprises fermenting syrup with a high glucose content in a final product.

15. In some embodiments, the method of paragraph 14, in which saccharification and fermentation are performed as a simultaneous saccharification and fermentation (SSF) process.

16. In some embodiments, the method of paragraph 14 or 15, in which the final product is alcohol, for example, ethanol.

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

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

19. In some embodiments, the method of any of paragraphs 11 to 18, in which the starch substrate is selected from wheat, barley, corn, rye, rice, sorghum, bran, cassava, milo, millet, potato, potato sweet, tapioca and any combination of them.

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

21. In some embodiments, the method of any one of paragraphs 11 to 20 which further comprises adding a hexokinase, xylanase, glucose isomerase, xylose isomerase, phosphatase, phytase, pululanase, beta-amylase, a glycoamylase, a protease, a cellulase, a hemicellulase, a lipase, a cutinase, a trehalase, an isoamylase, a redox enzyme, an esterase, a transferase, a pectinase, a hydrolase, an alpha-glucosidase, a beta-glucosidase or a combination of them to the starch substrate.

22. In another aspect, a method for applying the method of any of paragraphs 11 to 21 for production of saccharides.

23. In another aspect, a saccharide produced by the method of paragraph 22.

24. In another aspect, a method for saccharifying and fermenting a starch substrate to produce a final product which comprises putting the starch substrate in contact with the polypeptide that has the alpha-amylase activity of paragraph 1; saccharifying the starch substrate to produce the saccharides that comprise glucose; and placing the saccharides in contact with a fermentation organism to produce a final product.

25. In some embodiments, the method in paragraph 24, where fermentation is performed as a simultaneous saccharification and fermentation (SSF) process.

26. In some embodiments, the method of paragraph 24 or 25, in which the final product is alcohol, for example, ethanol.

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

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 represents the expression vector pZKY258 that houses the synthetic alpha amylase gene AspAmy14. 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 shows results at pH 4.5 Figure 3 (Panels A, B and C) shows an alignment of multiple MUSCLE protein sequences for AspAmy14 and several homologous fungal alpha amylases described in Example 11.

DETAILED DESCRIPTION

[0007] Aspergillus polypeptides having alpha-amylase activity and compositions comprising such polypeptides are described. The present invention also relates to polynucleotides that encode such polypeptides, genetically modified nucleic acid constructs, vectors and host cells that comprise genes that encode such polypeptides, which may also enable the production of such polypeptides. In addition, the description refers to methods for recombinantly producing such polypeptides or such compositions, as well as methods for using or applying the polypeptides or compositions thus produced in industrial environments, for example, for starch liquefaction, saccharification, fermentation and preparation of food or drink. These and other aspects of the compositions and methods are described in detail, below.

[0008] Before describing the various aspects and modalities of the present compositions and methods, the following definitions and abbreviations are described.

1. Definitions and Abbreviations

[0009] According to this detailed description, the following abbreviations and definitions apply. It is observed that the singular forms "um / um" and "o / a" include plural referents, unless the context clearly determines otherwise. Thus, for example, the reference to "an enzyme" includes a plurality of such enzymes, and the reference to "the dosage" includes reference to one or more dosages and their equivalents known to those skilled in the art, and so on.

[0010] This document is organized into several sections for ease of reading; however, the reader will understand that the statements made in one section may apply to other sections. Therefore, the headings used for different sections of the disclosure should not be interpreted as limiting.

[0011] Unless otherwise defined, all technical and scientific terms used in this document have the same meaning as is commonly understood by a person 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-bis-3-ethylbenzothiazoline-6-sulfonic acid cDNA Complementary DNA DNA deoxyribonucleic acid DPn degree of polymerization of saccharide that has n ds or DS subunits dry solids GA glycoamylase GAU / g ds unit of glycoamylase activity / gram dry solids IRS insoluble residual starch kDa kiloDalton MW molecular weight NCBI National Center for Biotechnology Information PAHBAH p-hydroxybenzoic acid hydrazide PEG polyethylene glycol pI isoelectric point PI performance index ppm parts per million, for example, μg of protein per gram of dry solid RNA ribonucleic acid SDS-PAGE polyacrylamide gel electrophoresis dodecyl sodium sulfate SSF simultaneous saccharification and fermentation of SSU / g of SSU / g solid soluble starch unit / gram of dry solids sp. TrGA species glycoamylase from Trichoderma reesei w / v weight / volume w / w weight / weight v / v volume / volume% by weight percent by weight C degrees Centigrade H2O dH2O water or DI deionized water dIH2O deionized water, Milli-Q filtration g or gm grams μg micrograms mg milligrams kg kilograms μL and µl microliters mL and ml milliliters M molar mM millimolar μM micromolar U units s seconds min minute / minutes h hour / hours OF dissolved oxygen ETOH ethanol eq. equivalents N normal PDB CAZy Protein Database Active Carbohydrate Enzyme Database Tris-HCl tris (hydroxymethyl) aminomethane hydrochloride HEPES 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid mS / cm milli-Siemens / cm CV column volumes

1.2. Definitions

[0013] The terms "amylase" or "amylolytic enzyme" refer to an enzyme that has, among other things, the ability to catalyze starch degradation. Alpha-amylases are hydrolases that cleave α-D- (1 → 4) O-glycosidic bonds in starch. Alpha-amylases (EC 3.2.1.1; alpha-D- (1 → 4) -glycan-glycan-hydrolase) are generally defined as endo-acting enzymes that cleave alpha- D- (1 → 4) O- glycosides within the starch molecule in a random manner yielding polysaccharides that contain three or more D-glucose (1-4) -alpha-linked units. In contrast, exo-acting amylolytic enzymes, such as beta-amylases (EC 3.2.1.2; alpha-D- (1 → 4) -glucan maltohydrolase) and some product-specific amylases, such as maltogenic 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; alpha-D- (1 → 4) -glucan glucohydrolase) and product-specific amylases , such as maltotetraosidases (EC 3.2.1.60) and maltohexaosidases (EC

3.2.1.98), can produce malto-oligosaccharides of a specific length or syrups enriched with specific malto-oligosaccharides.

[0014] The term "starch" refers to any material comprised of complex plant polysaccharide carbohydrates, comprised of amylose and amylopectin with the formula (C6H10O5) x, where 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, corn, rye, rice, sorghum, bran, cassava, millet, milo, potatoes, sweet potatoes and tapioca. The term "starch" includes granular starch. The term "granular starch" refers to raw starch, that is, uncooked, for example, starch that has not been subjected to gelatinization or that has been subjected to temperatures at or below the gelatinization temperature of the starch.

[0015] The terms, "wild type", "original" or "reference", in relation to a polypeptide, refer to a naturally occurring polypeptide that does not include a man-made substitution, insertion or deletion in one or more amino acid positions. Similarly, the terms "wild type", "original" or "reference", in relation to a polynucleotide, refer to a naturally occurring polynucleotide that does not include a man-made nucleoside change. However, note that a polynucleotide that encodes a wild-type, parent or reference polypeptide is not limited to a naturally occurring polynucleotide and covers any polynucleotide that encodes a wild-type, parental or reference polypeptide.

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

[0017] The term "variant", in relation to a polypeptide, refers to a polypeptide that differs from a wild type, original or reference polypeptide specified by the fact that it includes one or more substitutions, insertions or deletions of a naturally occurring or man-made amino acid. Similarly, the term "variant", in relation to a polynucleotide, refers to a polynucleotide that differs in sequence from nucleotides to a specified, original or reference wild-type polynucleotide. The identity of the wild type, original or reference polypeptide or polynucleotide will be evident from the context.

[0018] In the case of the present alpha-amylases, "activity" refers to the activity of alpha-amylase, which can be measured as described in this document.

[0019] The term "recombinant", when used in reference to a cell, nucleic acid, protein or vector in question, indicates that the individual has been modified from its native state. Thus, for example, recombinant cells express genes that are not found within the cell's native (non-recombinant) form or express native genes at different levels or under different conditions than is found in nature. Recombinant nucleic acids differ from a native sequence by one or more nucleotides and / or are functionally linked to heterologous sequences, for example, a heterologous promoter in an expression vector. Recombinant proteins can differ from a native sequence by one or more amino acids and / or are fused to heterologous sequences. A vector that comprises a nucleic acid that encodes an amylase is a recombinant vector.

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

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

[0022] The terms "thermostable" and "thermostability", in reference to an enzyme, refer to the enzyme's ability to maintain activity after exposure to an elevated temperature. The thermostability of an enzyme, such as an amylase enzyme, is measured by its half-life (t1 / 2) given in minutes, hours or days, during which half of the enzyme activity is lost under defined conditions. The half-life can be calculated by measuring residual alpha-amylase activity, for example, after exposure (i.e. challenge) to an elevated temperature.

[0023] A "pH range", in 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", in reference to an enzyme, refer to the enzyme's ability to retain activity over a wide range of pH values for a predetermined period of time ( for example, 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. When such amino acid sequences exhibit activity, they can be called "enzyme". Conventional one-letter or three-letter codes for amino acid residues are used, with the amino acid sequences being presented in the terminal amino orientation for standard carboxy (ie, N → C).

[0026] The term "nucleic acid" encompasses DNA, RNA, heteroduplexes and synthetic molecules with the ability to encode a polypeptide. Nucleic acids can be single-stranded or double-stranded and can be chemically modified. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Due to the fact that the genetic code is degenerate, more than one codon can 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 shown in the 5 ′ to 3 ′ orientation.

[0027] The term "hybridization" refers to the process by which a nucleic acid strand forms a duplex, that is, base pairs, with a complementary strand, as occurs during blot hybridization techniques and PCR techniques. The stringent hybridization conditions are exemplified by hybridization under the following conditions: 65 ° C and 0.1X SSC (where 1X SSC = 0.15 M NaCl, 0.015 M Na3 citrate, pH 7.0). Hybridized duplex nucleic acids are characterized by a melting temperature (Tm), in which half of the hybridized nucleic acids are not matched to the complementary strand. Unpaired nucleotides within the duplex reduce Tm.

[0028] A "synthetic" molecule is produced by chemical or enzymatic synthesis in vitro instead of by an organism.

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

[0030] A "host strain" or "host cell" is an organism in which an expression vector, phage, virus or other DNA construct, including a polynucleotide that encodes a polypeptide of interest (for example, an amylase) , It was introduced. Exemplary host strains are microorganism cells (for example, bacteria, filamentous fungi and yeast) with the ability to express the polypeptide of interest and / or fermentation saccharides. The term "host cell" includes protoplasts created from cells.

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

[0032] The term "endogenous", in 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 sequence of nucleic acids. The process includes both transcription and translation.

[0034] A "selective marker" or "selectable marker" refers to a gene that can be expressed in a host to facilitate the selection of host cells that carry 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,

bifunctional vectors (shuttle), plasmids, phage particles, cassettes and the like.

[0036] An "expression vector" refers to a DNA construct that comprises a DNA sequence that encodes a polypeptide of interest, such a coding sequence is functionally linked to a suitable control sequence with the ability to effect expression of DNA in a suitable host. Such control sequences can include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence that encodes suitable ribosome binding sites on the mRNA, enhancers and sequences that control the termination of transcription and translation.

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

[0038] 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 removed by cleavage during the secretion process.

[0039] "Biologically active" refers to a sequence that has a specified biological activity, such as an enzymatic activity.

[0040] The term "specific activity" refers to the number of moles of the substrate that can be converted into product by an enzyme or enzyme preparation per unit of time under specific conditions. Specific activity is usually 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 cellular material may be from a heterologous host that is cultured for the purpose of producing the amylase.

[0042] "Percentage of 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 with the use of the CLUSTAL W algorithm with standard parameters. See Thompson et al. (1994) Nucleic Acids Res. 22: 4,673 to 4,680. The standard parameters for the CLUSTAL W algorithm are: Gap opening penalty: 10.0 Gap extension penalty: 0.05 Protein weight matrix: series

BLOSUM DNA weight matrix: IUB% delay divergent sequence 40 Gap separation distance: 8 weight of DNA transitions: 0.50 List hydrophilic residues: GPSNDQEKR Use negative matrix: OFF Toggle specific residue penalties: ON Toggle penalties hydrophilic: ON Toggled end gap separation penalty OFF.

[0043] Deletions are counted as non-identical residues, compared to a reference sequence. Deletions that take place at terminals are included. For example, a variant polypeptide with 500 amino acid residues with a deletion of five C-terminus amino acid residues could have a 99% sequence identity percentage (495/500 identical residues × 100) to the original polypeptide. Such a variant would be covered by the term "a variant that has at least 99% sequence identity with the polypeptide".

[0044] "Fused" polypeptide sequences are connected, that is, linked in a functional way, by means of a peptide bond between the two polypeptide sequences in question.

[0045] The term "degree of polymerization" (DP) refers to the number (n) of anhydro-glucopyranose units in a given saccharide. Examples of DP1 are glucose and fructose monosaccharides. Examples of DP2 are the maltose and sucrose disaccharides. The term "DE", or "dextrose equivalent", is defined as the percentage of reducing sugar, that is, 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 on a dry weight percentage 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 biochemical products in which a microbial organism, such as an ethanological microorganism, and at least one enzyme, such as an amylase, are present during the same process step. SSF includes the contemporary hydrolysis of starch substrates (granular, liquefied or solubilized) in saccharides, including glucose, and the fermentation of saccharides in alcohol or another biochemical or biomaterial in the same reactor vessel.

[0048] An "ethanological microorganism" refers to a microorganism with the ability to convert a sugar or other carbohydrates into 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, glucone 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 drink" refers to any drink produced by a method that comprises a fermentation process, such as a microbial fermentation, for example, a bacterial and / or fungal fermentation. "Beer" is an example of such a fermented drink, and the term "beer" means to understand any fermented must produced by fermenting / infusing a plant material containing starch. Often, beer is produced exclusively from malt or adjunct or any combination of malt and adjunct.

[0051] The term "about" refers to ± 15% of the reported value.

2. Polypeptides that have alpha-amylase activity used in this invention

[0052] In a first aspect, the present invention relates to polypeptides that comprise a sequence of amino acids that preferably have 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 still at least 99% amino acid sequence identity with the polypeptide of SEQ ID NO: 3 and which has alpha-amylase activity.

[0053] In some embodiments, the polypeptides of the present invention are the homologous polypeptides comprising amino acid sequences that differ by ten amino acids, preferably by nine amino acids, preferably by eight amino acids, preferably by seven amino acids, preferably in six amino acids,

preferably, in five amino acids, more preferably, in four amino acids, even more preferably, in three amino acids, most preferably, in two amino acids, and even more preferably, in one amino acid of the polypeptide of SEQ ID NO: 3.

[0054] In some embodiments, the polypeptides of the present invention are the polypeptide variants of SEQ ID NO: 3, or a fragment thereof that has alpha-amylase activity.

[0055] In some embodiments, the polypeptides of the present invention are the catalytic regions comprising amino acids 22 to 499 of SEQ ID NO: 2, provided by Clustalx https://www.ncbi.nlm.nih.gov/pubmed/17846036 .

[0056] In some embodiments, the polypeptides of the present invention are the catalytic regions and linking regions that comprise amino acids 22 to 550 of SEQ ID NO: 2, provided by Clustalx https://www.ncbi.nlm.nih.gov/ pubmed / 17846036.

[0057] In a second aspect, the present alpha-amylases comprise conservative substitution of one or several amino acid residues in relation to the amino acid sequence of SEQ ID NO:

3. Exemplary conservative amino acid substitutions are listed in 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 For Amino Acid Code Replace with any of Alanine A D-Ala, Gly, beta-Ala, L-Cys, D-Cys Arginine R D-Arg, Lys, D-Lys, homo-Arg , D-homo-Arg, Met, Ile, D-Met, D-Ile, Orn, D-Orn Asparagine N D-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-Gln Aspartic Acid D D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln Cysteine C D-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr Glutamine Q D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp Glutamic Acid And D-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln

Glycine G Ala, D-Ala, Pro, D-Pro, b-Ala, Acp Isoleucine I D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met Leucine L D-Leu, Val, D-Val, Leu, D-Leu, Met, D-Met Lysine K D-Lys, Arg, D-Arg, homo-Arg, D-homo-Arg, Met, D-Met, Ile, D-Ile, Orn , D-Orn Methionine M D-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val Phenylalanine F D-Phe, Tyr, D-Thr, L-Dopa, His , D-His, Trp, D-Trp, Trans-3,4 or 5-phenylproline, cis-3,4, or 5-phenylproline Proline P D-Pro, L-1-thioazolidine-4-carboxylic acid, D acid - or L-1-oxazolidine-4-carboxylic Serine S D-Ser, Thr, D-Thr, alo-Thr, Met, D-Met, Met (O), D-Met (O), L-Cys , D-Cys Threonine T D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Met (O), D-Met (O), Val, D-Val Tyrosine Y D-Tyr, Phe , D-Phe, L-Dopa, His, D-His Valina V D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met

[0058] In some embodiments, the present alpha-amylase comprises a deletion, replacement, insertion or addition of one or a few amino acid residues in relation 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 more amino 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 amino acid residues in relation to the amino acid sequence of SEQ ID NO: 3. In in all cases, the term "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 variants of the carbohydrate binding domain of SEQ ID NO: 3 which comprise a substitution, deletion and / or insertion in one or more (for example, several) positions.

[0060] The present amylases can be "precursor", "immature" or "full-length", in such a case, they include a signal sequence, or "mature", in such a case, they do not have a sequence of signal. The mature forms of polypeptides are generally the most useful. Unless otherwise indicated, the numbering of amino acid residues used in this document refers to the mature forms of the respective amylase polypeptides. The present amylase polypeptides can also be truncated to remove the N or C terminations, as long as the resulting polypeptides maintain amylase activity.

[0061] Alternatively, the amino acid changes are such that the physicochemical properties of the polypeptides are altered. For example, changes in amino acids can improve the thermal stability of the polypeptide, change the specificity of the substrate, change the ideal pH and the like.

[0062] 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 revealed by Reidhaar-Olson and Sauer, 1988, Science 241: 53 to 57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2,152 to 2,156; WO 95/17413; or WO 95/22625. Other methods that can be used include error prone PCR, phage display (for example, Lowman et al., 1991, Biochem. 30: 10,832 to 10,837; US patent 5,223,409; document in WO 92/06204) and mutagenesis targeted region (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

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

[0064] The present amylase can be a "chimeric" or "hybrid" polypeptide, since it includes at least a portion of a first amylase, and at least a portion of a second amylase, glycoamylase, beta-amylase, alpha-glucosidase or other starch-degrading enzymes, or other glycosyl hydrolases, such as, without limitation, cellulases, hemicellulases, etc. (including such chimeric amylases have recently been "rediscovered" as domain exchange amylases). The present amylases can additionally include a heterologous signal sequence, an epitope to allow screening or purification or the like. Exemplary heterologous signal sequences are B. licheniformis amylase (LAT), B. subtilis (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 cellular material (for example, a total cell broth) comprising an alpha-amylase can be obtained after secretion of the alpha-amylase in the cell medium. Optionally, alpha-amylase can be isolated from host cells, or isolated from 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 cells (including yeast and filamentous fungi) and plants (including algae). Particularly useful host cells include Aspergillus niger, Aspergillus oryzae,

Trichoderma reesi or Myceliopthora thermophila. Other host cells include bacterial cells, for example, Bacillus subtilis or B. licheniformis, as well as Streptomyces.

[0066] Additionally, the host can express one or more enzymes, proteins or accessory peptides. They can benefit from liquefaction, saccharification, fermentation, SSF and downstream processes. In addition, the host cell can produce ethanol and other biochemical or biomaterials in addition to the enzymes used to digest the various raw materials. Such host cells can be useful for fermentation or saccharification and simultaneous fermentation processes to reduce the need for adding enzymes.

3.1. Vector

[0067] A DNA construct that comprises a nucleic acid that encodes alpha-amylases can be constructed to be expressed in a host cell. Due to the well-known degeneracy in the genetic code, different polynucleotides that encode an identical amino acid sequence can be designed and produced with common skill. Optimization of the use of codons for a particular host cell is also well known in the art. Nucleic acids encoding alpha-amylases can be incorporated into a vector. The vectors can be transferred to a host cell using well-known transformation techniques, such as those disclosed below.

[0068] The vector can 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 integration is generally considered to be an advantage, since the DNA sequence is more likely to be kept stable in the cell. A representative useful vector is p2JM103BBI (Vogtentanz, Protein Expr Purif, 55:40 to 52, 2007), which can be modified with routine skill and integrated into the chromosome of a host cell, so that it comprises other DNA fragments to improve alpha expression of the invention.

[0069] Host cells that serve as expression hosts can include filamentous fungi, for example. The Fungal Genetics Stock Center (FGSC) Catalog of Strains lists vectors suitable for expression in fungal host cells. See FGSC, Catalog of Strains, University of Missouri, at www.fgsc.net (last modified on January 17, 2007). A representative useful vector is pTrex3gM (see Published Patent Application No. US 20130323798) and pTTT (see Published Patent Application No. 20110020899), which can be inserted into the host genome. The pTrex3gM and pTTT vectors can both be modified with routine skill so that they understand and express a polynucleotide that encodes an alpha-amylase polypeptide of the invention.

[0070] A nucleic acid encoding an alpha-amylase can be functionally linked to a suitable promoter, which allows transcription in the host cell. The promoter can be any DNA sequence that shows transcriptional activity in the host cell of choice and can be derived from genes that encode proteins or homologues or heterologues to the host cell. Exemplary promoters to target transcription of the DNA sequence encoding an alpha-amylase, especially in a bacterial host, are promoters derived from the E. coli lac operon, the Streptomyces coelicolor dagA or celA agarase gene, the alpha-amyloid gene

Bacillus licheniformis (amyL), the Bacillus stearothermophilus (amyM) gene for maltogenic amylase (amyM), the Bacillus amyloliquefaciens (amyQ) alpha-amylase gene, the xylA and xylB genes of Bacillus subtilis, etc. For transcription in a fungal host, examples of useful promoters are those derived from the gene encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, A. niger acid-stable alpha-amylase, glycoamylase of A. niger, lipase of Rhizomucor miehei, protease of A. oryzae alkaline, triose phosphate isomerase of A. oryzae or acetamidase of A. nidulans. When a gene encoding an amylase is expressed in a bacterial species, such as an E. coli, a suitable promoter can be selected, for example, from a bacteriophage promoter that includes a T7 promoter and a phage lambda promoter. Examples of promoters suitable for expression in a yeast species include, but are not limited to, the Gal 1 and Gal 10 promoters of Saccharomyces cerevisiae and the AOX1 or AOX2 promoters of Pichia pastoris. Examples of a suitable promoter for expression in filamentous fungi include, but are not limited to, cbh1, an endogenous inducible promoter of T. reesei. See Liu et al. (2008) “Improved heterologous gene expression in Trichoderma reesei by cellobiohydrolase I gene (cbh1) promoter optimization,” Acta Biochim. Biophys. Sin (Shanghai) 40 (2): 158 to 165.

[0071] The coding sequence can be functionally linked to a signaling sequence. The DNA encoding the signal sequence may be the DNA sequence naturally associated with the gene for amylase to be expressed or from a different genus or species. A DNA construct or vector that comprises 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 cbh1 signal sequence that is functionally linked to a cbh1 promoter.

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

[0073] The vector can also comprise a DNA sequence that allows the vector to replicate in the host cell. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUB110, pE194, pAMB1 and pIJ702.

[0074] The vector may also comprise a selectable marker, for example, a product of a gene that complements a defect in the isolated host cell, such as the B. subtilis or B. licheniformis dal genes, or a gene that confers antibiotic resistance, such as, for example, resistance to ampicillin, kanamycin, chloramphenicol or tetracycline. In addition, the vector can comprise Aspergillus selection markers, such as amdS, argB, niaD and xxsC, a marker that generates resistance to hygromycin, or the selection can be performed by cotransformation, as known in the art. See, for example, International PCT Application WO 91/17243.

[0075] Intracellular expression can be advantageous in some ways, for example, when using bacteria or fungi as host cells to produce large amounts of amylase for subsequent enrichment and purification. The extracellular secretion of amylase in the culture medium can also be used to produce a cultured cell material that comprises isolated amylase.

[0076] The procedures used to link the DNA construct that encodes an amylase, the promoter, terminator and other elements, respectively, and to insert them into suitable vectors containing the necessary information for replication, are also well known to people skilled in technical (see, for example, Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition, Cold Spring Harbor, 1989, and 3rd edition, 2001).

3.2. Transformation and Culture of Host Cells

[0077] An isolated cell, comprising a DNA construct or an expression vector, is advantageously used as a host cell in the recombinant production of an amylase. The cell can be transformed with the DNA construct that encodes the enzyme, conveniently integrating the DNA construct (in one or more copies) into the host chromosome. This integration is generally considered to be an advantage since the DNA sequence is more likely to be kept stable in the cell. The integration of the DNA constructs into the host chromosome can be performed according to conventional methods, for example, by homologous or heterologous recombination. Alternatively, the cell can 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 amylolique, Bacillus amylolique coagulans, Bacillus lautus, Bacillus megaterium and Bacillus thuringiensis; Streptomyces species, such as Streptomyces murinus; bacterial species producing lactic acid, including Lactococcus sp., such as Lactococcus lactis; Lactobacillus sp., Including Lactobacillus reuteri; Leuconostoc sp .; Pediococcus sp .; eStreptococcus sp.

Alternatively, strains of a Gram-negative bacterial species that belong to Enterobacteriaceae, including E. coli, or Pseudomonadaceae can be selected as the host organism.

[0079] A suitable yeast host organism can be selected from among biotechnologically relevant yeast species, such as, but not limited to, yeast species such as Pichia sp., Hansenula sp., Or Kluyveromyces, Yarrowinia, Schizosaccharomyces species or a species Saccharomyces, which includes 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 may be a species of Hansenula.

[0080] Suitable host organisms among filamentous fungi include species of Aspergillus, for example, Aspergillus niger, Aspergillus oryzae, Aspergillus tubigensis, Aspergillus awamori or Aspergillus nidulans. A suitable procedure for transforming Aspergillus host cells includes, for example, that described in the document in EP 238023. Alternatively, strains of a species of Fusarium, for example, Fusarium oxysporum or a species of Rhizomucor, such as Rhizomucor miehei, can be used as the host organism. Other suitable strains include species of Myceliopthora, Thermomyces and Mucor. Additionally, Trichoderma sp. can be used as a host. A suitable procedure for transforming Trichoderma host cells includes, for example, that described by Penttilä et al. [Gene 61 (1987) 155 to 164]. An amylase expressed by a fungal host cell can be glycosylated, that is, it will comprise a glycosyl chemical moiety. The glycosylation pattern can be the same or different from that present in wild-type amylase. The type and / or degree of glycosylation can confer changes in enzymatic and / or biochemical properties.

[0081] It is advantageous to delete genes from the expression hosts, where the gene deficiency can be cured by the transformed expression vector. The known methods can be used to obtain a fungal host cell that has one or more inactivated genes. Gene inactivation can be accomplished by complete or partial deletion, by inactivation by insertion or by any other means that gives rise to a non-functional gene for its intended purpose so that the gene is prevented against the expression of a functional protein. Any gene of a Trichoderma sp. or another filamentous fungal host that has been cloned can be deleted, for example, genes cbh1, cbh2, egl1 and egl2. The deletion of genes can be performed by inserting a form of the desired gene to be inactivated in a plasmid by methods known in the art.

[0082] The appropriate host cell may be the ethanological microbial cell, which may express one or more of the amylase homologs described herein, and / or other bacillus amylases (including from B. licheniformis, B. stearothermophilus, B. substilis and other Bacillus species), and / or amylases from other sources. They can also express homologous or heterologous starch degrading enzymes, such as glycoamylase, that is, a glycoamylase that is not the same species as the host cell. In addition, the host can express one or more enzymes, proteins and / or accessory peptides. They can benefit the pre-treatment, liquefaction, saccharification, fermentation, SSF, vinasse, soluble products or condensed distillate syrups, etc. processes. In addition, the host cell can produce ethanol and other biochemical or biomaterials in addition to the enzymes used to digest the various raw materials. Such host cells can be useful for fermentation or saccharification and simultaneous fermentation processes to reduce the need for adding enzymes.

[0083] The introduction of a vector or DNA construct into a host cell includes techniques, such as transformation; electroporation; nuclear microinjection; transduction; transfection, for example, DEAE-Dextrin-mediated lipofection and transfection; incubation with calcium phosphate DNA precipitate; high-speed pumping with DNA-coated microinjections and protoplast fusion. General transformation techniques are known in the art. See, for example, Sambrook et al. (2001), supra. The expression of heterologous protein in Trichoderma is described, for example, in U.S. Patent 6,022,725. Reference is also made to Cao et al. (2000) Science 9: 991 to 1,001 for transformation of Aspergillus strains.

3.3. Expression and Fermentation

[0084] A method for producing an amylase can comprise culturing 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 grow the cells can be any conventional medium suitable for culturing the host cell and obtaining the expression of an alpha-amylase polypeptide. Suitable media and media components are available from commercial suppliers or can be prepared according to published recipes (for example, as described in American Type Culture Collection catalogs).

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

3.4. Amylase Activity Identification

[0087] To evaluate the expression of an amylase in a host cell, the assays can measure the expressed protein, corresponding mRNA or alpha-amylase activity. For example, suitable assays include Northern blotting transfer, polymerase chain reaction with reverse transcriptase and in situ hybridization, using an appropriately identified hybridization probe. Suitable assays also include measuring amylase activity in a sample, for example, by assays that directly measure reducing sugars, such as glucose, in the culture media. For example, the glucose concentration can be determined using a 15-UV glucose reagent kit (Sigma Chemical Co.) or an instrument, such as a Technicon autoanalyzer. Alpha-amylase activity can also be measured by any known method, such as the PAHBAH or ABTS assays, described below.

3.5. Methods to Enrich and Purify Alpha-Amylases

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

[0089] After fermentation, a fermentation broth is obtained, microbial cells and various suspended solids, including residual crude fermentation materials, are removed by conventional separation techniques in order to obtain an alpha-amylase solution. Filtration, centrifugation, microfiltration, vacuum drum filtration, ultrafiltration, centrifugation followed by ultrafiltration, extraction or chromatography or the like are generally used.

[0090] Sometimes it may be desirable to concentrate a solution or broth that comprises an alpha-amylase polypeptide to optimize recovery. The use of broth or non-concentrated solutions would typically increase the 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 starch degradation processes, particularly in gelatinized starch liquefaction, simultaneous liquefaction and saccharification, simultaneous fermentation and / or saccharification and fermentation (SSF).

[0092] The starch conversion process can be a precursor to, or concurrent with, a fermentation process designed to produce alcohol for fuel or drink (ie, potable alcohol) or other biochemical or biomaterials. One skilled in the art is aware of various fermentation conditions that can be used in the production of these final 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 the methods available that can be used to prepare starch substrates for use in the processes disclosed in this document. For example, a useful starch substrate can be obtained from tubers, roots, stems, vegetables, cereals or whole grains. More specifically, granular starch can be obtained from corn, cobs, wheat, barley, rye, triticale, milo, sago, millet, tapioca, sorghum, rice, peas, beans, bananas or potatoes. Corn contains about 60 to 68% starch; barley contains about 55 to 65% starch; millet contains about 75 to 80% starch; wheat contains about 60 to 65% starch; and polished rice contains 70 to 72% starch. The starch substrates specifically contemplated are corn starch and wheat starch. Starch from a grain can be crushed or whole and includes corn solids such as crumbs, bran and / or cobs. Starch can also be highly refined crude starch or raw material from starch refinery processes. Various starches are also commercially available. For example, corn starch is available from Cerestar, Sigma and Katayama Chemical Industry 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 may be a raw, whole-grain starch that contains fractions other than starch, for example, germ and fiber residues. The grinding may comprise dry grinding or wet grinding or crushing. In wet milling, the whole grain is soaked in water or diluted acid to separate the grain into its component parts, for example, starch, protein, germ, oil, crumb fibers. Wet grinding effectively separates the germ and bran (ie starch granules and protein) and is especially suitable for the production of syrups. The starch to be processed can 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 grinding or grinding, the whole brains are ground into a fine powder and often processed without fractioning the grain into its component parts. In some cases, oils and / or fiber from the brains are recovered. The dry-ground grain will thus comprise significant amounts of non-starch carbohydrate compounds, in addition to the starch. Dry grinding of the starch substrate can be used to produce ethanol and other biochemicals and biomaterials.

4.2. Starch Gelatinization and Liquefaction

[0095] As used herein, the term "liquefaction" or "liquefy" means a process by which gelatinized starch is converted into less viscous liquid containing shorter-chain soluble dextrins, liquefying and / or saccharine-inducing enzymes - fication can optionally be added. In some embodiments, the starch substrate prepared as described above is made into a slurry with water. The starch slurry may contain starch as a dry weight solids percentage of about 10 to 55%, about 20 to 45%, about 30 to 45%, about 30 to 40% or about 30 to 35 %. Alpha-amylase (EC 3.2.1.1) can be added to the slurry, with a metering pump, for example. The alpha-amylase typically used for this order is a thermostable bacterial alpha-amylase, such as a Geobacillus stearothermophilus alpha-amylase, Cytophaga alpha-amylase, etc., for example, SPEZYME® RSL (DuPont product), SPEZYME® AA (product DuPont), SPEZYME® Fred (DuPont product), CLEARFLOW® AA (DuPont product), SPEZYME® Alpha PF (DuPont product), SPEZYME® Powerliq (DuPont product) can be used here. Alpha-amylase can be supplied, for example, at about 1,500 units per kg of dry starch matter. To optimize the stability and activity of alpha-amylase, the pH of the slurry is typically adjusted to about pH 5.5 to 6.5 or a more suitable pH for the amylase to be added and about 1 mM calcium ( about 40 ppm of free calcium ions) can also be added. Several alpha-amylases may require different conditions. The liquefaction alpha-amylase remaining in the slurry after liquefaction can be deactivated by several methods, including reducing the pH in a subsequent reaction step or removing calcium from the slurry in cases where the enzyme is calcium dependent .

[0096] Starch slurry plus alpha-amylase can be pumped continuously through a jet cooker, which is heated by steam to 80 to 110 ° C, depending on the source of the raw material containing starch. Gelatinization occurs rapidly under these conditions, and the enzymatic activity, combined with the significant shear forces, begins the hydrolysis of the starch substrate. The time spent in the jet cooker is brief. The partially gelatinized starch can then be passed through a series of holding tubes maintained at 105 to 110 ° C and held for 5 to 8 min to complete the gelatinization process ("primary liquefaction"). Hydrolysis to the required DE is completed in holding tanks at 85 to 95 ° C or higher temperatures for about 1 to 2 hours ("secondary liquefaction"). These tanks may contain baffles to discourage re-mixing. As used in this document, the term "minutes of secondary liquefaction" refers to the time that elapsed from the start of the 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, for example, 90 minutes to 120 minutes. The liquefied starch is typically in the form of a slurry that has a dry solids (w / w) content of about 10 to 50%; about 10 to 45%; about 15 to 40%; about 20 to 40%; about 25 to 40%; or about 25 to 35%.

[0097] Liquefaction with alpha-amylases can be advantageously conducted at a low pH, eliminating the requirement to adjust the pH to about pH 5.5 to 6.5. Alpha-amylases can be used for liquefaction at a pH range of 2 to 7, for example, pH 3.0 to 7.5, pH 4.0 to 6.0 or pH 4.5 to 5.8. Alpha-amylases can maintain liquefaction activity in a temperature range of about 70 ° C to 140 ° C, for example, 85 ° C, 90 ° C or 95 ° C. For example, liquefaction can be conducted with 800 µg of an amylase in a 25% solution of DS corn starch for 10 min at pH 5.8 and 85 ° C, or pH 4.5 and 95 ° C. Liquefaction activity can be assessed using any one of several test methods known in the art.

[0098] In particular modalities with the use of the present alpha-amylases, the liquefaction of starch is carried out at a temperature range of 90 to 115 ° C, for the purpose of producing high purity glucose syrups, HFCS, maltodextrins, etc. .

4.3. Saccharification

[0099] The liquefied starch can be saccharified in a syrup rich in lower DP saccharides (for example, DP1 + DP2), with the use of alpha-amylases, optionally in the presence of another enzyme (or enzymes). The exact composition of saccharification products depends on the combination of enzymes used, as well as the type of processed starch. Advantageously, the syrup obtainable using the supplied alpha-amylases can contain a percentage by weight of DP2 of the total oligosaccharides in the saccharified starch that exceeds 30%, for example, 45% to 65% or 55% to 65%. The weight percentage of (DP1 + DP2) in the saccharified starch can exceed about 70%, for example, 75% to 85% or 80% to 85%. The present amylases also produce a relatively high glucose yield, for example, DP1> 20%, in the syrup product.

[0100] Although liquefaction is generally performed as a continuous process, saccharification is often conducted as a batch process. Saccharification conditions depend on the nature of the liquefied and the type of enzymes available. In some cases, a saccharification process may involve temperatures of about 60 to 65 ° C and a pH of about 4.0 to 4.5, for example, pH 4.3. Saccharification can be carried out, for example, at a temperature between about 40 ° C, about 50 ° C or about 55 ° C to about 60 ° C or about 65 ° C, requiring cooling of the Liquefied. The pH can also be adjusted as needed. Saccharification is usually carried out in agitated tanks, which can take several hours to fill or empty. Enzymes are typically added or at a fixed rate to dry solids as the tanks are filled or added as a single dose at the beginning of the filling stage. A saccharification reaction to compose a syrup is typically carried out over about 24 to 72 hours, for example, 24 to 48 hours. When a maximum or desired DE has been achieved, the reaction is stopped by heating to 85 ° C for 5 min, for example. The further incubation will result in a lower DE, possibly around 90 DE, as the accumulated glucose polymerizes again into isomaltose and / or other reversion products through an enzymatic reversal reaction and / or with the thermodynamic equilibrium approach. Preferably, saccharification is ideally conducted over a temperature range of about 30 ° C to about 75 ° C, for example, 45 ° C to 75 ° C or 47 ° C to 75 ° C. Saccharification can be carried out over a pH range of about pH 3 to about pH 7, for example, pH 3.0 to pH 6.5, pH 3.5 to pH 5.5, pH 3 , 5, pH 3.8 or pH 4.5.

[0101] An amylase can be added to the slurry as a composition. Amylase can be added to a granular starch substrate slurry. An amylase can be added as a whole, clarified, enriched, partially purified or purified broth enzyme. Amylase can also be added as a whole stock product.

[0102] An amylase can 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 that express such an amylase. An amylase can also be secreted by a host cell in the reaction medium during the fermentation or SSF process, so that the enzyme is supplied continuously to the reaction. The host cell that produces and secretes amylase can also express an additional enzyme, such as a glycoamylase. For example, U.S. Patent No. 5,422,267 discloses the use of a glycoamylase in yeast for the production of alcoholic drinks. For example, a host cell, for example, Trichoderma reesei or Aspergillus niger, Myceliopthora thermophila or Yeast, can be genetically modified to coexpress an amylase and a glycoamylase, for example, Humicola GA, Trichoderma GA, or variants thereof, during saccharification. The host cell can be genetically modified so as not to express its endogenous glycoamylase 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 glycoamylase, an alpha-glycosidase, beta-amylase, an enzyme that uses pentose sugar, an alpha-amylase, a pullulanase, an isoamylase, an isopululanase, a phytase, a protease and / or other enzymes. See, for example, WO 2011/153516 A2.

4.4. Hydrolysis of raw starch

[0103] The present alpha-amylases can also be used in a process of raw starch hydrolysis (RSH) or granular starch hydrolysis (GSH) to produce desired sugars and fermentation products. The term "granular starch" means raw uncooked starch, that is, starch in its natural form found in cereal, tubers or grains. A "raw starch hydrolysis" (RSH) process differs from conventional starch treatment processes, including liquefying high temperature gelatinized starch with the typical use of a bacterial alpha-amylase, followed by simultaneous saccharification and fermentation. in the presence of a glycoamylase and a fermentation organism and possibly other enzymes. The 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 one amylase and / or glycoamylase. The gelatinization temperature can vary according to the plant species, with the particular variety of the plant species as well as the growing conditions.

[0104] A fungal alpha-amylase described in this document expressed in ethanological bacterial, fungal, yeast or microbial cells can be used in the process of hydrolysis of raw starch, described in this document.

[0105] Additionally, the alpha-amylase that is different from the alpha-amylase described in this invention, glycoamylase, hexokinase, xylanase, glucose isomerase, xylose isomerase, phosphatase, phytase, pullulanase, beta-amylase, protease, cellulase, hemicellulase, lipase, cutinase, isoamylase, redox enzyme, esterase, transferase, pectinase, α-glycosidase, beta-glycosidase or a combination thereof can also be used in the raw starch hydrolysis process described in this document. Said enzymes can be coexpressed with alpha-amylase in this invention or directly added in the process of hydrolysis of raw starch.

4.5. Fermentation

[0106] Soluble starch hydrolyzate, particularly a glucose-rich syrup, can be fermented by placing the starch hydrolyzate in contact with a fermenting organism typically at a temperature around 32 ° C, such as 30 ° C to 35 ° C for alcohol-producing yeast. The temperature and pH of the fermentation will depend on 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 fatty acid

3, butanol, isoprene, 1,3-propanediol, vitamins and other biomaterials.

[0107] Ethanological microorganisms include yeasts, such as Saccharomyces cerevisiae and bacteria, for example, Zymomonas moblis, which express alcohol dehydrogenase and pyruvate decarboxylase. The ethanological microorganism can express xylose reductase and xylitol dehydrogenase, which convert xylose into xylulose. Improved strains of ethanological microorganisms, which can withstand higher temperatures, for example, are known in the art and can be used. See Liu et al. (2011) Sheng Wu Gong Cheng Xue Bao 27: 1,049 to 1,056 Commercial yeast sources include ethanol RED (LeSaffre); THERMOSACC (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 in the art. See, for example, Papagianni (2007) Biotechnol. Adv. 25: 244 to 263; John et al. (2009) Biotechnol. Adv. 27: 145 to 152.

[0108] The saccharification and fermentation processes can be performed as an SSF process. Fermentation can comprise subsequent enrichment, purification and recovery of ethanol, for example. During fermentation, the ethanol content of the broth or "beer" can reach about 8 to 18% in v / v, for example, 14 to 15% in v / v. The broth can be distilled to produce enriched ethanol solutions, for example, 96% pure. In addition, the CO2 generated by fermentation can be collected with a CO2 purifier, compressed and marketed for other uses, for example, carbonation of beverages or production of dry ice. The solid refuse from the fermentation process can be used as products rich in protein, for example, cattle feed.

[0109] As mentioned above, an SSF process can be conducted with fungal cells that continuously express and secrete amylase throughout the SSF. Fungal cells that express amylase can also be the fermentation microorganism, for example, an ethanological microorganism. The production of ethanol can thus be carried out using a fungal cell that expresses sufficient amylase so that less or no enzymes have to be added exogenously. The fungal host cell can be an appropriately genetically modified fungal strain. Fungal host cells that express and secrete other enzymes, in addition to amylase, can also be used. Such cells can express glycoamylase and / or a pullulanase, phytase, alpha-glucosidase, isoamylase, beta-amylase cellulase, xylanase, other hemicellulases, protease, beta-glucosidase, pectinase, esterase, oxidase enzymes, transferase or other enzymes.

4.6. Post-fermentation and post-fermentation products

[0110] Fermentation products, such as ethanol, are produced by first degrading material containing starch in fermentable sugar by liquefaction and saccharification, or liquefaction followed by SSF, or saccharification followed by fermentation (fermentation process). raw starch), and converting the sugars directly or indirectly into the desired fermentation product using a fermentation organism. Liquid fermentation products, such as ethanol, are recovered from fermented porridge (often called "beer" or "beer wort"), for example, by distillation, which separates the desired fermentation product from other liquids and / or solids. The remaining fraction, called "whole vinasse", is separated into a solid and a liquid phase, for example, by centrifugation. The solid phase is called "wet cake" (or "wet grains" or "WDG") and the liquid phase (supernatant) is called "fine vinasse". The wet cake is dried to provide "Dry Distiller Grains" (DDG) used as a nutrient in animal feed. Fine vinasse is typically evaporated to provide condensate and syrup (or "thick vinasse") or can alternatively be recycled directly to the slurry tank as a "setback". The condensate can be sent to a methanator before being discharged or it can be recycled to the slurry tank. The syrup that consists mainly of limit dextrins and non-fermentable sugars can be mixed in DDG or added to the wet cake before drying to produce DDGS (Dry Grain from Distillers with Soluble).

[0111] It is known that commercially using the various by-products and residues derived from fermentation processes such as the ethanol production process. Distiller residues or by-products, as well as cereal by-products and other food industry manufactures, are known to have a certain value as sources of protein and energy for animal feed. In addition, oil from by-products such as Vinhaça Inteira, Torta Humida, Vinhaça Fina, DDG and / or DDGS can be recovered as a separate by-product for use in the production of biodiesel or other products.

[0112] By-products such as DDG, DDGS or WDG comprise proteins, fibers, fat and unconverted starch. Wet Pie can be used in dairy feedlots. Dry DDGs can be used in livestock, for example, dairy, meat and pig feed and poultry feed. Although the protein content is high, the amino acid composition is not well suited for monogastric animals if used as animal feed. In addition, the by-products contain significant levels of Crude Fiber (CF), which are structural carbohydrates that consist of cellulose, hemicellulose and indigestible materials, such as lignin. The proportion of cellulose and lignin in the crude fiber fraction also determines the digestibility of crude fibers and their solubility in the intestine. Soluble non-amidic polysaccharides (NSP) cannot be digested by monogastric animals, such as pigs and birds, and can cause an increase in viscosity, due to their ability to bind to water, which can result in sticky, moist and wet debris. Another effect of NSP is the so-called "Nutrient Encapsulation". Essentially, starch, protein, oil and other nutrients are encapsulated within the plant cell, which is an impermeable barrier that prevents full utilization of nutrients within the cell.

[0113] In addition, soluble NSP can cause an increase in viscosity during fermentation and can influence the separation and drying conditions of fermentation by-products such as DDGS in the production process.

[0114] Therefore, several specific treatment processes or methods have been used and are being investigated to improve the quality of the by-products of fermentation processes. For example, the addition of enzymes to liquefaction, saccharification, fermentation or SSF, whole vinasse, moist cake and / or fine vinasse, etc., in the ethanol production process has been used to improve the separation between solid and liquid in the process and / or change or improve the throughput and / or quality of by-products. In addition, enzymes are also being used or investigated as a way to access residual starch and, in some cases, access cellulosic and / or hemicellulosic sugars associated with corn fiber. These sugars can then be used by suitable hosts to produce fermentation products, including ethanol. The present amylases can be used in these processes, as well as other starch degrading enzymes, such as alpha-amylase which is different from the alpha-amylase described in this invention, glycoamylase, hexokinase, xylanase, glucose isomerase, xylose isomerase, phosphatase, phytase, pullulanase, beta-amylase, protease, cellulase, hemicellulase, lipase, cutinase, isoamylase, redox enzyme, esterase, transferase, pectinase, α-glucosidase, beta-glucosidase or a combination thereof, even hemicellulases, cellulases. Enzymes can be added at any stage in the process.

5. Compositions That Include Alpha Amylases

[0115] 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 also be used in the enzyme composition.

[0116] Alpha-amylases (EC 3.2.1.1) can be combined with a glucoamylase (EC 3.2.1.3), for example, a Trichoderma glycoamylase or variant thereof. An exemplary glycoamylase is Trichoderma reesei glycoamylase (TrGA) and variants of it that have superior specific activity and thermal stability. See Published Orders No. U.S. 2006/0094080, 2007/0004018 and 2007/0015266 (Danisco US Inc.). Suitable variants of TrGA include those with glycoamylase activity and at least 80%, at least 90% or at least 95% sequence identity with wild-type TrGA. Alpha-amylases advantageously increase the glucose yield produced in a TrGA-catalyzed saccharification process.

[0117] Alternatively, the glycoamylase can be another glycoamylase derived from plants (including algae), fungi or bacteria. For example, glycoamylases can be Aspergillus niger glycoamylase G1 or G2 or variants thereof (for example, Boel et al. (1984) EMBO J. 3: 1.097 a

1,102; WO 92/00381; WO 00/04136 (Novo Nordisk A / S)); and A glycoamylase. awamori (e.g., WO 84/02921 (Cetus Corp.)). Other contemplated glycoamylases of Aspergillus include variants with increased thermal stability, for example, G137A and G139A (Chen et al. (1996) Prot. Eng. 9: 499 to 505); D257E and D293E / Q (Chen et al. (1995) Prot. Eng. 8: 575 to 582); No. 182 (Chen et al. (1994) Biochem. J. 301: 275 to 281); A246C (Fierobe et al. (1996) Biochemistry, 35: 8,698 to 8,704); and variants with Pro residues in positions A435 and S436 (Li et al. (1997) Protein Eng. 10: 1,199 to 1,204). Other glycoamylases contemplated include Talaromyces glycoamylases, in particular derived from T. emersonii (for example, WO 99/28448 (Novo Nordisk A / S), T. leycettanus (for example, US Patent No. RE 32,153 (CPC International, Inc.) ), T. duponti or T. thermophilus (for example, US Patent No. 4,587,215). Bacterial glycoamylases contemplated include glycoamylases of the genus Clostridium, in particular C. thermoamylolyticum (for example, EP 135138 (CPC International, Inc.) and C. thermohydrosulfuricum (eg WO 86/01831 (Michigan Biotechnology Institute)) Suitable glycoamylases include glycoamylases derived from Aspergillus oryzae, such as a glycoamylase shown in SEQ ID NO: 2 in WO 00/04136 (Novo Nordisk Commercial glycoamylases such as AMG 200L; AMG 300 L; SAN ™ SUPER and AMG ™ E (Novozymes); OPTIDEX® 300 and OPTIDEX L-400 (Danisco US Inc.); AMIGASE ™ and AMIGASE are also suitable. ™ PLUS (DSM); G-ZYME® G900 (Enzyme Bio-Systems); and G-ZYME® G990 ZR (A. niger glycoamylase with a low protease content). Still other suitable glycoamylases include Aspergillus fumigatus glycoamylase, Talaromyces glycoamylase, Thielavia glycoamylase, Trametes glycoamylase, Thermomyces glycoamylase, Athelia glycoamylase, Pycnoporus glycoamylase, for example, Penicillin glycosylate. Glycoamylases are typically added in an amount of about 0.1 to 2 units of glycoamylase (GAU) / g ds, for example, about 0.16 GAU / g ds, 0.23 GAU / g ds or 0.33 GAU / g of ds.

[0118] Other suitable enzymes that can be used with amylase include phytase, protease, pullulanase, beta-amylase, isoamylase, a different alpha-amylase, alpha-glycosidase, cellulase,

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), can be added in effective amounts well known to the person skilled in the art. A pullulanase (EC 3.2.1.41), for example, PROMOZYME®, is also suitable. Additional suitable enzymes include proteases, such as fungal and bacterial proteases. Fungal proteases include those obtained from Aspergillus, such as A. niger, A. awamori, A. oryzae; Mucor (for example, M. miehei); Rhizopus; and Trichoderma.

[0119] Beta-amylases (EC 3.2.1.2) are exo-acting maltogenic amylases that catalyze the hydrolysis of 1,4-alpha-glycosidic bonds in amylopectin and related glucose polymers, thus releasing maltose. Beta-amylases have been isolated from various plants and microorganisms. See Fogarty et al. (1979) in Progress in Industrial Microbiology, Volume 15, pages 112 to 115. These beta-amylases have ideal temperatures in the range of 40 ° C to 65 ° C and ideal pH in the range of about 4.5 to about 7, 0. The contemplated beta-amylases include, but are not limited to, barley beta-amylases SPEZYME® BBA 1500, SPEZYME® DBA, OPTIMALT ™ ME, OPTIMALT ™ BBA (Danisco US Inc.); and NOVOZYM ™ WBA (Novozymes A / S).

[0120] The compositions comprising the present amylases can be aqueous or non-aqueous formulations, granules, powders, gels, slurries, pastes, etc. which may additionally comprise any one or more of the additional enzymes listed in this document, together with buffers, salts, preservatives, water, cosolvents, surfactants and the like.

[0121] All references cited in this document are incorporated into this document as a reference in their entirety for all purposes. In order to further illustrate the compositions and methods, and their advantages, the following specific examples are given with the understanding that they are illustrative rather than limiting.

EXAMPLES EXAMPLE 1 Alpha-amylase sequence from Aspergillus sp. (AspAmy14)

[0122] The protein sequence of a fungal alpha-amylase called AspAmy14 was identified from a strain of Aspergillus sp. based on sequence homology. A synthetic gene encoding AspAmy14 was ordered as a codon gene optimized for expression in Trichoderma reesei. The synthetic gene with codon optimized that encodes AspAmy14 is presented as SEQ ID NO: 1:

ATGAAGTGGACCGTCTCTCTCTTCCCTTTGCTGTCCTTGTTCGGTC AGACAGCCCATGCCCTCACCCCAGCACAATGGCGCAGCCAGTCA ATCTACTTCCTGATGACCGACCGCTTCGGTCGAACGGACAATTCT ACAACTGCCGCCTGCAACACTGCTGACAGAGTTTGTACTTCGATA ACGGCACTCGGGTGCATGTACTGATGTGTGCAGGTATACTGCGGT GGTAGCTGGCAGGGGATCATCAATCATGTATGAGTGGATTATGAT GGATATTCTCTGTTTGATACTAACGCCACCAGCTCGATTACATCCA AGGAATGGGATTCACTGCCATCTGGATCACCCCAGTCACAGAGCA GTTCTATGAAGACACCGGCGACGGCACCTCCTACCATGGGTACTG GCAGCAGAACATGTAGGCATTCGTCCTCGTTTCGTGTTCGGTGCT AATGCATGCAGCTACAATGTCAATTCCAACTACGGAACGGCGCAA GACCTCAAGAATCTCGCCAGTGCGTTGCACGCGCGCGGCATGCA CCTGATGGTCGATGTGGTTGCCAACCACATGGTAAGCTGTCTCTT CATGGAAATATAATAGAAACGAACTGAACTGGCGTAGGGCTACGA CGGAGCCGGAAACTCCGTCGACTACGGCGTTTTCGATCCGTTTTC CTCTTCGAGCTACTTCCACCCATACTGTCTCATCTCCGACTACAAC AACCAGACCAACGTCGAAGACTGCTGGCTCGGAGATACCACTGTT TCGTTACCTGATCTTGACACGACAAGCACAGACGTACGAAATATCT GGTACGACTGGGTTGAGGAACTGGTTGCCAACTATTCCAGTCAGT AGCCCGCATCATATGAGTAGGGGGCGTACTGACAGCCATAGTCG ATGGCCTGCGGGTCGACACGGTAAAACATGTTGAGAAGGACTTTT GGCCCGGCTACAACAGCGCAGCAGGCGTCTACTGTGTCGGTGAG GTGTTCTCGGGCGATCCGGCATACACATGTCCATACCAGAACTAC ATGGACGGTGTGCTCAACTACCCAATGTGAACATGCCTACCTTCC AGAAAACCCCAGAGGCTGACACACCGCAGCTACTACCAACTCCTC TATGCGTTCGAGTCAACCAGCGGCAGCATGAGCAACCTGTACAAC ATGATCAACTCGGTTGCCTCCGACTGCAAGGATCCCACCCTACTG GGCAACTTTATCGAGAACCACGACAACCCGCGCTTTGCTTCGTAA GTCTTTCTTCCTCTATTCGTGCAGTCCATGCTAAATCCCGCAGCTA CACGAGTGACTACTCGCAAGCGAAGAATGTGATCTCGTTTATCTT CCTCACCGATGGCATCCCCATCGTCTACGCCGGACAGGAACAGC ACTACAGCGGCGGCAGCGACCCAGCCAACCGCGAGGCCACCTG GCTATCCGCATACTCAACCGGCGCCACGCTGTACACCTGGATCG CGTCGACAAACAAGATCCGCAAGCTGGCGATATCCAAGGACACG GGATACGTGGAGGCCAAGGTATGCGCACACCCCCGGCTCTGTAG CTCACGCTAACGCGGACAGAACAACCCCTTCTACTACGACTCCAA TACGATCGCCATGCGCAAGGGAACCACCGCCGGTGCGCAGGTCA TCACCGTCTTGAGCAACAAGGGCGCGTCGGGTAGCTCCTACACC CTCTCCTTGAGCGGTACGGGCTACGCCGCCGGCGCGACCCTGGT CGAGATGTACACCTGCACCACGGCCACTGTAGACTCAAGCGGCA ACCTCCCGGTTCTAATGACATCCGGTTTGCCCAGAGTGTTTCTAC CGTCGTCTTGGGTAAGTGGCAGCGGTCTTTGCGGCTCCGCTGTC TCTACTACACTCACGACAGTTTCCACTACGCTCACGACAGTCGCC GCGACCACGACGTCGACCACGACATCGACCACGACATCGACCAC GACATCGACCACGACATCGACCACGACATCGACCACGACATCGA CCACGACATCGACAACATGCACGGCCGCCACAGCCCTTCCCATTC TCTTCGAGGAACTCGTCACGACAACCTACGGAGAGAACATCTTCC TGACCGGCTCGATCAGCCAACTGGGCAGCTGGAACACCGCCTCG GCCGTTGCCTTGTCGGCGAGTAAGTACACCGCTTCCAAGCCGGA ATGGTACGTGACCGTGACCTTGCCCGTGGGCACCACGTTCCAGT ACAAGTTTATCAAGAAAGAGGCGGACGGGAGTGTGGCGTGGGAG AGTGATCCGAACCGATCGTACACGGTTCCGAGTGGCTGTGCGGG TGCGACAGTGACGGTTGTTGATACTTGGAGGTGA

[0123] The amino acid sequence of the precursor protein AspAmy14 is presented as SEQ ID NO: 2. The native signal peptide is shown in italics and underlined.

MKWTVSLFPLLSLFGQTAHALTPAQWRSQSIYFLMTDRFGRTDNSTT AACNTADRVYCGGSWQGIINHLDYIQGMGFTAIWITPVTEQFYEDTG DGTSYHGYWQQNIYNVNSNYGTAQDLKNLASALHARGMHLMVDVV ANHMGYDGAGNSVDYGVFDPFSSSSYFHPYCLISDYNNQTNVEDC WLGDTTVSLPDLDTTSTDVRNIWYDWVEELVANYSIDGLRVDTVKHV EKDFWPGYNSAAGVYCVGEVFSGDPAYTCPYQNYMDGVLNYPIYY QLLYAFESTSGSMSNLYNMINSVASDCKDPTLLGNFIENHDNPRFAS YTSDYSQAKNVISFIFLTDGIPIVYAGQEQHYSGGSDPANREATWLSA YSTGATLYTWIASTNKIRKLAISKDTGYVEAKNNPFYYDSNTIAMRKG TTAGAQVITVLSNKGASGSSYTLSLSGTGYAAGATLVEMYTCTTATV DSSGNLPVLMTSGLPRVFLPSSWVSGSGLCGSAVSTTLTTVSTTLTT VAATTTSTTTSTTTSTTTSTTTSTTTSTTTSTTTSTTCTAATALPILFEE LVTTTYGENIFLTGSISQLGSWNTASAVALSASKYTASKPEWYVTVTL PVGTTFQYKFIKKEADGSVAWESDPNRSYTVPSGCAGATVTVVDTW R

[0124] The amino acid sequence of the mature form of AspAmy14 conformed by LC MS / MS is presented as SEQ ID NO: 3:

LTPAQWRSQSIYFLMTDRFGRTDNSTTAACNTADRVYCGGSWQGII NHLDYIQGMGFTAIWITPVTEQFYEDTGDGTSYHGYWQQNIYNVNSN YGTAQDLKNLASALHARGMHLMVDVVANHMGYDGAGNSVDYGVFD PFSSSSYFHPYCLISDYNNQTNVEDCWLGDTTVSLPDLDTTSTDVRNI WYDWVEELVANYSIDGLRVDTVKHVEKDFWPGYNSAAGVYCVGEV FSGDPAYTCPYQNYMDGVLNYPIYYQLLYAFESTSGSMSNLYNMINS VASDCKDPTLLGNFIENHDNPRFASYTSDYSQAKNVISFIFLTDGIPIV YAGQEQHYSGGSDPANREATWLSAYSTGATLYTWIASTNKIRKLAIS KDTGYVEAKNNPFYYDSNTIAMRKGTTAGAQVITVLSNKGASGSSYT LSLSGTGYAAGATLVEMYTCTTATVDSSGNLPVLMTSGLPRVFLPSS WVSGSGLCGSAVSTTLTTVSTTLTTVAATTTSTTTSTTTSTTTSTTTS TTTSTTTSTTTSTTCTAATALPILFEELVTTTYGENIFLTGSISQLGSWN TASAVALSASKYTASKPEWYVTVTLPVGTTFQYKFIKKEADGSVAWE

SDPNRSYTVPSGCAGATVTVVDTWR EXAMPLE 2 Expression of alpha-amylase from Aspergillus sp. (AspAmy14)

[0125] The DNA sequence of AspAmy14 was optimized for expression of AspAmy14 in 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).

[0126] Plasmid pZKY258 was transformed into a suitable Trichoderma reesei strain (method described in PCT Application in published WO 05/001036) using protoplastic transformation (Te'o et al. (2002) J. Microbiol. Methods 51 : 393 to 399). Transformants were selected on a solid medium containing acetamide as the only source of nitrogen. After 5 days of growth on acetate plates, transformants were collected and subjected to fermentation in 250 ml shake flasks in defined media that contain a mixture of glucose and sophorosis. EXAMPLE 3 Purification of AspAmy14

[0127] The AspAmy14 crude sample from the fermentation was concentrated and ammonium sulfate was added to the concentrated sample to the final concentration of 1 M. The solution was then loaded onto a 20 ml HiPrepTM Fenil FF 16/10 column pre-balanced with 20 mM sodium acetate (pH 5.0) supplemented with 1 NH (NH4) 2SO4. Elution was performed using 6 column volumes of 0.75 M ammonium sulfate. Fractions were collected and passed in SDS-PAGE. Fractions containing the target protein were pooled, concentrated and exchanged with the 20 mM NaH2 PO4 buffer (pH 7.0). The solution was then loaded onto a 20 ml HiPrepTM Q FF 16/10 column pre-equilibrated with 20 mM NaH2PO4 (pH 7.0). Elution was performed using 6 column volumes of 0.3 NaCl. The fractions were collected and passed on SDS-PAGE. The fractions containing the target protein were pooled, concentrated and exchanged with the buffer to 20 mM sodium acetate pH 5.0 using the Amicon Ultra-15 device with 10 K MWCO. The purified sample is around 90% pure and stored in 40% glycerol at -20oC until use. EXAMPLE 4 AspAmy14 potato amylopectin hydrolyzation activity

[0128] Alpha-amylase activity was determined using a calorimeric assay to monitor the release of amylopectin-reducing sugars from potato. Activity is reported as glucose equivalents released per minute. Substrate solutions were prepared by mixing 9 ml of 1% (w / w, in water) of potato amylopectin (Sigma, catalog number 10118), 1 ml of 0.5 M buffer (sodium acetate at pH 5 , 0 or HEPES at pH 8.0) and 40 µl of 0.5 M CaCl2 in a 15 ml conical tube. The purified alpha-amylase sample stock solution was produced by diluting the original sample at 20 ppm in water. Serial dilutions of enzyme sample and glucose standard were prepared in water on non-binding microtiter plates (MTP, Corning 3641). Then, 90 µl of substrate solution (pre-incubated at 50oC for 5 min at 600 rpm) and 10 µl of the enzyme serial dilution were added and mixed in non-binding microtiter plates (MTP, Corning 3641). All incubations were performed at 50oC for 10 min at 600 rpm in a thermomixer (Eppendorf). After incubation, 50 µl of 0.5 N NaOH was added to each well to stop the reaction.

The total reducing sugars present in each well were measured using a PAHBAH method: 80 µl of 0.5 N NaOH were divided into aliquots on a microtiter plate, followed by the addition of 20 µl of PAHBAH reagent [5% in w / v 4-hydroxybenzoic acid hydrazide in 0.5 N HCl] and 10 µl of each reaction mixture.

The plates were incubated at 95 ° C for 5 min and cooled to 4 ° C for 5 seconds.

The samples (80 µl) were then transferred to polystyrene microtiter plates (Costar 9017) and the absorbance was read at 410 nm.

The resulting absorbance values were plotted against enzyme concentration and linear regression was used to determine the angular coefficient of the linear region of the plot.

Using the methanol mentioned above, the specific activity of AspAmy14 was determined and compared with a reference fungal alpha-amylase, AcAA (described in U.S. Patent 8,945,889). The results for both enzymes are shown in Table 2. Specific Activity (U / mg) = Linear Coefficient (enzyme) / linear coefficient (standard) * 100 Definition: 1 U = 1 µmol of glucose equivalent / min Table 2. Activity specific AspAmy14 and AcAA in potato amylopectin Specific activity (U / mg) Enzyme name pH 5 pH 8 AspAmy14 501 165 AcAA 608 107 EXAMPLE 5 AspAmy14 pH profile

[0129] The effect of pH (from 3.0 to 10.0) on the activity of AspAmy14 was monitored using the PAHBAH assay protocol as described in Example 4. The working buffer solutions consisted of a combination of glycine / sodium acetate / HEPES (250 mM), with pH ranging from 3.0 to 10.0. The substrate solutions were prepared by mixing 896 µl of 1% (w / w, in water) of potato amylopectin (Sigma, catalog number 10118), 100 µl of 250 mM buffer working solutions ( pH 3.0 to 10.0) and 4 µl of 0.5 M CaCl2.

The enzyme working solution was prepared in water at a certain dose (which shows a signal within the linear range as per the dose response curve). All incubations were performed at 50 ° C for 10 min following the same protocol as described above for specific AspAmy activity14. The absorbance of a control (water only) was subtracted, and the resulting values were converted into percentages of relative activity, defining the activity at the ideal pH as 100%. As shown in Table 3, AspAmy14 showed a pH profile similar to AcAA, with an ideal pH of 4.0. The pH range, within which the enzyme maintained more than 70% of maximum activity, was pH 3.3 to 6.4. Table 3. pH profile of AspAmy14 and AcAA Relative activity (%) pH AspAmy14 AcAA 3 60 59 4 100 100 5 83 91 6 74 85 7 63 61 8 33 16 9 6 3 10 0 1 EXAMPLE 6

AspAmy14 temperature profile

[0130] The temperature effect (from 40 to 90oC) on the alpha-amylase activity was monitored using the PAHBAH assay protocol as described in Example 4. The substrate solutions were prepared by mixing 3.6 ml of 1% (w / w, in water) of potato amylopectin (Sigma, catalog number 10118), 0.4 ml of 0.5 M sodium acetate buffer pH 5.0 and 16 µl of 0.5 CaCl2 M in a 15 ml conical tube. The enzyme working solution was prepared in water at 2.5 ppm. Before the reaction, 90 µl of substrate solution was added to PCR plates (Axygen, PCR-96-HS-C) and incubated in Peltier Thermal Cyclers (BioRad) at the desired temperatures (ie, 40 to 90oC) for 5 min . Then, 10 µl of diluted enzyme was added to the substrate to start the reaction. After 10 min of incubation in the PCR machines, the reactions were abruptly cooled and measured using the same protocol as described above for specific AsAmy activity14. The absorbance of a control (water only) was subtracted, and the resulting values were converted into percentages of relative activity, setting the activity at the ideal temperature to 100%. As shown in Table 4. AspAmy14 showed an ideal temperature at 70 ° C and maintained over 70% of maximum activity between 54 ° C and 75 ° C, while AcAA exhibited an ideal temperature of 63 ° C and maintained over 70 % of maximum activity between 49 ° C and 71 ° C. Table 4. Temperature profile of AspAmy14 and AcAA Relative activity (%) Temperature (oC) AspAmy14 AcAA 40 44 46 42 48 50 45 54 59 49 61 70 55 71 85

EXAMPLE 7 Thermostability of AspAmy14

[0131] AspAmy14 alpha-amylase thermostability was determined by measuring enzyme activity before and after enzyme samples pre-incubated at temperatures of 40 to 90 C for 2 h. The enzyme was diluted in 50 mM sodium acetate buffer (pH 5.0) containing 2 mM CaCl2 at 10 ppm and 50 μl were aliquoted in PCR strip tubes. The tubes were transferred to PCR machines at the desired temperature of 40 to 90oC. After 2 h pre-incubation, the enzymes were diluted to 2.5 ppm in water and evaluated for their residual activities using the amylopectin / PAHBAH methods, as described in Example 4. The residual activities were converted into percentages of relative activity, defining the activity of the sample kept on ice as 100%. Thermostability was defined as the temperature at which the sample maintained 50% of activity. As shown in Table 5, AspAmy14 maintained over 60% of initial activity after 2 h of incubation at 60oC, while AcAA only maintained 5% of residual activity under the same incubation conditions.

Table 5. Thermostability of AspAmy14 and AcAA Residual Activity (%) Temperature (oC) AspAmy14 AcAA 40 87 87 42 80 80 45 83 70 49 84 53 55 72 23 60 62 5 63 27 1 65 11 3 65 2 0 67 4 2 70 0 1 74 2 1 80 0 1 85 0 0 90 2 1 EXAMPLE 8 pH stability of AspAmy14

[0132] SSF is typically conducted at pH 3.8 to 4.8, 32oC for 55 hours, and the enzymes used in the process must be able to maintain their activity under their condition throughout the entire process. Thus, it is very useful to know the enzyme's low pH stability. The pH stability is assessed by measuring the residual enzyme activity after pre-incubating the enzyme at pH 3.7 and pH 4.5, respectively, for a determined time interval. The residual enzymatic activity is assessed using the amylopectin / PAHBAH method, as described in Example 4. The enzyme stock solutions were prepared by diluting the samples to 400 ppm in water and stored at 4oC. At each point in time, 97.5 μl of dilution buffer (50 mM sodium acetate buffer, pH 3.7 or 4.5 with 2 mM CaCl2) was added to PCR strip tubes, followed by adding 2.5 μl of enzyme stock solution (400 ppm) and mix well. After incubation at 32oC for different points in time, the enzymes were further diluted to 2.5 ppm in water and evaluated for their residual activities. The residual activity was converted into a percentage of relative activity, defining the activity without pre-incubation at pH 3.7 or 4.5 as 100%. As shown in Table 6, AspAmy14 shows pH stability greater than AcAA. After 24 hours at pH 3.7, AspAmy14 maintained almost 100% of original activity, while AcAA only maintained 41%. At pH 4.5 and 48 hours of incubation, AspAmy14 maintained almost 100% of original activity, while AcAA maintained 81%. Table 6. pH stability of AspAmy14 and AcAA Residual Activity (%) Incubation time pH 3.7 pH 4.5 (h) AspAmy14 AcAA AspAmy14 AcAA 0.5 106 102 109 103 1 103 102 106 105 2 106 102 104 103 6 102 78 104 100 24 99 41 97 96 48 87 19 99 81 EXAMPLE 9 Starch Solubilization Assay

[0133] The purpose of the starch solubilization assay is to assess the enzyme's ability to remove residual insoluble starch by measuring the remaining insoluble starch at the end of the assay. This is done by determining the optical density (OD) of each well at 260 nm. The large starch granules will spread the light that passes through the well; therefore, the higher the concentration of insoluble starch, the higher the OD. The substrate for the solubilization test was a mixture of amyl gel (70% Hylon VII amylose content) and insoluble corn starch (Sigma-aldrich, Lot no: 129K0076) that mimics the real substrate used in SSF. It was prepared by repeated washing of corn starch and amylogel with water 10 times through successive centrifugation / decanting and then suspended in 100 mM sodium acetate buffer (pH 3.7 and pH 4.5, respectively). 30% (w / w). Equal proportions of corn starch slurry and amylogel were mixed and diluted 25 times in 100 mM sodium acetate (pH 3.7 and pH 4.5, respectively), which were then autoclaved for 60 minutes at 121oC with a stir bar. As the mixture cooled, it was stirred overnight on a shaking plate to prevent gelation. After that, the substrate was stored at 4 oC and ready for use. The solubilization substrate (150 µl) was mixed well (stirring) while being added to the UV / vis plate (MTP, Corning 3635). Wider orifice tips were needed to transfer the substrate. The enzyme solution (10 µl) was added to each well with a final concentration of 0 to 12.5 ppm. The plate was then incubated for 24 hours at 32oC while shaking at 250 rpm. After 24 hours, the plate was briefly mixed to ensure that the particles were suspended. Then, the plate was read at 260 nm. As shown in Figure 2, AspAmy14 showed better performance compared to AcAA in terms of removing insoluble starch at pH both 3.7 and 4.5, especially at lower enzyme dosages. EXAMPLE 10 Evaluation of alpha-amylase by simultaneous saccharification and fermentation (SSF)

[0134] AspAmy14 and AcAA were evaluated for their performance under conditions (at pH 4.4, 32 ° C) that are intended to represent industrial simultaneous saccharification and fermentation (SSF) conditions. Liquefied corn (34.85% dry solids) was stored at -20oC until thawed for use. H2SO4 was added to adjust the pH to 4.4 and solid urea was added at 600 ppm. Dry yeast (Ethanol Red, Lesaffre Advanced Fermentations, France, no 42138) was hydrated by adding 1 g to 4 ml of water and incubated for 10 min before resuspension and addition to liquefied at a dilution of 1: 200. The liquefied (0.4 ml) was added to each well of a 96-well microtiter plate containing a protease (Fermgen, Dupont, 0.124 SAPU / g dry solids), a variant of T. reesei glycoamylase (3, 5 µg / g dry solid) and an alpha-amylase (0 to 36 µg / g dry solids). The plate was sealed to allow the gas to escape, but not to enter the wells, and placed in a forced air incubator at 32 ° C while shaking at 300 rpm.

[0135] The reaction was stopped at 47 or 69 hours by adding 0.4 ml. H2SO4 0.02 N with stirring, followed by centrifugation and collection of the supernatant which was filtered through 0.2 micron membranes. The ethanol content was determined by HPLC using a Rezex RFQ-Fast Acid column (Phenomenex) with a 0.01 N H2SO4 mobile phase. The ethanol produced during fermentation is given in table 7.

[0136] SAPU: A unit of spectrophotometric acid protease (SAPU) is the enzymatic activity that will release 1μmol of tyrosine per minute under the specified conditions (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 by spectrophotometry.

[0137] Under the conditions of this assay, AspAmy14 performs better than AcAA under most concentrations, especially at the highest concentrations. Table 7. SSF performance of AspAmy14 and AcAA

% EtOH (v / v) 47 hours 69 hours µg / ml AspAmy14 AcAA AspAmy14 AcAA 12.5 10.5 9.1 12.0 11.3 6.3 10.9 8.7 13.1 11.2 3 , 1 9.5 7.3 12.5 10.1 1.6 8.1 6.6 11.5 8.8 0.8 6.7 5.9 10.0 7.9 0.4 5.9 5.5 8.7 7.5 0.2 5.0 4.9 7.6 7.1 0.0 4.4 4.7 6.6 6.6 6.8 EXAMPLE 11 Sequence analyzes of alpha-amylase proteins mature As- pAmy14

[0138] Related proteins have been identified by a BLAST survey (Altschul et al., Nucleic Acids Res, 25: 3,389 to 3,402, 1997) using the mature amino acid sequences for AspAmy14 (SEQ ID NO: 3) against banks of Public and Genome Quest Patent data with search parameters set to default values and a subset is shown in Tables 8A and 8B. The percent identity (PID) for both sets of research is defined as the number of identical residues divided by the number of residues aligned in the alignment of sequence pairs. The values identified with "Sequence length" in the tables correspond to the length (in amino acids) for the proteins named with the accession numbers listed, while "Aligned length" refers to the sequence used for PID alignment and calculation. Table 8A. List of sequences with percent identity to the mature AspAmy14 sequence identified from the NCBI non-redundant protein database

Length of Access Length to the NCBI PID Organism Sequence Alignment XP_001209405.1 81.7 Aspergillus terreus NIH2624 607 638 GAQ07461.1 82.3 Aspergillus lentulus 630 638 EDP53736.1 80.6 Aspergillus fumigatus A1163 630 638 XP_749208.1 80.6 fumigatus Af293 630 638 XP_001265628.1 80.7 Aspergillus fischeri NRRL 181 632 638 OXN35790.1 80.6 Aspergillus turcosus 631 638 OXN21095.1 80.7 Aspergillus turcosus 631 638 OXS03711.1 80.7 Aspergillus 638 thermomatus 638 , 3 Aspergillus udagawae 620 638 Table 8B. List of sequences with percent identity to the mature AspAmy14 sequence identified from the Genome Quest database Identifier GQ PID Organism Length Length of Individual alignment WO2014099415-BBI91800 90.5 Aspergillus terreus NIH2624 477 476 WO2014093125-BBI74751 87.1 Aspergillus fumigatus 479 479 WO2014078588-0160 82.5 Aspergillus terreus 587 638 WO2017112631-0022 82.1 Aspergillus terreus 587 638 WO2015094809-0014 82.1 Chimeric alpha-amylase from Aspergillus niger and Aspergillus terreus 607 638 WO2017112635-246 US20150240223-0007 81.8 Neosartorya fischeri NRRL 181 632 655 US20150337277-0004 81.7 Artificial Sequence 643 638 WO2014058572-0004 81.4 Aspergillus fumigatus Af293 607 638 US8343747-0052 80.7 Aspergillus terreus 607 65200262660 terreus 607 652 WO2015094809-0014 80.2 chimeric alpha-amylase from Aspergillus niger and Aspergillus terreus 606 652 US20160010128-0007 79.8 Neosartorya fisc hero NRRL 181 632 655 US20160032338-0001 79.2 Aspergillus fumigatus Af293 630 655 US20150337277-0006 76.1 Artificial Sequence 628 657

[0139] An alignment of the mature AspAmy14 sequences (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); OXS03711.1 (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 standard parameters using the MUSCLE program from the Geneious software (Biomatters Ltd.) (Robert C.

E d g a r.

MUSCLE: multiple sequence alignment with high accuracy and high throughput Nucl.

Acids Res. (2004) 32 (5): 1,792 to 1,797. The alignment of multiple sequences of mature AspAmy14 alpha amylase and several other homologous sequences is shown in Figure 3.

Claims (27)

1. Polypeptide characterized by the fact that it has alpha-amylase activity selected from the group consisting of: (a) a polypeptide that comprises an amino acid sequence that preferably has at least 91% identity with the SEQ polypeptide ID NO: 3; (b) a polypeptide comprising an amino acid sequence that preferably has at least 91% identity to a catalytic domain of SEQ ID NO: 3; (c) a polypeptide comprising an amino acid sequence that preferably has at least 91% identity with 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 to high stringency conditions, with maximum preference, at least high stringency and, even more preferably, at least very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, (ii) the genomic DNA sequence that comprises 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 that preferably has at least 91% identity with the polypeptide coding sequence of SEQ ID NO: 3; (f) a variant comprising a substitution, deletion and / or insertion of one or more (for example, several) amino acids of the polypeptide of SEQ ID NO: 3; (g) a mature polypeptide produced by processing the polypeptide of SEQ ID NO: 2 by a post-translational signaling or modification peptidase during secretion from an expression host; and (h) a fragment of a polypeptide from (a), (b), (c), (d), (e), (f) or (g) that has alpha-amylase activity. 2. Polynucleotide characterized by the fact that it comprises a nucleotide sequence that encodes the polypeptide, as defined in claim 1. 3. Vector characterized by the fact that it comprises the polynucleotide, as defined in claim 2, functionally linked to one or more control sequences that control the production of the polypeptide in an expression host. 4. Recombinant host cell characterized by the fact that it comprises the polynucleotide, as defined in the claim two. 5. Host cell, according to claim 4, characterized by the fact that it is an ethanological microorganism. 6. Host cell according to claim 4 or 5, characterized by the fact that it also expresses and secretes one or more additional enzymes selected from the group comprising a protease, hemicellulase, cellulase, peroxidase, lipolytic enzyme, xylan- whether, lipase, phospholipase, esterase, perhydrolase, cutinase, pectinase, lactate, mannase, keratinase, reductase, oxidase, phenoloxidase, lyase poxygenase, ligninase, glycoamylase, pullulanase, phytase, tanase, pento-sanase, malanase, beta-glucanase, arabinosidase, hyaluronidase, chondroitininase, laccase, transferase or a combination thereof. 7. Composition characterized by the fact that it comprises the polypeptide, as defined in claim 1. 8. Composition according to claim 7, characterized by the fact that it comprises a protease, hemicellulase, cellulose, peroxidase, lipolytic enzyme, xylanase, lipase, phospholipase, esterase, peridrolase, cutinase, pectinase, pectate lyase, mannanase, keratinase, reductase, oxidase, phenoloxidase, lipoxygenase, ligninase, glycoamine, pululanase, phytase, tanase, pentosanase, malanase, beta-glucinase, arabinosidase, hyaluronidase, chondroitinase, laccase, transferase or the same. 9. Method for producing a polypeptide that has alpha-amylase activity characterized by the fact that it comprises: (a) cultivating the host cell, as defined in claim 4, under conditions conducive to the production of the polypeptide; and (b) recovering the polypeptide. 10. Method characterized by the fact that it is intended to treat a material containing starch with the polypeptide that has alpha-amylase activity, as defined in claim 1. 11. Method for saccharifying a starch substrate characterized by the fact that it comprises a) putting the starch substrate in contact with the polypeptide that has alpha-amylase activity, as defined in claim 1; and b) saccharify the starch substrate to produce the saccharides that comprise glucose. 12. Method according to claim 11, characterized by the fact that saccharifying the starch substrate results in a syrup with a high glucose content. 13. Method according to claim 11 or 12, characterized by the fact that 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. Method according to any one of claims 10 to 12, characterized by the fact that it also comprises fermenting syrup with a high glucose content in a final product. 15. Method, according to claim 14, characterized by the fact that saccharification and fermentation are carried out as a simultaneous saccharification and fermentation (SSF) process. 16. Method according to claim 14 or 15, characterized by the fact that the final product is alcohol, for example, ethanol. 17. Method according to claim 14 or 15, characterized by the fact that the final product is a biochemical product selected from the group consisting of an amino acid, an organic acid, citric acid, lactic acid , succinic acid, monosodium glutamate, glyconic acid, sodium gluconate, calcium gluconate, potassium gluconate, delta-lactone glucone, sodium erythorbate, omega 3 fatty acid, butanol, lysine, itaconic acid, 1,3-propanediol , biodiesel and isoprene. 18. Method according to any one of claims 11 to 17, characterized in that the starch substrate is about 5% to 99%, 15% to 50% or 40 to 99% dry solid (DS). 19. Method according to any of the claims 11 to 18, characterized by the fact that the starch substrate is selected from wheat, barley, corn, rye, rice, sorghum, bran, cassava, milo, millet, potato, sweet potato, tapioca and any combination thereof . 20. Method according to any one of claims 11 to 19, characterized in that the starch substrate comprises liquefied starch, gelatinized starch or granular starch. 21. Method according to any one of claims 11 to 20, characterized in that it further comprises adding a hexokinase, a xylanase, a glucose isomerase, a xylose isomerase, a phosphatase, a phytase, a pullulanase, a beta-amylase, a glycoamylase, a protease, a cellulase, a hemicellulase, a lipase, a cutinase, a trehalase, an isoamylase, a redox enzyme, an esterase, a transferase, a pectinase, a hydrolase, an alpha-glucosidase, a beta-glycosidase or a combination of them to the starch substrate. 22. Method for applying the method, as defined in any one of claims 11 to 21, characterized by the fact that it is intended for the production of saccharides. 23. Saccharide characterized by the fact that it is produced by the method, as defined in claim 22. 24. Method for saccharifying and fermenting a starch substrate to produce a final product characterized by the fact that it comprises a) putting the starch substrate in contact with the polypeptide that has alpha-amylase activity, as defined in claim 1; b) saccharify the starch substrate to produce the saccharides that comprise glucose; and c) placing the saccharides in contact with a fermentation organism to produce a final product. 25. Method according to claim 24, characterized by the fact that fermentation is carried out as a process of simultaneous stripping and fermentation (SSF). 26. Method, according to claim 24 or 25, characterized by the fact that the final product is alcohol, for example, ethanol. 27. Method according to claim 24 or 25, characterized by the fact that the final product is a biochemical product selected from the group consisting of an amino acid, an organic acid, citric acid, lactic acid , succinic acid, monosodium glutamate, glyconic acid, sodium gluconate, calcium gluconate, potassium gluconate, delta-lactone glucone, sodium erythorbate, omega 3 fatty acid, butanol, lysine, itaconic acid, 1,3-propanediol , biodiesel and isoprene.