Amylase variants

Abstract

Genetically engineered enzymes having amylase activity, compositions containing the enzymes, and methods of making and using the enzymes. Genetically engineered amylase enzymes are useful in laundry detergents, dishwashing detergents, and in ethanol production, starch processing, paper processing, and pulp, animal feed, baking, car care, industrial, and household cleaning products. It is useful in many different applications, such as:

Description

[Technical Field]

[0001] The present invention provides novel amylase enzymes, more specifically, genetically engineered amylase enzymes, compositions containing the enzymes, methods of using the enzymes, or compositions containing the enzymes. [Background technology]

[0002] Genetically engineered amylase enzymes are useful in many different applications, such as laundry detergents, dish detergents, and cleaning products for ethanol production, starch processing, paper processing and pulp, animal feed, baking, car care, industrial, and household use. Amylases are used in the removal of starch stains and are added to various compositions, such as cleaning products. Many of these applications require the use of amylases that are stable at high temperatures or under denaturing conditions. Thus, there is a need for genetically engineered amylase enzymes with improved properties, particularly improved stability and improved performance. Summary of the Invention

[0003] The present invention relates to an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprising A and B domains and a C domain, wherein the amino acid sequences of the A and B domains are at least 75% identical to the amino acid sequence of SEQ ID NO:42 and the amino acid sequence of the C domain is at least 75% identical to the amino acid sequence of SEQ ID NO:44.

[0004] Preferably, the polypeptide having alpha-amylase activity consists of A and B domains and a C domain, wherein the amino acid sequences of the A and B domains are at least 75% identical to the amino acid sequence of SEQ ID NO: 42, and the amino acid sequence of the C domain is at least 75% identical to the amino acid sequence of SEQ ID NO: 44.

[0005] Preferably, the A and B domains of the polypeptide having alpha-amylase activity have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the A and B domains having the amino acid sequence of SEQ ID NO:42.

[0006] Preferably, the C domain of the polypeptide having alpha-amylase activity has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to a C domain having the amino acid sequence of SEQ ID NO:44.

[0007] Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity contains substitutions, deletions, and / or insertions at one or more positions.

[0008] Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises the sequence: TQXDYLDHPDVIGWTREGDXXHXXSGLAXLMSDGPXGXKWMXVGKNNAGEXWXDITGNQTNTVTINXDGXGQFXVXXGSXSIYXQX, where X can be any amino acid.

[0009] Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity is selected from 402R,H; 419S,G,D; 420V,I; 422A,V; 423N,D,G,K; 428T,A; 435G,R,E; 437S,A; 441N,E,D; 444K,E; 450V,I; 452Y,H,R; 466K,R; 469W,S; 473H,Q,R; 475S,N; 476G,E; 479V,A; 483V,I; and 485R,Q,K according to the numbering of SEQ ID NO:39, preferably 402R; 419D; 420V; 422A; and 485Q.

[0010] Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises a substitution at one or more positions selected from the group consisting of 430 and 454 according to the numbering of SEQ ID NO: 39, and preferably the amylase of the invention comprises a substitution resulting in amino acid residues 430M and / or 430I, preferably 430M and / or 451I, preferably a substitution selected from the group consisting of I430M and M454I.

[0011] Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid residue at one or more amino acid positions selected from the group consisting of 401, 403, 405, 411, 413, 415, 424, 426, 428, 432, 455, 477, 479, and 481 (according to the numbering of SEQ ID NO:39), as present in SEQ ID NO:39.

[0012] Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid residue at one or more amino acid positions selected from the group consisting of 309, 313, 347, 348, 350, 351, 354, 355, 358, 359, 388, 389, 392 and 396 (according to the numbering of SEQ ID NO:39), as present in SEQ ID NO:40.

[0013] Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises a deletion of one or more amino acids corresponding to positions 181, 182, 183, and 184 according to the numbering of SEQ ID NO:39, preferably a deletion of two or more amino acids corresponding to positions 181, 182, 183, and 184, preferably a deletion of amino acids corresponding to positions 181 and 182, 182 and 183, or 183 and 184 (according to the numbering of SEQ ID NO:39).

[0014] Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises a substitution at one or more positions selected from the group consisting of 9, 130, 195, 206, 244, 202, 179, 181, 186, and 190 according to the numbering of SEQ ID NO:39, preferably one or more substitutions selected from the group consisting of M9L, E130V, N195F, I206L, S244Q, M202L, K179L, R181E, G186E / N / Q / S and E190P according to the numbering of SEQ ID NO:39.

[0015] Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity is (a) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37; (b) an amino acid sequence encoded by a polynucleotide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:55, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38; (c) under high stringency conditions; (i) a sequence encoding SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37; or (ii) a polynucleotide set forth in SEQ ID NO:55, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38 an amino acid sequence encoded by a polynucleotide that hybridizes to the complement of; or (d) a fragment of (a), (b), or (c) having amylase activity. Includes:

[0016] Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises increased expression, activity, thermostability, stability, laundry performance, specific activity, substrate specificity, pH-dependent activity, pH-dependent stability, oxidative stability, Ca2+ dependence, or any combination thereof, compared to the amylase set forth in SEQ ID NO:39 or SEQ ID NO:40, and preferably the amylase has increased thermostability compared to the amylase set forth in SEQ ID NO:39 or SEQ ID NO:40.

[0017] The present invention also provides (a) a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:55, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38, wherein the nucleic acid encodes a polypeptide having amylase activity; (b) a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, wherein the polypeptide has amylase activity, or a nucleic acid sequence encoding any polypeptide described herein with amylase activity; (c) under high stringency conditions; (i) a sequence encoding SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37; or (ii) a polynucleotide set forth in SEQ ID NO:55, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38 a polynucleotide that hybridizes to the complement of; (d) a fragment of (a), (b), or (c), wherein the fragment encodes a polypeptide having amylase activity; or (e) a nucleic acid sequence that is completely complementary to any of (a) to (d) The present invention relates to an isolated, synthetic, or recombinant nucleic acid comprising:

[0018] The present invention also relates to nucleic acid constructs comprising the polynucleotides described herein.

[0019] The present invention also relates to expression vectors comprising the polynucleotides or nucleic acid constructs described herein.

[0020] The present invention also relates to a host cell comprising a polynucleotide described herein, a nucleic acid construct described herein, or an expression vector described herein.

[0021] The present invention also relates to compositions comprising isolated, synthetic, or recombinant polypeptides having alpha-amylase activity as described herein.

[0022] Preferably, the composition further comprises at least one second enzyme selected from the group consisting of a second amylase, a lipase, a protease, a cellulase, a laccase, a mannase, a pectinase, a xylanase, and a nuclease.

[0023] The present invention also relates to methods for making an isolated, synthetic, or recombinant polypeptide having an alpha-amylase as described herein, comprising providing a nucleic acid sequence encoding the polypeptide, transforming the nucleic acid sequence into an expression host, culturing the expression host to produce the polypeptide, and, optionally, purifying the polypeptide.

[0024] The present invention also relates to methods for preparing dough or baked products prepared from dough, comprising adding an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity described herein to dough and baking it.

[0025] The present invention also relates to methods of using the isolated, synthetic, or recombinant polypeptides with alpha-amylase activity described herein to process starch, clean or wash textiles, hard surfaces, or tableware, make ethanol, process pulp or paper, or feed animals.

[0026] The present invention also relates to a method for making an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity, comprising the step of making a hybrid from at least two different amylases, wherein the hybrid comprises A and B domains and a C domain, and wherein the amino acid sequences of the A and B domains are at least 75% identical to the amino acid sequence of SEQ ID NO:42 and the amino acid sequence of the C domain is at least 75% identical to the amino acid sequence of SEQ ID NO:44.

[0027] The present invention also relates to a method of use of a C domain of a first amylase, wherein the C domain has an amino acid sequence at least 75% identical to the amino acid sequence of SEQ ID NO: 44, for improving one or more properties selected from the group consisting of stability, pH profile, expression, activity, thermostability, specific activity, substrate specificity, pH-dependent activity, pH-dependent stability, oxidative stability, Ca2+ dependence, performance in laundry, processing starch, cleaning textiles, cleaning hard surfaces, cleaning dishware, making ethanol, processing pulp or paper, and feeding animals, of a second alpha-amylase having A and B domains at least 75% identical to the amino acid sequence of SEQ ID NO: 42, wherein the use comprises replacing the C domain of the second alpha-amylase with the C domain of the first alpha-amylase. [Brief explanation of the drawings]

[0028] [Figure 1] % remaining activity after challenge for SEQ ID NOs: 40, 41, 54, and 9 is shown. DETAILED DESCRIPTION OF THE INVENTION

[0029] An enzyme is a biological molecule (polypeptide) that contains a sequence of amino acid residues and can catalyze a reaction. Therefore, an enzyme is a catalytically active protein or polypeptide. The name of an enzyme is determined based on the recommendations of the Nomenclature Commission of the International Union of Biochemistry and Molecular Biology (IUBMB). An enzyme is defined by its Enzyme Commission (EC) number, recommended name, synonyms (if any), catalytic activity, and other factors. Enzymes herein can be identified by their polypeptide sequence (also referred to herein as amino acid sequence). The polypeptide sequence specifies the three-dimensional structure of the enzyme, including its "active site," which in turn determines its catalytic activity. The polypeptide sequence can be identified by its SEQ ID NO.

[0030] Enzymes can be obtained or derived from many different sources, including plants, animals, bacteria, archaea, fungi, yeast, and environmental samples that contain enzyme-encoding DNA, or they can be synthetically produced in the laboratory. For example, bacterial sources of enzymes include enzymes from Bacillus, Streptomyces, E. coli, and Pseudomonas; fungal sources of enzymes include enzymes from Aspergillus, Fusarium, Thermomyces, and Trichoderma; yeast sources of enzymes include enzymes from Pichia and Saccharomyces.

[0031] The World Intellectual Property Organization (WIPO) Standard ST.25 (1998) states that the following three-letter symbols, with the first letter capitalized, should be used to represent amino acid residues in sequence listings: The following table provides an overview of amino acid identifiers and the corresponding DNA codons that encode the amino acids, using standard genetic standards: The DNA codons that encode amino acid residues may differ depending on the organism used, and slightly different tables for translating the genetic code may apply. A compilation of such non-standard code translation tables is maintained at NCBI.

[0032] A "parent" polypeptide amino acid sequence is a starting sequence for introducing mutations into the sequence (e.g., by introducing one or more amino acid substitutions, insertions, deletions, or a combination thereof) that result in a "variant" of the parent polypeptide amino acid sequence. Parents include wild-type or synthetically produced polypeptide amino acid sequences that are used as starting sequences for introducing (further) changes.

[0033] A "variant polypeptide" refers to an enzyme whose amino acid sequence differs from its parent. The difference between the parent and variant polypeptides can be one single amino acid residue, or more than one amino acid residue (two or more amino acid residues). The more than one amino acid residue can be consecutive amino acid residues or non-conserved amino acid residues. The consecutive amino acid residues can be four consecutive amino acid residues; five consecutive amino acid residues; eight consecutive amino acid residues; nine consecutive amino acid residues; eleven consecutive amino acid residues; thirteen consecutive amino acid residues; or fourteen consecutive amino acid residues. While the following definition describes variant in the context of amino acid changes, nucleic acids can be similarly modified, for example, by substitution.

[0034] "Mature polypeptide" refers to an enzyme in its final form, including any post-translational modifications, glycosylation, phosphorylation, truncation, N-terminal modifications, C-terminal modifications, signal sequence deletion, etc. The mature polypeptide may vary depending on the expression system, vector, promoter, and / or production process.

[0035] A "synthetic" or "man-made" compound is produced by chemical or enzymatic synthesis in vitro.

[0036] The term "non-naturally occurring" refers to a (poly)nucleotide, amino acid, (poly)peptide, enzyme, protein, cell, organism, or other material that is not found in its original, naturally occurring environment or source. Preferably, the amylase of the present invention is a non-naturally occurring amylase.

[0037] Variant polynucleotide and variant polypeptide sequences can be defined by their sequence identity when compared with parent sequence.Sequence identity is usually provided as "sequence identity%" or "identity%".For calculating sequence identity, the first step is to make sequence alignment.According to the present invention, pairwise overall alignment is made, which means that two sequences are aligned over their entire length, and is usually made by using a mathematical method called alignment algorithm.

[0038] According to the present invention, alignments are generated by using the algorithm of Needleman and Wunsch (J. Mol. Biol. (1979) 48, pp. 443-453). Preferably, the "NEEDLE" program (The European Molecular Biology Open Software Suite (EMBOSS)) is used for the purposes of the present invention, using the program's default parameters (polynucleotides: gap open=10.0, gap extension=0.5, and matrix=EDNAFULL; polypeptides: gap open=10.0, gap extension=0.5, and matrix=EBLOSUM62).

[0039] After aligning the two sequences, in a second step an identity score is determined from the resulting alignment.

[0040] For this purpose, % identity is calculated by dividing the number of identical residues by the length of the alignment region showing the corresponding sequence of the invention in its entirety and multiplying by 100: % identity = (identical residues / length of alignment region showing the corresponding sequence of the invention in its entirety). * 100.

[0041] To calculate the percent identity of two nucleic acid sequences, the same applies as for calculating the percent identity of two amino acid sequences, with some specifications. For nucleic acid sequences encoding proteins, pairwise alignment should be performed over the full length of the coding region of the sequence of the present invention, from the start codon to the stop codon, excluding introns. Introns present in the other sequence to which the sequence of the present invention is compared may be removed for pairwise alignment. The percent identity is then calculated as follows: % identity = (identical residues / length of the aligned region showing the sequence of the present invention over its full length, from the start codon to the stop codon, excluding introns). *100. After aligning the two sequences, in a second step an identity value is determined from the resulting alignment.

[0042] Furthermore, a preferred alignment program for nucleic acid sequences that implements the Needleman and Wunsch algorithm (J. Mol. Biol. (1979) 48, pp. 443-453) is "NEEDLE" (The European Molecular Biology Open Software Suite (EMBOSS)) using the program's default parameters (gap open=10.0, gap extension=0.5, and matrix=EDNAFULL).

[0043] Sequences having regions identical or similar to the sequences of the present invention and to be compared to the sequences of the present invention to determine percent identity can be readily identified by a variety of methods within the skill in the art, for example, using publicly available computer methods and programs such as BLAST, BLAST-2, etc., available at NCBI.

[0044] Variant polypeptides can be defined by their sequence similarity when compared to a parent sequence. Sequence similarity is usually provided as "sequence similarity %" or "similarity %". Sequence similarity % takes into account that a defined set of amino acids share similar properties, for example, by their size, by their hydrophobicity, by their charge, or by other characteristics. Herein, the replacement of an amino acid with a similar amino acid can be referred to as a "conservative mutation". Similar amino acids according to the present invention are defined as follows and should also be applied to the determination of similarity % according to the present invention, for example, according to the BLOSUM62 matrix used by the "NEEDLE" program, which is one of the most used amino acid similarity matrices for database searching and sequence alignment: Amino acid A is similar to amino acid S Amino acid D is similar to amino acid E; N Amino acid E is similar to amino acids D; K; and Q Amino acid F is similar to amino acid W; Y Amino acid H is similar to amino acid N; Y Amino acid I is similar to amino acids L; M; and V The amino acid K is similar to the amino acids E, Q, and R. Amino acid L is similar to amino acids I; M; and V Amino acid M is similar to amino acids I; L; and V The amino acid N is similar to the amino acids D; H; and S. The amino acid Q is similar to the amino acids E; K; and R. The amino acid R is similar to the amino acids K and Q. The amino acid S is similar to the amino acids A, N, and T. The amino acid T is similar to the amino acid S Amino acid V is similar to amino acids I; L; M Amino acid W is similar to amino acid F; Y The amino acid Y is similar to the amino acids F; H; W.

[0045] Conservative amino acid substitutions may occur throughout the entire polypeptide sequence of a functional protein, such as an enzyme. In one embodiment, such mutations do not involve functional domains of the enzyme. In one embodiment, conservative mutations do not involve the catalytic center of the enzyme.

[0046] To calculate sequence similarity, in the first step, a sequence alignment is generated as described above. After aligning the two sequences, in the second step, a similarity value is determined from the generated alignment.

[0047] For this purpose, % similarity is calculated by dividing the number of identical residues plus the number of similar residues by the length of the alignment region showing the sequence of the invention in its entirety and multiplying by 100: % similarity = [(identical residues + similar residues) / length of the alignment region showing the sequence of the invention in its entirety]. * 100.

[0048] The present invention relates to polypeptides having amylase activity comprising an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to any one of the full-length amino acid sequences of SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37.

[0049] The present invention further relates to polynucleotides encoding the variant polypeptides of the present invention. The terms "polynucleotide," "nucleic acid sequence," "nucleotide sequence," "nucleic acid," and "nucleic acid molecule" are used interchangeably herein and refer to a polymeric, unbranched form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, or a combination of both. A "gene" is a DNA segment that carries a specific genetic information.

[0050] A "parent" polynucleotide acid sequence is a starting sequence for introducing mutations into the sequence that result in a "variant" of the parent polynucleotide sequence. A "variant polynucleotide" refers to a polynucleotide that encodes an enzyme, where the variant polynucleotide differs in its nucleic acid sequence from its parent polynucleotide.

[0051] In one aspect, the polynucleotides of the present invention have a nucleic acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical when compared to any one of the full-length polynucleotide sequences of SEQ ID NO:55, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38.

[0052] Preferably, the polynucleotide is a codon-optimized polynucleotide for improved expression in a particular host cell.

[0053] A "substitution" is described by providing the original amino acid, followed by the position number within the amino acid sequence, followed by the substituted amino acid. A particular amino acid residue may be substituted with any of 19 amino acid residues different from the original. For example, a substitution of histidine at position 120 with alanine is designated "His120Ala" or "H120A." Alternative substitutions at amino acid positions are designated as follows: "His120Ala,Leu" or "H120A,L." It is understood herein that alternative substitutions using conservative amino acid substitutes can be used in place of the specific substitutions shown.

[0054] Amino acid deletions are indicated by the original amino acid of the parent enzyme, followed by the position number in the amino acid sequence, followed by * Thus, the deletion of glycine at position 150 is described as "Gly150 * " or "G150 * Alternatively, the deletion may be indicated by, for example, "deletion of D183 and G184."

[0055] Amino acid insertions are described by providing the original amino acid of the parent enzyme, then the position number in the amino acid sequence, followed by the original amino acid and the additional amino acid. For example, an insertion of a lysine at position 180 following a glycine would be designated "Gly180GlyLys" or "G180GK." If more than one amino acid residue is inserted, for example, a Lys and an Ala after Gly180, this can be designated as Gly180GlyLysAla or G195GKA.

[0056] If the substitution and insertion occur at the same position, this can be designated as S99SD+S99A or abbreviated as S99AD.

[0057] One or more amino acid substitutions in a variant polypeptide may be one or more conservative amino acid substitutions. "Conservative amino acid substitution" or "related amino acid" refers to the replacement of an amino acid residue in an amino acid sequence with a different amino acid residue that has similar properties at the same position compared to the parent amino acid sequence. Some examples of conservative amino acid substitutions include, but are not limited to, replacing a positively charged amino acid residue with a different positively charged amino acid residue; replacing a polar amino acid residue with a different polar amino acid residue; replacing a non-polar amino acid residue with a different non-polar amino acid residue; replacing a basic amino acid residue with a different basic amino acid residue; or replacing an aromatic amino acid residue with a different aromatic amino acid residue.

[0058] "Enzyme activity" refers to at least one catalytic effect exhibited by an enzyme. Enzyme activity is expressed as units per milligram of enzyme (specific activity) or molecules of substrate converted per minute per molecule of enzyme (molar activity). Enzyme activity can be specified by the actual function of the enzyme, such as proteases, which exhibit proteolytic activity by catalyzing the hydrolytic cleavage of peptide bonds; lipases, which exhibit lipolytic activity by hydrolytic cleavage of ester bonds; and amylase activity, which involves the (internal) hydrolysis of glycosidic bonds in polysaccharides.

[0059] Enzyme activity may change during storage or operational use of the enzyme. The term "enzyme stability" relates to the retention of enzyme activity as a function of time during storage or operation. The term "storage" herein is meant to refer to the fact that a product or composition or formulation is stored from the time it is manufactured to the time it is used in its final application. The retention of enzyme activity as a function of time during storage can be referred to herein as "storage stability."

[0060] To determine and quantify changes in the catalytic activity of an enzyme stored or used under certain conditions over time, the "initial enzyme activity" is measured under the specified conditions at time zero (100%) and at a certain later time point (x%). By comparing the measured values, the extent of potential loss of enzyme activity can be determined. The extent of enzyme activity loss determines the enzyme stability or instability.

[0061] Parameters that influence the enzymatic activity and / or storage and / or operational stability of the enzyme are, for example, pH, temperature, chelating agents, and the presence of oxidizing substances.

[0062] The variant polypeptides may be active over a wide range of pH, at any single point within the range of about pH 4.0 to about pH 12.0. The variant polypeptide enzymes may be active over the ranges of pH 5.0 to pH 11.0, pH 6.0 to pH 10.0, and pH 7.0 to pH 9.0. In other embodiments, the variant polypeptide enzymes may be active over the ranges of pH 7.1 to pH 8.9, pH 7.2 to pH 8.8, pH 7.3 to pH 8.7, pH 7.4 to pH 8.6, and pH 7.5 to pH 8.5. Variant polypeptides have pH 4.0, pH 4.1, pH 4.2, pH 4.3, pH 4.4, pH 4.5, pH 4.6, pH 4.7, pH 4.8, pH 4.9, pH 5.0, pH 5.1, pH 5.2, pH 5.3, pH 5.4, pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, pH 8.0, pH 8.1, pH 8.2, pH 8.3, pH 8.4, pH 8.5, pH 8.6 pH 8.7, pH 8.8 pH 8.9, pH 9.0, pH 9.1, pH 9.2, pH 9.3, pH 9.4, pH 9.5, pH 9.6, pH 9.7, pH 9.8, pH 9.9, pH 10.0, pH 10.1, pH 10.2, pH 10.3, pH 10.4, pH 10.5, pH 10.6, pH 10.7, pH 10.8, pH 10.9, pH 11.0, pH 11.1, pH 11.2, pH 11.3, pH 11.4, pH 11.5, pH 11.6, pH The compound may be active at pH 11.7, pH 11.8, pH 11.9, pH 12.0, pH 12.1, pH 12.2, pH 12.3, pH 12.4, and at pH 12.5, pH 12.6, pH 12.7, pH 12.8, pH 12.9, or higher.

[0063] "pH stability" refers to the ability of an enzyme to exhibit enzymatic activity over a particular pH range.

[0064] The variant polypeptides may be active over a wide range of temperatures, with temperatures anywhere within the range of about 10° C. to about 95° C. The variant polypeptides may be active at temperatures ranging from 10° C. to 55° C., 10° C. to 50° C., 10° C. to 45° C., 10° C. to 40° C., 10° C. to 35° C., 10° C. to 30° C., or 10° C. to 25° C. The variant polypeptides may be active at temperatures ranging from 20° C. to 55° C., 20° C. to 50° C., 20° C. to 45° C., 20° C. to 40° C., 20° C. to 35° C., 20° C. to 30° C., or 20° C. to 25° C. The variant polypeptide may be at least 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, and are active at temperatures of 3°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, or higher.

[0065] The terms "thermostable" and "thermostable" refer to the ability of a protein to exhibit catalytic activity over a particular temperature range. Enzyme thermostability is defined as the ability of a protein to exhibit catalytic activity over a particular temperature range. 50 It can be characterized by what is known as the T 50 indicates the temperature at which 50% residual enzyme activity is still present after heat inactivation for a certain time, when compared to a reference sample that has not undergone heat treatment.

[0066] In one embodiment, the variant polypeptide has improved thermal stability compared to the parent molecule. In another embodiment, the variant polypeptide has improved thermal stability by 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, or more degrees Celsius compared to the parent polypeptide. In another embodiment, the increase in thermal stability is measured at a temperature between 65°C and 100°C. The increase in thermal stability can be measured at 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, and / or 100°C. In another embodiment, the increase in thermal stability is measured at a temperature of 70°C. In another embodiment, the increase in thermal stability is measured at a temperature of 80°C. In another embodiment, the increase in thermal stability is measured at a temperature of 90°C. In one embodiment, thermal stability is improved at 70°C, 80°C, or 90°C, preferably 70°C. In another embodiment, the thermal stability is improved in the temperature range of 65°C to 90°C, preferably 70°C to 85°C, preferably 70°C to 80°C.

[0067] In one embodiment, the variant polypeptide is a fragment of the full-length amino acid sequence, wherein the fragment has amylase activity.

[0068] As used herein, a "fragment" or "subsequence" is a portion of a polynucleotide or amino acid sequence.

[0069] The term "functional fragment" refers to any nucleic acid or amino acid sequence, respectively, that comprises only a portion of a full-length amino acid sequence but still possesses the same or a similar activity and / or function. Preferably, the functional fragment is at least 75% identical, at least 76% identical, at least 77% identical, at least 78% identical, at least 79% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 98.5% identical, at least 99% identical, or at least 99.5% identical to the original sequence of the full-length amino acid sequence. A functional fragment comprises contiguous nucleic acids or amino acids compared to the original nucleic acid or original amino acid sequence, respectively.

[0070] A-, B- and C-domains: The structure of alpha-amylases comprises three distinct domains, A, B, and C; see, e.g., Machius et al., 1995, J. Mol. Biol. 246: 545-559. The term "domain" refers to a region of a polypeptide that forms a distinct and independent substructure of the whole molecule. Alpha-amylases consist of a beta / alpha-8 barrel carrying the active site residues, called the A domain, a fairly long loop between the beta sheet and alpha helix 3, called the B domain (together, the "A and B domains"), and a C domain and, in some cases, also additional carbohydrate-binding domains (e.g., WO2005 / 001064; Machius et al., supra).

[0071] The domains of an alpha-amylase can be determined by structural analysis, such as using crystallographic techniques. An alternative method for determining the domains of an alpha-amylase is by sequence alignment of the amino acid sequence of the alpha-amylase with another alpha-amylase whose domains have been determined. For example, a sequence that aligns with the C domain sequence in an alpha-amylase whose C domain has been determined can be considered the C domain of the given alpha-amylase.

[0072] A and B domains: As used herein, the term "A and B domains" means that these two domains are taken as one unit, while the C domain is a separate unit of the alpha-amylase. Thus, the amino acid sequence of the "A and B domains" is understood as one continuous sequence or a portion of a sequence of an alpha-amylase that includes the "A and B domains" and other additional domains (such as the C domain). Thus, the term "A and B domains have at least 75% sequence identity to SEQ ID NO: 42" means that the amino acid sequences forming the A and B domains have at least 75% sequence identity to SEQ ID NO: 42. As used herein, the "A and B domains" of an alpha-amylase correspond to amino acids 1 to 399 of SEQ ID NO: 39.

[0073] AB domain donor: The term "AB domain" as used herein refers to the alpha-amylase from which the A and B domains are derived. Thus, for an A and B domain having the amino acid sequence of SEQ ID NO:42, the AB domain donor is the alpha-amylase of SEQ ID NO:39.

[0074] C domain: As used herein, the "C domain" of an alpha-amylase corresponds, for example, to amino acids 400-485 of SEQ ID NO: 39. Thus, the C domain of an alpha amylase can be found by aligning said alpha amylase with the alpha amylase of SEQ ID NO: 39. The portion of said alpha amylase that aligns with amino acids 400-485 of SEQ ID NO: 39 is, according to the present invention, the "C domain" of the alpha amylase. Thus, for example, the C domain of an alpha amylase having the amino acid sequence of SEQ ID NO: 40 is composed of amino acids 401-486, which are disclosed herein as SEQ ID NO: 44.

[0075] Carbohydrate-binding domain or carbohydrate-binding module (CBM): Amylases containing catalytic modules (A, B, and C domains) may further contain one or more non-catalytic CBMs (carbohydrate-binding modules, also called carbohydrate-binding domains, or specifically, amylase starch-binding domains). The CBMs can improve the binding of the enzyme to the substrate. The CBMs are attached to the C domain.

[0076] The alpha-amylases of the present invention comprise three domains, the A, B and C domains. Preferably, the amylases of the present invention do not comprise a carbohydrate-binding domain. Preferably, the alpha-amylases of the present invention consist of only three domains, the A, B and C domains.

[0077] The inventors have surprisingly found that a polypeptide which is a hybrid of A and B domains from a first alpha-amylase of SEQ ID NO: 39 or a variant thereof (the "AB domain donor") and a C domain from a second alpha-amylase of SEQ ID NO: 40 or a variant thereof (the "C domain donor") has improved properties compared to the AB domain donor alpha-amylase (SEQ ID NO: 39 or a variant thereof) and the C domain donor alpha-amylase (SEQ ID NO: 40 or a variant thereof), and / or to the alpha-amylase of SEQ ID NO: 41, which is an alpha-amylase of SEQ ID NO: 39 having a stability-improving mutation, i.e., a deletion at amino acid positions 182 and 183 according to the numbering of SEQ ID NO: 39.

[0078] The A and B domains of the alpha-amylase having the amino acid sequence of SEQ ID NO:39 were determined to correspond to amino acids 1-399. This sequence is also disclosed herein as SEQ ID NO:42. The C domain of the amino acid sequence of SEQ ID NO:39 was determined to correspond to amino acids 400-485 (disclosed herein as SEQ ID NO:43). The C domain of the alpha-amylase having the amino acid sequence of SEQ ID NO:40 was determined to correspond to amino acids 401-486 of SEQ ID NO:40, also disclosed herein as SEQ ID NO:44. Thus, in one embodiment of the present invention, a polypeptide having alpha-amylase activity is a hybrid of amino acids 1-399 of SEQ ID NO:39, or a variant thereof, and amino acids 401-486 of SEQ ID NO:40, or a variant thereof.

[0079] AB domain donor: In one embodiment, the A and B domains are obtained from an alpha-amylase comprising the amino acid sequence of SEQ ID NO: 39, the A and B domains of which are also disclosed herein as SEQ ID NO: 42. In one embodiment of the invention, the amino acid sequences forming the A and B domains have at least 75% identity, such as at least 78%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 42.

[0080] Other suitable AB domain donors are alpha-amylases closely related to the alpha-amylase of SEQ ID NO: 39. Preferably, the AB domain donor is an alpha-amylase having at least 75% identity, such as at least 78%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the alpha-amylase of SEQ ID NO: 39. Preferably, the AB domain donor is an alpha-amylase having at least 75% identity, such as at least 78%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the alpha-amylase of SEQ ID NO: 41.

[0081] Alternatively, the AB domain donor is an alpha-amylase having at least 75% identity, such as at least 78%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the alpha-amylase of SEQ ID NO:40.

[0082] C domain donor: The most preferred C domain donor is the alpha-amylase disclosed as SEQ ID NO:40, where the C domain is determined to correspond to amino acids 401-486, also disclosed herein as SEQ ID NO:44. Thus, in a most preferred embodiment, the present invention relates to the A and B domains disclosed above fused to the C domain disclosed as SEQ ID NO:44 or a C domain having at least 75% sequence identity thereto. In another embodiment, the present invention relates to an alpha-amylase comprising the A and B domains disclosed above fused to a C domain having a sequence at least 80% identical to the sequence of SEQ ID NO:44. In another embodiment, the present invention relates to an alpha-amylase comprising the A and B domains disclosed above fused to a C domain having a sequence at least 85% identical to the sequence of SEQ ID NO:44. In another embodiment, the present invention relates to an alpha-amylase comprising the A and B domains disclosed above fused to a C domain having a sequence at least 90% identical to the sequence of SEQ ID NO:44. In another embodiment, the present invention relates to an alpha-amylase comprising the A and B domains disclosed above fused to a C domain having a sequence at least 95% identical to the sequence of SEQ ID NO: 44. In another embodiment, the present invention relates to an alpha-amylase comprising the A and B domains disclosed above fused to a C domain having a sequence at least 97% identical to the sequence of SEQ ID NO: 44. In another embodiment, the present invention relates to an alpha-amylase comprising the A and B domains disclosed above fused to a C domain having a sequence at least 98% identical to the sequence of SEQ ID NO: 44. In another embodiment, the present invention relates to an alpha-amylase comprising the A and B domains disclosed above fused to a C domain having a sequence at least 99% identical to the sequence of SEQ ID NO: 44. In another embodiment, the present invention relates to an alpha-amylase comprising the A and B domains disclosed above fused to a C domain having a sequence 100% identical to the sequence of SEQ ID NO: 44.

[0083] Suitable C domains that are at least 75% identical to the C domain of SEQ ID NO: 44 are the two C domains disclosed herein as SEQ ID NO: 46 and SEQ ID NO: 48. The corresponding hybrid amylases are set forth in SEQ ID NO: 49 and SEQ ID NO: 50.

[0084] Preferably, the C domain donor is an alpha-amylase having at least 75% identity, such as at least 78%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the alpha-amylase of SEQ ID NO:40.

[0085] Alternatively, the C domain donor is an alpha-amylase having at least 75% identity, such as at least 78%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, to the alpha-amylase of SEQ ID NO: 39.

[0086] Thus, in alternative embodiments, if the AB domain donor is an alpha-amylase having at least 75% identity, such as at least 78%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, to the alpha-amylase of SEQ ID NO:40, the C domain donor is an alpha-amylase having at least 75% identity, such as at least 78%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% to the alpha-amylase of SEQ ID NO:39.

[0087] hybrid: The present invention relates to amylases (i.e., polypeptides having amylase activity) that can be considered hybrids of the amylase set forth in SEQ ID NO: 39 and the amylase set forth in SEQ ID NO: 40, and variants thereof with amylase activity. In one embodiment, the present invention relates to hybrid amylases comprising the A and B domains from the amylase set forth in SEQ ID NO: 39 and the C domain of the amylase set forth in SEQ ID NO: 40, and variants thereof with amylase activity.

[0088] In an alternative embodiment, the present invention relates to a hybrid amylase comprising the A and B domains from the amylase set forth in SEQ ID NO: 40 and the C domain of the amylase set forth in SEQ ID NO: 39, and variants thereof having amylase activity.

[0089] Thus, the polypeptides of the present invention can be described as hybrid or fusion polypeptides in which a region of one polypeptide is fused at the N-terminus or C-terminus to a region of another polypeptide. Techniques for producing fusion polypeptides are known in the art and involve ligating coding sequences encoding the polypeptides so that they are in-frame and expression of the fusion polypeptide is under the control of the same promoter and terminator. Fusion polypeptides can also be constructed using intein technology, in which the fusion polypeptide is generated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266:776-779). Alternatively, polypeptides according to the present invention can be produced by synthetic gene construction, by means known to those skilled in the art. Thus, the A and B domains of one of the claimed polypeptides and the C domain of the other need not be derived from different alpha-amylases and fused together. They can also be produced synthetically, for example, by creating a variant amylase equivalent to the hybrid sequence, for example, by introducing corresponding amino acid substitutions into the parent amylase sequence. Thus, the amylases of the invention can be obtained by a method for making an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity, comprising the step of making a hybrid from at least two different amylases, wherein the hybrid comprises A and B domains and a C domain, and wherein the amino acid sequences of the A and B domains are at least 75% identical to the amino acid sequence of SEQ ID NO:42 and the amino acid sequence of the C domain is at least 75% identical to the amino acid sequence of SEQ ID NO:44.Alternatively, the amylase of the present invention can be prepared by modifying a parent amylase (preferably as set forth in SEQ ID NO: 39 or SEQ ID NO: 40 or a variant thereof) and adding, to the parent amylase, at certain positions, preferably, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68 , 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, or 123, preferably at positions 1 to 123, preferably at positions 1 to 29, more preferably at positions 1 to 20, or even more preferably at positions 1 to 10.

[0090] Preferably, the parent amylase is the amylase set forth in SEQ ID NO: 39, and one or more amino acid substitutions are introduced within the C domain of SEQ ID NO: 39, preferably at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29, preferably at positions 1 to 29, more preferably at positions 1 to 10, to convert the amino acid sequence at these positions to SEQ ID NO: 40.

[0091] Preferably, the alpha-amylase has at least 75% identity, such as at least 78%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the alpha-amylase set forth in SEQ ID NO: 39, and includes the C domain of SEQ ID NO: 39. In SEQ ID NO: 39, the amino acid residues at one or more amino acid positions selected from the group consisting of 400, 402, 408, 409, 410, 418, 419, 420, 422, 423, 429, 430, 437, 441, 444, 446, 449, 452, 454, 458, 459, 460, 466, 471, 473, 475, 482, 484, and 485 according to the numbering of SEQ ID NO: 39 are preferably replaced with amino acid residues present in SEQ ID NO: 40.

[0092] Preferably, the alpha-amylase has at least 75% identity, such as at least 78%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, to the alpha-amylase set forth in SEQ ID NO:39, wherein within the C domain of SEQ ID NO:39, the amino acid sequence is and one or more substitutions selected from the group consisting of K400T, N402R, H408P, N409D, M410V, N418D, T419G, A420V, P422A, N423D, I429L, M430I, N437S, Y441E, R444K, K446N, Q449E, R452Y, I454M, R458Q, S459T, G460N, A466K, N471Q, S473H, N475S, W482Y, N484Q, and N485Q according to numbering in reference number 39.

[0093] Alternatively, the parent amylase is the amylase set forth in SEQ ID NO: 40, and preferably includes within the AB domain of SEQ ID NO: 40: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, One or more amino acid substitutions are introduced at positions 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, or 123, preferably at positions 1-123, more preferably at positions 1-100, and even more preferably at positions 1-50, to convert the amino acid sequence at these positions to SEQ ID NO:39.

[0094] Preferably, the alpha-amylase is at least 75% identical, such as at least 78%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the alpha-amylase set forth in SEQ ID NO:40. and within the A and B domains of SEQ ID NO: 40, 1, 2, 3, 4, 5, 9, 17, 25, 28, 29, 32, 35, 36, 41, 48, 51, 52, 82, 83, 86, 87, 89, 90, 93, 94, 95, 96, 98, 113, 116, 118, 123, 124, 125, 129, 136, 138, 142, 144, 150, 158, 165, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 2 2, 174, 183, 186, 192, 193, 206, 208, 212, 214, 217, 218, 222, 225, 227, 229, 235, 242, 243, 244, 245, 246, 250, 251, 255, 256, 260, 263, 267, 269, 273, 274, 275, 276, 280, 282, 284, 286, 291, 297, 298, 299, 302, 303, 304, 311 , 313, 318, 320, 323, 324, 328, 330, 337, 338, 339, 343, 345, 346, 355, 356, 360, 361, 374, 375, 376, 377, 378, 379, 382, ​​384, 391, 394, 395, and 396 are preferably replaced with amino acid residues present in SEQ ID NO: 39.

[0095] Preferably, the alpha-amylase has at least 75% identity, such as at least 78%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the alpha-amylase set forth in SEQ ID NO:40, wherein within the A and B domains of SEQ ID NO:40, the amino acid sequence is A1HH, A2HH, A3HH, A4HH, A5HH, A6HH, A7HH, A8HH, A9HH, A1 ... H, T3N, I4G, N5T, L9M, A17L, K25N, H28R, T29S, G32S, A35K, Q36D, S41A, Y48W, T51A, T52S, K82R, A83N, K86Q, S87A, I89V, E90T, H93K, K94S, Q95N , N96G, N98Q, Y113A, T116W, T118R, D123N, R124P, N125S, I129Q, E136T, N138E, G142K, N144D, D150N, K158R, T165V, E169Q, G170S, K172Q, L173L Q, I183D, A186G, S192D, S193T, L206I, F208M, D212E, A214V, M217L, K2 18R, T222V, A225T, E227T, N229G, L235I, D242K, H243Y, E244S, Y245F, L 246T, V250L, N251T, Q255N, Q256T, E260N, T263A, Y267F, Q269K, Q273G , T274A, L275I, N276E, A280S, V282T, Y284W, Q286H, A291V, F297L, H298 and one or more substitutions selected from the group consisting of positions Y, Y299N, K302R, G303S, N304G, N311Q, L313F, M318V, N320R, A323T, L324H, L328F, E330D, G337E, Q338E, S339A, V343F, S345E, P346E, F355L, I356T, A360D, E361Q, TSGN374I, S375P, S376T, Y377H, E378G, I379V, L382M, D384S, M391E, K394Q, N395K, and F396Y.

[0096] In one embodiment, the hybrid amylase comprises A and B domains derived from an amylase having an amino acid sequence with at least 75% sequence identity to the amylase set forth in SEQ ID NO: 39, and a C domain derived from an amylase having an amino acid sequence with at least 75% sequence identity to the amylase set forth in SEQ ID NO: 40.

[0097] In one embodiment of the invention, the amino acid sequences forming the A and B domains have at least 75% identity, such as at least 78%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:42, and the amino acid sequence forming the C domain has at least 75% identity to SEQ ID NO:44.

[0098] Alpha-amylases can be produced by substituting the C domain, or a portion thereof, of one alpha-amylase with the C domain, or a portion thereof, of another alpha-amylase. When producing hybrid alpha-amylases, amino acids should not be deleted or inserted in the linker region, which is herein understood to be amino acids 380-420 according to the numbering of SEQ ID NO: 39. Preferably, amino acids 380-420 according to the numbering of SEQ ID NO: 39 of the amylase comprise amino acid residues present in either SEQ ID NO: 39 and / or SEQ ID NO: 40. Preferably, amino acids 390-410 according to the numbering of SEQ ID NO: 39 of the amylase comprise amino acid residues present in either SEQ ID NO: 39 and / or SEQ ID NO: 40. Preferably, amino acids 395-405 according to the numbering of SEQ ID NO: 39 of the amylase comprise amino acid residues present in either SEQ ID NO: 39 and / or SEQ ID NO: 40. Preferably, the amylase comprises the amino acid residues of SEQ ID NO: 39 at amino acid positions 380 to 399 (according to the numbering of SEQ ID NO: 39). Preferably, the amylase comprises the amino acid residues of SEQ ID NO: 40 at amino acid positions 400 to 420 (according to the numbering of SEQ ID NO: 39).

[0099] Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity contains substitutions, deletions, and / or insertions at one or more positions.

[0100] Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises the sequence: TQXDYLDHPDVIGWTREGDXXHXXSGLAXLMSDGPXGXKWMXVGKNNAGEXWXDITGNQTNTVTINXDGXGQFXVXXGSXSIYXQX, where X can be any amino acid.

[0101] Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity is selected from 402R,H; 419S,G,D; 420V,I; 422,A,V; 423N,D,G,K; 428T,A; 435G,R,E; 437S,A; 441N,E,D; 444K,E; 450V,I; 452Y,H,R; 466K,R; 469W,S; 473H,Q,R; 475S,N; 476G,E; 479V,A; 483V,I; and 485R,Q,K according to the numbering of SEQ ID NO:39, preferably 402R; 419D; 420V; 422A; and 485Q.

[0102] In further embodiments, the present invention relates to variants of the above-disclosed polypeptides. The amylases of the present invention may contain additional substitutions, deletions, and / or insertions at one or more positions. The polypeptides can be mutated (substituted, deleted, and / or inserted) in only the A and B domains, or only the C domain, or in both the A and B domains and the C domain. The polypeptides can be mutated (substituted, deleted, and / or inserted) with additional residues, for example, outside the A and B domains and the C domain, if a carbohydrate-binding domain is present. Amino acid changes can be small, conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; typically, small deletions of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering net charge or another function, such as a polyhistidine sequence, antigenic epitope, or binding domain.

[0103] In one embodiment, a variant of the amylase of SEQ ID NO: 54, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37 containing substitutions at one or more positions and having amylase activity contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 substitutions, preferably conservative substitutions, at positions 1-20, more preferably positions 1-10.

[0104] Essential amino acids in a polypeptide can be identified using procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resulting mutant molecules are tested for alpha-amylase activity to identify amino acid residues that are important for the activity of the molecule. See also Hilton et al., 1996, J. Bio / . Chem. 271:4699-4708. The active site of an enzyme or other biological interactions can also be determined by physical analysis of the structure, as determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, along with mutations of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from alignment with related polypeptides. The essential amino acids in the amino acid sequence of SEQ ID NO: 39 are located at positions D236, E266, and D333, which are catalytic residues. These should preferably not be mutated. 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 associated screening procedures such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95 / 17413; or WO 95 / 22625.Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409; WO 92 / 06204), and region-specific mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

[0105] Mutagenesis / shuffling methods can be combined with highly efficient automated screening methods to detect the activity of cloned mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules encoding active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow for the rapid determination of the importance of individual amino acid residues in a polypeptide.

[0106] Preferred mutations are deletions of one or more, preferably at least two, amino acid positions selected from 181, 182, 183, and 184, such as amino acids 181+182, or 182+183, or 181+183, or 181+184 of SEQ ID NO: 39, whereby the molecule is significantly stabilized. In a preferred embodiment of the invention, the amino acids corresponding to 182 and 183 in SEQ ID NO: 39 are deleted. In one embodiment, a hybrid amylase of the invention comprises the A and B domains of SEQ ID NO: 39, or a variant disclosed herein, and the C domain derived from the alpha amylase of SEQ ID NO: 40, or a variant disclosed herein, and further comprises a deletion of amino acids corresponding to 182 and 183 in SEQ ID NO: 39. Such a variant is, for example, disclosed herein as SEQ ID NO: 54.

[0107] In another embodiment of the invention, the hybrid amylase contains amino acid substitutions within the interface between domain C and the A and B domains to avoid steric clashes. In one embodiment, preferred substitutions are in the amino acid of the C domain (e.g., SEQ ID NO: 43) to be replaced at the equivalent position in the C domain (e.g., SEQ ID NO: 44) of the donor amylase. Equivalent positions can be defined by aligning both sequences. Preferred positions for these mutations are analyzed by inspection of the structural model. Preferred are, but are not limited to, substitutions at one or more of the following amino acid positions in the donor C domain: 401, 403, 405, 411, 413, 415, 424, 426, 428, 430, 432, 454, 455, 477, 479, and 481 (numbered according to SEQ ID NO: 39). Preferably, the polypeptide having amylase activity comprises an amino acid residue at one or more amino acid positions selected from the group consisting of 401, 403, 405, 411, 413, 415, 424, 426, 428, 430, 432, 454, 455, 477, 479 and 481 (according to the numbering of SEQ ID NO:39) present in SEQ ID NO:39.

[0108] In another embodiment, preferred substitutions are made in amino acids of the A and B domains of the C domain donor amylase (SEQ ID NO:51) that are present at equivalent positions in the A and B domains of the C domain acceptor amylase (such as SEQ ID NO:42). Equivalent positions can be defined by aligning both sequences. Preferred positions for these mutations are analyzed by inspection of structural models. Preferred are, but are not limited to, substitutions within the A and B domains of the C domain acceptor amylase; preferably, at one or more of the following amino acid positions in the A and B domains of SEQ ID NO:39: 309, 313, 347, 348, 350, 351, 354, 355, 358, 359, 388, 389, 392, and 396 (numbered according to SEQ ID NO:39). Preferably, the polypeptide having amylase activity comprises an amino acid residue at one or more amino acid positions selected from the group consisting of 309, 313, 347, 348, 350, 351, 354, 355, 358, 359, 388, 389, 392 and 396 (according to the numbering of SEQ ID NO:39) present in SEQ ID NO:40.

[0109] An example for adapting the interface within a hybrid amylase consisting of other than the A and B domains given in SEQ ID NO: 42 and the C domain given in SEQ ID NO: 44, resulting in the hybrid amylase given in SEQ ID NO: 52, is the I430M and M454I mutations (numbering according to SEQ ID NO: 39) resulting in the amylase given as SEQ ID NO: 53 or in SEQ ID NO: 54. Thus, preferably, amylases of the invention comprise substitutions at one or more positions selected from the group consisting of 430 and 454 according to the numbering of SEQ ID NO: 39, preferably amylases of the invention comprise substitutions resulting in amino acid residues 430M and / or 430I, preferably 430M and / or 451I, preferably selected from the group consisting of I430M and M454I.

[0110] The polypeptides having amylase activity of the present invention may comprise a substitution at one or more positions selected from the group consisting of 9, 130, 195, 206, 244, 202, 179, 181, 186, and 190, preferably at one or more positions selected from the group consisting of 9, 179, 186, 195, and 206, more preferably at one or more positions selected from the group consisting of 179, 195, and 206, according to the numbering of SEQ ID NO: 39, preferably at one or more positions selected from the group consisting of The amylase may further comprise one or more substitutions selected from the group consisting of M9L, E130V, N195F, I206L, S244Q, M202L, K179L, R181E, G186E / N / Q / S, and E190P, preferably one or more substitutions selected from the group consisting of M9L, K179L, G186E / N / Q / S, N195F, and I206L, more preferably one or more substitutions selected from the group consisting of K179L, G186E, N195F, and I206L. In a further embodiment, the present invention also relates to polynucleotides encoding the amylases of the present invention. In a further embodiment, the present invention also relates to polypeptides encoded by polynucleotides that hybridize under high stringency conditions to (i) the mature polypeptide coding sequence described herein or (ii) the full-length complement of (i). The term "hybridization" as defined herein is the process by which substantially complementary nucleotide sequences anneal to each other. The hybridization process can occur entirely in solution, i.e., both complementary nucleic acids can occur in solution. The hybridization process can also occur with one of the complementary nucleic acids immobilized on a matrix such as magnetic beads, sepharose beads, or any other resin. The hybridization process can also occur with one of the complementary nucleic acids immobilized on a solid support such as nitrocellulose or nylon membrane, or immobilized on a carrier, including but not limited to, silica glass support, for example, by photolithography (the latter known as a nucleic acid array, microarray, or nucleic acid chip).To allow hybridization to occur, nucleic acid molecules are generally denatured thermally or chemically to melt duplexes into two single strands and / or remove hairpins or other secondary structures from single-stranded nucleic acids.

[0111] This formation or melting of hybrids depends on various parameters, including but not limited to temperature. An increase in temperature promotes melting, while a decrease in temperature promotes hybridization. However, this hybridization process does not follow a linear change in temperature; the hybridization process is dynamic, and already formed nucleotide pairs also support the pairing of adjacent nucleotides. Therefore, to a close approximation, hybridization is a yes-or-no process, and temperature essentially defines the boundary between hybridization and non-hybridization. This temperature is the melting temperature (Tm). Tm is the temperature in degrees Celsius at which 50% of all molecules of a given nucleotide sequence hybridize into double strands and 50% exist as single strands.

[0112] The melting temperature (Tm) depends on the physical properties of the nucleic acid sequence being analyzed, and can therefore indicate the relationship between two different sequences. However, the melting temperature (Tm) is also affected by various other parameters that are not directly related to the sequence, and the application conditions of the hybridization experiment must be taken into account. For example, an increase in salt (e.g., monovalent cations) leads to a higher Tm.

[0113] Although the Tm for given hybridization conditions can be determined by performing physical hybridization experiments, the Tm can also be estimated in silico for a given pair of DNA sequences. In this embodiment, the formula of Meinkoth and Wahl (Anal. Biochem., 138:267-284, 1984) is used for stretches having a length of 50 or more bases: Tm=81.5°C+16.6(logM)+0.41(%GC)-0.61(%form)-500 / L.

[0114] where M is the molar concentration of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA stretch, %form is the percentage of formamide in the hybridization solution, and L is the hybrid length in base pairs. This formula is for a salt range of 0.01 to 0.4 M and a %GC range of 30% to 75%.

[0115] The Tm is the temperature for a perfectly matched probe, but the Tm decreases by approximately 1°C for each 1% mismatch (Bonner et al., J. Mol. Biol. 81:123-135, 1973): Tm = [81.5°C + 16.6(log M) + 0.41 (% GC) - 0.61 (% formamide) - 500 / L] - % non-identity.

[0116] This formula is useful for probes having 35 or more nucleotides and is widely referenced in scientific methodology literature (e.g., "Recombinant DNA Principles and Methodologies," James Greene, Chapter "Biochemistry of Nucleic Acids," Paul S. Miller, page 55; 1998, CRC Press), in many patent applications (e.g., US 7026149), and also in commercial company data sheets (e.g., "Equations for Calculating Tm" from www.genomics.agilent.com).

[0117] Other formulas for calculating Tm, which are less preferred in this embodiment, can be used only in the cases indicated: For DNA-RNA hybrids (Casey, J. and Davidson, N. (1977) Nucleic Acids Res., 4:1539): Tm = 79.8°C +18.5 (log M) + 0.58 (% GC) + 11.8 (%GC * % GC) -0.5 (% form) - 820 / L.

[0118] For RNA-RNA hybrids (Bodkin, DK and Knudson, DL (1985) J. Virol. Methods, 10: 45): Tm = 79.8°C +18.5 (log M) + 0.58 (% GC) + 11.8 (%GC * % GC)-0.35 (% form) - 820 / L.

[0119] For oligonucleotide probes less than 20 bases long (Wallace, RB et al. (1979) Nucleic Acid Res. 6: 3535): Tm = 2 x n(A+T) + 4 x n(G+C), where n is the number of each base in the probe that hybridizes.

[0120] For oligonucleotide probes of 20-35 nucleotides, a modified Wallace calculation can be applied: Tm = 22 + 1.46 n(A+T) + 2.92 n(G+C), where n is the number of bases in the probe that hybridize.

[0121] For other oligonucleotides, the neighbor model for melting point calculation should be used along with appropriate thermodynamic data: [Number 1] Tm = (Σ(ΔHd)+ΔHi) / (Σ(ΔSd)+ΔSi+ΔSself + R×ln(cT / b) ) + 16.6log[Na +] - 273.15 (Breslauer, KJ, Frank, R., Blocker, H., Marky, LA 1986 Predicting DNA duplex stability from the base sequence. Proc. Natl Acad. Sci. USA 833746-3750; Alejandro Panjkovich, Francisco Melo, 2005. Comparison of different melting temperature calculation methods for short DNA sequences. Bioinformatics, 21 (6): 711-722) (where Tm is the melting point in degrees Celsius; Σ(ΔHd) and Σ(ΔSd) are the sums of the enthalpy and entropy (accordingly) calculated over all internal adjacent doublets; ΔSself is the entropy penalty for self-complementary sequences; ΔHi and ΔSi are the sums of the initiation enthalpy and entropy, respectively; R is the gas constant (fixed at 1.987 cal / K mol); cT is the total strand concentration in molar units; and the constant b takes the value 4 for non-self-complementary sequences or is equal to 1 for self-complementary duplexes or duplexes where one strand is in significant excess).

[0122] Thermodynamic calculations predict that annealing occurs in buffered solutions at a pH close to 7.0, resulting in a two-state transition.

[0123] Thermodynamic values ​​for the calculations were taken from Table 1 in (Alejandro Panjkovich, Francisco Melo, 2005. Comparison of different melting temperature calculation methods for short DNA sequences. Bioinformatics, 21 (6): 711-722) or from the original research papers (Breslauer, KJ, Frank, R., Blocker, H., Marky, LA 1986 Predicting DNA duplex stability from the base sequence. Proc. Natl Acad. Sci. USA 833746-3750; SantaLucia, J., Jr, Allawi, HT, Seneviratne, PA 1996 Improved nearest-neighbor parameters for predicting DNA duplex stability. Biochemistry 353555-3562; Sugimoto, N., Nakano, S., Yoneyama, M., Honda, K. 1996 Improved thermodynamic parameters and helix initiation factor to predict The stability of DNA duplexes. Nucleic Acids Res. 244501-4505).

[0124] For in silico estimation of Tm according to this embodiment, first, a set of bioinformatics sequence alignments between two sequences is generated. Such alignments can be generated by various tools known to those skilled in the art, such as "Blast" (NCBI), "Water" (EMBOSS), or "Matcher" (EMBOSS), which generate partial alignments, or "Needle" (EMBOSS) programs, which generate global alignments. These tools should be applied not only with their default parameter settings, but also with some parameter modifications. For example, the "MATCHER" program is applied with various parameters for gap opening / gap extension (such as 14 / 4; 14 / 2; 14 / 5; 14 / 8; 14 / 10; 20 / 2; 20 / 5; 20 / 8; 20 / 10; 30 / 2; 30 / 5; 30 / 8; 30 / 10; 40 / 2; 40 / 5; 40 / 8; 40 / 10; 10 / 2; 10 / 5; 10 / 8; 10 / 10; 8 / 2; 8 / 5; 8 / 8; 8 / 10; 6 / 2; 6 / 5; 6 / 8; 6 / 10), and the "WATER" program is applied with various parameters for gap opening / gap extension (such as 10 / 0,5; 10 / 1; 10 / 2; 10 / 3; 10 / 4; 10 / 6; 15 / 1; 15 / 2; 15 / 3; 15 / 4; 15 / 6; 20 / 1; 20 / 2; 20 / 3; 20 / 4; 20 / 6; 30 / 1; 30 / 2; 30 / 3; 30 / 4; 30 / 6; 45 / 1; 45 / 2; 45 / 3; 45 / 4; 45 / 6; 60 / 1; 60 / 2; 60 / 3; 60 / 4; 60 / 6), and these programs should be applied not only by using both nucleotide sequences as given, but also by using one of the sequences in its reverse complementary form. For example, BlastN (NCBI) can also be applied with an increased e-value cutoff (e.g., e+1 or even e+10) to identify very short alignments, especially in small databases.

[0125] Because hybridization does not necessarily occur over the full length of two sequences, but may be best in different regions, it is important to consider partial alignment, and then determine actual melting point.Therefore, from all the generated alignments, it is necessary to determine the length of alignment, the GC content (%) of alignment (more precisely, the GC content (%) of the base that matches in alignment) and the identity of alignment.Then, it is necessary to calculate the predicted melting point (Tm) for each alignment.Using the highest calculated Tm value to predict actual melting point.

[0126] As defined herein, the term "hybridization across the entire sequence of the present invention" means that if a sequence of the present invention is fragmented into pieces of approximately 300-500 bases in length, all fragments should hybridize for sequences longer than 300 bases. For example, DNA can be fragmented into small pieces using one or a combination of restriction enzymes. Bioinformatic in silico calculations of Tm are then performed using the same procedure described above for all fragments. Physical hybridization of individual fragments can be analyzed by standard Southern analysis or equivalent methods known to those skilled in the art.

[0127] The term "stringency" as defined herein describes the ease with which hybridization between two nucleotide sequences can occur. Conditions of "higher stringency" require more bases of one sequence to pair with the other sequence (the melting temperature Tm decreases under "higher stringency" conditions), while conditions of "lower stringency" require somewhat more bases not to pair. Therefore, the degree of relationship between two sequences can be estimated by the actual stringency conditions under which they can still form a hybrid. Increased stringency can be achieved by keeping the experimental hybridization temperature constant and decreasing the salt concentration, or by keeping the salt constant and increasing the experimental hybridization temperature, or by a combination of these parameters. Also, increasing formamide will increase stringency. Those skilled in the art are aware of additional parameters that may be altered during hybridization that will maintain or change stringency conditions (Sambrook et al. (2001) Molecular Cloning: a laboratory manual, 3rd ed., Cold Spring Harbor Laboratory Press, CSH, New York or to Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989 and annual updates)).

[0128] A typical hybridization experiment involves an initial hybridization step, followed by one to several washing steps. The solutions used for these steps may contain additional components known to those skilled in the art, such as EDTA, SDS, fragmented sperm DNA, or similar reagents, to prevent degradation of the analyzed sequence and / or to prevent nonspecific background binding of the probe (Sambrook et al. (2001) Molecular Cloning: a laboratory manual, 3rd ed., Cold Spring Harbor Laboratory Press, CSH, New York, or Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989 and annual updates)).

[0129] Typical probes for hybridization experiments are generated by the random-primed labeling method, originally developed by Feinberg and Vogelstein (Anal. Biochem., 132 (1), 6-13 (1983); Anal. Biochem., 137 (1), 266-7 (1984)), which relies on hybridization of a mixture of all possible hexanucleotides with the DNA to be labeled. The labeled probe product may actually be a collection of fragments of variable length, typically 100-1000 nucleotides in length, with the highest fragment concentration typically being approximately 200-400 bp. The actual size range of the probe fragments ultimately used as probes for hybridization experiments can also be affected by the parameters of the labeling method used, subsequent purification of the generated probe (e.g., agarose gel), and the size of the template DNA used for labeling (larger templates can be restriction digested, for example, with a 4-bp cutter, e.g., HaeIII, prior to labeling).

[0130] For the present invention, the sequences described herein are analyzed by hybridization experiments in which probes are generated from other sequences, using standard random-primed labeling techniques. For the present invention, the probes consist of a set of labeled oligonucleotides with a size of approximately 200-400 nucleotides. Hybridization of the sequences of the present invention with other sequences means that the hybridization of the probe occurs across the entire sequence of the present invention, as defined above. Hybridization experiments are performed with the highest stringency achieved by the stringency of the final wash step. The final wash step may be at least Wash Condition 1: 1.06 x SSC, 0.1% SDS, 0% formamide at 50°C; in another embodiment, at least Wash Condition 2: 1.06 x SSC, 0.1% SDS, 0% formamide at 55°C; in another embodiment, at least Wash Condition 3: 1.06 x SSC, 0.1% SDS, 0% formamide at 60°C; in another embodiment, at least Wash Condition 4: 1.06 x SSC, 0.1% SDS, 0% formamide at 65°C; in another embodiment, at least Wash Condition 5: 0.52 x SSC, 0.1% SDS, 0% formamide at 65°C; in another embodiment, at least Wash Condition 6: 0.25 x SSC, 0.1% SDS, 0% formamide at 65°C; and in another embodiment, at least Wash Condition 7: 0.12 x SSC, 0.1% SDS, 0% formamide at 65°C. % formamide, and in another embodiment, have stringency conditions at least equivalent to the stringency conditions of wash conditions 8: 0.07 x SSC, 0.1% SDS, 0% formamide at 65°C.

[0131] A "low stringency wash" has stringency conditions at least equivalent to wash conditions 1 but less stringent than wash conditions 3, where the wash conditions are as described above.

[0132] A "high stringency wash" has stringency conditions equivalent to the stringency conditions of at least wash condition 4, in another embodiment at least wash condition 5, in another embodiment at least wash condition 6, in another embodiment at least wash condition 7, and in another embodiment at least wash condition 8, where the wash conditions are as described above.

[0133] The amylases of the present invention exhibit improved properties. Preferably, the improved properties relate to the properties of the amylases set forth in SEQ ID NO: 39 and / or SEQ ID NO: 40. Preferably, the cleaning performance is improved at 15°C. In one embodiment, the improved property is detergent stability. In another embodiment, the improved property is specific activity. In another embodiment, the improved property is temperature stability. In another embodiment, the improved property is pH-dependent stability. In another embodiment, the improved property is oxidative stability. In another embodiment, the improved property is reduced Ca2+ dependency. In yet another embodiment, the improved property is cleaning performance at low temperatures. In one embodiment of the present invention, the polypeptide has improved cleaning performance at low temperatures, such as at or below 40°C, or at or below 30°C, or at or below 25°C, or at or below 20°C, or at or below 15°C, or at or below 10°C. In a preferred embodiment, the improved property is temperature stability.

[0134] Preferred polypeptides having amylase activity (amylases): Preferably, the present invention relates to an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprising A and B domains and a C domain, wherein the amino acid sequences of the A and B domains are at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:42, and the amino acid sequence of the C domain is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:44.

[0135] Preferably, the polypeptide having alpha-amylase activity consists of A and B domains and a C domain, wherein the amino acid sequences of the A and B domains are at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:42, and the amino acid sequence of the C domain is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:44.

[0136] Preferably, the present invention provides a method for the preparation of a polypeptide comprising a polypeptide comprising: an amino acid sequence of the A and B domains that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:42; and an amino acid sequence of the C domain that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:44. The present invention relates to an isolated, synthetic, or recombinant polypeptide with alpha-amylase activity comprising A and B domains and a C domain that are 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical, the polypeptide comprising the sequence TQXDYLDHPDVIGWTREGDXXHXXSGLAXLMSDGPXGXKWMXVGKNNAGEXWXDITGNQTNTVTINXDGXGQFXVXXGSXSIYXQX, where X can be any amino acid.

[0137] Preferably, the present invention provides a method for the preparation of a polypeptide comprising a polypeptide comprising: an amino acid sequence of the A and B domains that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:42; and an amino acid sequence of the C domain that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:44. The present invention relates to an isolated, synthetic, or recombinant polypeptide with alpha-amylase activity comprising A and B domains and a C domain that are 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical, the polypeptide comprising the sequence TQXDYLDHPDVIGWTREGDXXHXXSGLAXLMSDGPXGXKWMXVGKNNAGEXWXDITGNQTNTVTINXDGXGQFXVXXGSXSIYXQX, where X can be any amino acid.

[0138] Preferably, the present invention relates to an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprising A and B domains and a C domain, wherein the amino acid sequences of the A and B domains are at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:42, and the amino acid sequence of the C domain is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:44, wherein the amino acid sequence of the C domain is 402R,H; 419S,G,D; 420V,I; according to the numbering of SEQ ID NO:39. and 485R,Q,K, preferably selected from the group consisting of 402R; 419D; 420V; 422A; 423D; 428A,T; 435R; 437S; 441E; 444K; 450V; 452Y; H; R; 466K; 469W; The present invention relates to an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity, having one or more amino acid residues selected from the group consisting of 473R; 475S; 476G; 479V; 483V; and 485Q, and preferably selected from the group consisting of 435R, 437A, 441D, and 485K according to the numbering of SEQ ID NO: 39.

[0139] Preferably, the present invention relates to an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprising A and B domains and a C domain, wherein the amino acid sequences of the A and B domains are at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:42, and the amino acid sequence of the C domain is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:44, wherein the amino acid sequence of the C domain is 402R,H; 419S,G,D; 420V,I; according to the numbering of SEQ ID NO:39. and 485R,Q,K, preferably selected from the group consisting of 422,A,V; 423N,D,G,K; 428T,A; 435G,R,E; 437S,A; 441N,E,D; 444K,E; 450V,I; 452Y,H,R; 466K,R; 469W,S; 473H,Q,R; 475S,N; 476G,E; 479V,A; 483V,I; and 485R,Q,K, according to the numbering of SEQ ID NO: 39. The present invention relates to an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or all amino acid residues selected from the group consisting of 469W; 473R; 475S; 476G; 479V; 483V; and 485Q.

[0140] Preferably, in the present invention, the amino acid sequences of the A and B domains are at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 42, and the amino acid sequence of the C domain is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 44, an isolated, synthetic or recombinant polypeptide having alpha -amylase activity and comprising the A and B domains and the C domain, characterized in that it comprises substitutions at one or more positions selected from the group consisting of 430 and 454 according to the numbering of SEQ ID NO: 39, preferably, the amylase comprises the amino acid residues 430M and / or 430I, preferably, the substitutions are selected from the group consisting of I430M and M454I, preferably consisting of I430M and M454I, relating to an isolated, synthetic or recombinant polypeptide having alpha -amylase activity.

[0141] Preferably, the present invention relates to an isolated, synthetic, or recombinant antibody having alpha-amylase activity comprising A and B domains and a C domain, wherein the amino acid sequences of the A and B domains are at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:42, and the amino acid sequence of the C domain is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:44. The present invention relates to an isolated, synthetic or recombinant polypeptide having alpha-amylase activity, wherein the polypeptide comprises a substitution at one or more positions selected from the group consisting of 430 and 454 according to the numbering of SEQ ID NO: 39, preferably wherein the amylase comprises a substitution selected from the group consisting of amino acid residues 430M and / or 430I, preferably I430M and M454I, preferably I430M and M454I, and the polypeptide having alpha-amylase comprises a deletion of one or more amino acids corresponding to positions 181, 182, 183 and 184 according to the numbering of SEQ ID NO: 39, preferably a deletion of two or more amino acids corresponding to positions 181, 182, 183 and 184, preferably a deletion of amino acids corresponding to positions 181 and 182, 182 and 183, or 183 and 184 (according to the numbering of SEQ ID NO: 39).

[0142] Preferably, the present invention relates to an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprising A and B domains and a C domain, wherein the amino acid sequences of the A and B domains are at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:42, and the amino acid sequence of the C domain is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:44, wherein the amino acid sequence of the C domain is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:44, and a substitution selected from the group consisting of I430M and M454I, preferably I430M and M454I, wherein the polypeptide having alpha-amylase has one or more deletions of amino acids corresponding to positions 181, 182, 183 and 184 according to the numbering of SEQ ID NO: 39, preferably two or more deletions of amino acids corresponding to positions 181, 182, 183 and 184, preferably 181 and 182, 182 and 183, or 183 and 184. and wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises a deletion of the amino acids corresponding to (according to the numbering of SEQ ID NO: 39) the sequence TQXDYLDHPDVIGWTREGDXXHXXSGLAXLMSDGPXGXKWMXVGKNNAGEXWXDITGNQTNTVTINXDGXGQFXVXXGSXSIYXQX (where X can be any amino acid), preferably the polypeptide comprises a deletion of 402R,H according to the numbering of SEQ ID NO: 39; 419S,G,D; 420V,I; 422,A,V; 423N,D,G,K; 428T,A; 435G,R,E; 437S,A; 441N,E,D; 444K,E; 450V,I; 452Y,H,R; 466K,R; 469W,S;The present invention relates to an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity, having one or more amino acid residues selected from the group consisting of 473H,Q,R; 475S,N; 476G,E; 479V,A; 483V,I; and 485R,Q,K, preferably selected from the group consisting of 402R; 419D; 420V; 422A; 423D; 428A,T; 435R; 437S; 441E; 444K; 450V; 452Y; 466K; 469W; 473R; 475S; 476G; 479V; 483V; and 485Q according to the numbering of SEQ ID NO: 39.

[0143] Preferably, the present invention relates to an isolated, synthetic, or recombinant polypeptide with alpha-amylase activity comprising A and B domains and a C domain, wherein the amino acid sequences of the A and B domains are at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:42, and the amino acid sequence of the C domain is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:44, and preferably the amino acid sequence of the C domain comprises a substitution at one or more positions selected from the group consisting of 430 and 454 according to the numbering of SEQ ID NO:39. wherein the polypeptide having alpha-amylase activity comprises a deletion of one or more amino acids corresponding to positions 181, 182, 183 and 184, preferably a deletion of two or more amino acids corresponding to positions 181, 182, 183 and 184, preferably a deletion of amino acids corresponding to positions 181 and 182, 182 and 183, or 183 and 184 (according to the numbering of SEQ ID NO: 39).

[0144] Preferably, the present invention relates to an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 52, wherein the polypeptide comprises a substitution at one or more positions selected from the group consisting of 430 and 454 according to the numbering of SEQ ID NO: 39, preferably wherein the amylase comprises a substitution resulting in amino acid residues 430M and / or 430I, preferably 430M and 454I, preferably selected from the group consisting of I430M and M454I. The present invention relates to an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity, wherein the polypeptide comprises a deletion of one or more amino acids corresponding to positions 181, 182, 183, and 184, preferably two or more amino acids corresponding to positions 181, 182, 183, and 184, preferably a deletion of amino acids corresponding to positions 181 and 182, 182 and 183, or 183 and 184 (according to the numbering of SEQ ID NO: 39).

[0145] Preferably, the present invention relates to an isolated, synthetic or recombinant polypeptide with alpha-amylase activity which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 53, wherein the polypeptide with alpha-amylase activity comprises a deletion of one or more amino acids corresponding to positions 181, 182, 183 and 184 according to the numbering of SEQ ID NO: 39, preferably a deletion of two or more amino acids corresponding to positions 181, 182, 183 and 184, preferably a deletion of amino acids corresponding to positions 181 and 182, 182 and 183, or 183 and 184 (according to the numbering of SEQ ID NO: 39).

[0146] Preferably, the present invention provides a method for the preparation of a polypeptide comprising a polypeptide comprising: an amino acid sequence of the A and B domains that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:42; and an amino acid sequence of the C domain that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:44. 96%, at least 97%, at least 98%, at least 99% or 100% identical, isolated, synthetic or recombinant polypeptides with alpha-amylase activity comprising A and B domains and a C domain, wherein the amino acid residues are at one or more amino acid positions selected from the group consisting of 401, 403, 405, 411, 413, 415, 424, 426, 428, 430, 432, 454, 455, 477, 479 and 481 (according to the numbering of SEQ ID NO:39) as present in SEQ ID NO:39.

[0147] Preferably, the present invention provides a polypeptide comprising an A and B domain and a C domain, wherein the amino acid sequences of the A and B domains are at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:42, and the amino acid sequence of the C domain is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:44. The present invention relates to an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprising an amino acid residue at at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, or at least thirteen, or all, of the amino acid positions selected from the group consisting of 401, 403, 405, 411, 413, 415, 424, 426, 428, 430, 432, 454, 455, 477, 479, and 481 (according to the numbering of SEQ ID NO:39), as present in SEQ ID NO:39.

[0148] Preferably, the present invention provides a method for the preparation of a polypeptide comprising a polypeptide having an amino acid sequence of the A and B domains that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:42, and a polypeptide having an amino acid sequence of the C domain that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% or 100% identical to the amino acid sequence of SEQ ID NO:44. 392 and 396 (according to the numbering of SEQ ID NO:39), as present in SEQ ID NO:40.

[0149] Preferably, the present invention provides A and B domains wherein the amino acid sequences of the A and B domains are at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 42, and wherein the amino acid sequence of the C domain is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 44; The present invention relates to an isolated, synthetic, or recombinant polypeptide with alpha-amylase activity comprising a C domain, the polypeptide comprising amino acid residues at at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, or at least thirteen, or all, of the amino acid positions selected from the group consisting of 309, 313, 347, 348, 350, 351, 354, 355, 358, 359, 388, 389, 392, and 396 (according to the numbering of SEQ ID NO:39) as present in SEQ ID NO:40.

[0150] Preferably, the present invention provides a method for the preparation of a polypeptide comprising a polypeptide comprising: an amino acid sequence of the A and B domains that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:42; and an amino acid sequence of the C domain that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:44. The present invention relates to an isolated, synthetic, or recombinant polypeptide with alpha-amylase activity comprising the A and B domains and the C domain, which are at least 99% or 100% identical to the polypeptide of SEQ ID NO: 39, and which contains a deletion of one or more amino acids corresponding to positions 181, 182, 183, and 184, preferably two or more amino acids corresponding to positions 181, 182, 183, and 184, preferably a deletion of amino acids corresponding to positions 181 and 182, 182 and 183, or 183 and 184, preferably a deletion of amino acids corresponding to positions 182 and 183.

[0151] Preferably, the present invention relates to an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprising A and B domains and a C domain, wherein the amino acid sequences of the A and B domains are at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:42, and the amino acid sequence of the C domain is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:44, wherein the amino acid sequence of the C domain is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:44, Substitutions at one or more positions selected from the group consisting of 195, 206, 244, 202, 179, 181, 186, and 190, preferably at one or more positions selected from the group consisting of 9, 179, 186, 195, and 206, more preferably at one or more positions selected from the group consisting of 179, 195, and 206, preferably at M9L, E130V, N195F, I206L, S244Q, M202L, K179L, R1 according to the numbering of SEQ ID NO: 39. The present invention relates to an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity, comprising one or more substitutions selected from the group consisting of 81E, G186E / N / Q / S, and E190P, preferably one or more substitutions selected from the group consisting of M9L, K179L, G186E / N / Q / S, N195F, and I206L, more preferably one or more substitutions selected from the group consisting of K179L, G186E, N195F, and I206L.

[0152] Also provided are isolated, synthetic, or recombinant polypeptides having alpha-amylase activity that are at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:39 or SEQ ID NO:40, wherein the amino acid sequence is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:39 or SEQ ID NO:40, and ... Also disclosed herein are isolated, synthetic, or recombinant polypeptides having alpha-amylase activity that include substitutions at 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or at least 13, or all, amino acid residue positions, preferably one or more substitutions selected from the group consisting of M9L, E130V, N195F, I206L, S244Q, M202L, K179L, R181E, G186E / N / Q / S, and E190P according to the numbering of SEQ ID NO:39.

[0153] Preferably, the present invention relates to an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprising A and B domains and a C domain, wherein the amino acid sequences of the A and B domains are at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:42, and the amino acid sequence of the C domain is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:44, wherein the amino acid sequence of the C domain is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:44, Substitutions at at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, or at least thirteen, or all, amino acid residue positions selected from the group consisting of 179, 181, 186, and 190, preferably M9L, E130V, N195F, I206L, S244Q, M202L, K according to the numbering of SEQ ID NO: 39. The present invention relates to an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity that comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, or at least thirteen, or all, amino acid substitutions selected from the group consisting of 179L, R181E, G186E / N / Q / S, and E190P.

[0154] Preferably, the present invention provides a method for the preparation of a polypeptide comprising a polypeptide comprising: an amino acid sequence of the A and B domains that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:42; an amino acid sequence of the C domain that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:44; The present invention relates to an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprising A and B domains and a C domain that are at least 99% or 100% identical, and which comprises an increase in expression, activity, thermostability, stability, performance in laundry, specific activity, substrate specificity, pH-dependent activity, pH-dependent stability, oxidative stability, Ca2+ dependence, or any combination thereof, compared to the amylase set forth in SEQ ID NO:39 or SEQ ID NO:40, and preferably the amylase has increased thermostability compared to the amylase set forth in SEQ ID NO:39 or SEQ ID NO:40.

[0155] Preferably, the present invention provides a method for the preparation of a polypeptide comprising a polypeptide having an amino acid sequence of the A and B domains that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:42, and a polypeptide having an amino acid sequence of the C domain that is at least 75%, at least 80%, at least 85%, at least 90%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:44. The present invention relates to an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprising an A and B domain and a C domain that are 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical, wherein the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises increased thermostability compared to the amylase set forth in SEQ ID NO:39 or SEQ ID NO:40.

[0156] In one embodiment, the present invention provides (a) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, preferably SEQ ID NO:54, SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:17, or SEQ ID NO:27; (b) an amino acid sequence encoded by a polynucleotide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:55, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38, preferably SEQ ID NO:55, SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:18, or SEQ ID NO:29; (c) under high stringency conditions; (i) a sequence encoding SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, preferably SEQ ID NO:54, SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:17, or SEQ ID NO:27; or (ii) a polynucleotide set forth in SEQ ID NO:55, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38, preferably SEQ ID NO:55, SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:18, or SEQ ID NO:29 an amino acid sequence encoded by a polynucleotide that hybridizes to the complement of; or (d) a fragment of (a), (b), or (c) having amylase activity. The present invention relates to an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity, comprising:

[0157] Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119 ... An isolated, synthetic, or recombinant polypeptide comprising an amino acid sequence having 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity and having alpha-amylase activity comprises the sequence TQXDYLDHPDVIGWTREGDXXHXXSGLAXLMSDGPXGXKWMXVGKNNAGEXWXDITGNQTNTVTINXDGXGQFXVXXGSXSIYXQX (where X can be any amino acid).

[0158] Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 39, and the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises the sequence TQXDYLDHPDVIGWTREGDXXHXXSGLAXLMSDGPXGXKWMXVGKNNAGEXWXDITGNQTNTVTINXDGXGQFXVXXGSXSIYXQX (where X may be any amino acid), and preferably the polypeptide is 402R,H; 419S,G,D; 420V,I; 422,A,V; according to the numbering of SEQ ID NO: 39. and 485R,Q,K, preferably selected from the group consisting of 423N,D,G,K; 428T,A; 435G,R,E; 437S,A; 441N,E,D; 444K,E; 450V,I; 452Y,H,R; 466K,R; 469W,S; 473H,Q,R; 475S,N; 476G,E; 479V,A; 483V,I; and 485R,Q,K, according to the numbering of SEQ ID NO: 39. and 485Q.

[0159] Preferably, the isolated, synthetic, or recombinant polypeptide with alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 54, and the isolated, synthetic, or recombinant polypeptide with alpha-amylase activity comprises the sequence TQXDYLDHPDVIGWTREGDXXHXXSGLAXLMSDGPXGXKWMXVGKNNAGEXWXDITGNQTNTVTINXDGXGQFXVXXGSXSIYXQX (where X can be any amino acid).

[0160] Preferably, an amylase of the invention contains at least one amino acid substitution as described herein and comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% identical to the amino acid sequence of SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, preferably SEQ ID NO:54, SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:17, or SEQ ID NO:27.

[0161] Preferably, the amylases of the invention contain at least one amino acid substitution as described herein and comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% identical to the amino acid sequence of SEQ ID NO:54, preferably at least 95%, at least 96%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% identical to the amino acid sequence of SEQ ID NO:54.

[0162] Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, and the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity is 402R,H; 419S,G,D; 420V,I; 422,A,V; according to the numbering of SEQ ID NO:39. and 485R,Q,K, preferably having one or more amino acid residues selected from the group consisting of 435R, 437A, 441D, and 485K, and having one or more amino acid residues selected from the group consisting of 423N,D,G,K; 428T,A; 435G,R,E; 437S,A; 441N,E,D; 444K,E; 450V,I; 452Y,H,R; 466K,R; 469W,S; 473H,Q,R; 475S,N; 476G,E; 479V,A; 483V,I; and 485R,Q,K.

[0163] Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:39, and the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity is selected from the group consisting of: 402R,H; 419S,G,D; 420V,I; 422,A,V; 423N,D,G,K; 428T,A; 435G,R,E; 437S,A; 441N,E,D; 444K,E; 450V,I; 452Y,H,R; 466K,R; It has one or more amino acid residues selected from the group consisting of 469W,S; 473H,Q,R; 475S,N; 476G,E; 479V,A; 483V,I; and 485R,Q,K, preferably selected from the group consisting of 402R; 419D; 420V; 422A; 423D; 428A,T; 435R; 437S; 441E; 444K; 450V; 452Y; 466K; 469W; 473R; 475S; 476G; 479V; 483V; and 485Q according to the numbering of SEQ ID NO: 39.

[0164] Preferably, the isolated, synthetic, or recombinant polypeptide with alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:54, and the isolated, synthetic, or recombinant polypeptide with alpha-amylase activity is 402R,H; 419S,G,D; 420V,I; 422,A,V; 423N,D,G,K; 428T,A; 435G,R,E; 437S,A; 441N,E,D; 444K,E; 450V,I; 452Y,H,R; 466K,R; according to the numbering of SEQ ID NO:39. and 485R,Q,K, preferably selected from the group consisting of 402R; 419D; 420V; 422A; 423D; 428A,T; 435R; 437S; 441E; 444K; 450V; 452Y; 466K; 469W; 473R; 475S; 476G; 479V; 483V; and 485Q, according to the numbering of SEQ ID NO: 39, preferably selected from the group consisting of 435R, 437S,A, 441E,D, and 485Q,K.

[0165] Preferably, the isolated, synthetic, or recombinant polypeptide with alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:54, and the isolated, synthetic, or recombinant polypeptide with alpha-amylase activity is 402R,H; 419S,G,D; 420V,I; 422,A,V; 423N,D,G,K; 428T,A; 435G,R,E; 437S,A; 441N,E,D; 444K,E; 450V,I; 452Y,H,R; 466K,R; according to the numbering of SEQ ID NO:39. and 485R,Q,K, preferably selected from the group consisting of 402R; 419D; 420V; 422A; 423D; 428A,T; 435R; 437S; 441E; 444K; 450V; 452Y; 466K; 469W; 473R; 475S; 476G; 479V; 483V; and 485Q.

[0166] Preferably, the isolated, synthetic, or recombinant polypeptide with alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:54, and the isolated, synthetic, or recombinant polypeptide with alpha-amylase activity comprises amino acid residues 402R,H; 419S,G,D; 420V,I; 422,A,V; 423N,D,G,K; 428T,A; 435G,R,E; 437S,A; 441N,E,D; 444K,E; 450V,I; 452Y,H,R; according to the numbering of SEQ ID NO:39. and 485R,Q,K, preferably having amino acid residues selected from the group consisting of 402R; 419D; 420V; 422A; 423D; 428A,T; 435R; 437S; 441E; 444K; 450V; 452Y; 466K; 469W; 473R; 475S; 476G; 479V; 483V; and 485Q according to the numbering of SEQ ID NO: 39.

[0167] Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity has a sequence similar to that of SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134 An isolated, synthetic, or recombinant polypeptide comprising an amino acid sequence having 5%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity and having alpha-amylase activity comprises a substitution at one or more positions selected from the group consisting of 430 and 454 according to the numbering of SEQ ID NO: 39, preferably the amylase comprises a substitution resulting in amino acid residues 430M and / or 430I, preferably 430M and / or 454I, preferably a substitution selected from the group consisting of I430M and M454I.

[0168] Preferably, the isolated, synthetic, or recombinant polypeptide with alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 54, and the isolated, synthetic, or recombinant polypeptide with alpha-amylase activity comprises a substitution at one or more positions selected from the group consisting of 430 and 454 according to the numbering of SEQ ID NO: 39, preferably the amylase comprises a substitution resulting in amino acid residues 430M and / or 430I, preferably 430M and / or 454I, preferably a substitution selected from the group consisting of I430M and M454I.

[0169] Preferably, the isolated, synthetic, or recombinant polypeptide with alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 54, and the isolated, synthetic, or recombinant polypeptide with alpha-amylase activity comprises substitutions at positions 430 and 454 according to the numbering of SEQ ID NO: 39, preferably substitutions resulting in 430M and / or 454I, preferably a substitution selected from the group consisting of I430M and M454I.

[0170] Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 54, and the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity is selected from the group consisting of 430 and 454 according to the numbering of SEQ ID NO: 39. The polypeptides having alpha-amylase activity comprise a substitution at positions 181, 182, 183 and 184, preferably a substitution resulting in 430M and / or 454I, preferably a substitution selected from the group consisting of I430M and M454I, and comprise a deletion of one or more amino acids corresponding to positions 181, 182, 183 and 184 according to the numbering of SEQ ID NO: 39, preferably a deletion of two or more amino acids corresponding to positions 181, 182, 183 and 184, preferably a deletion of amino acids corresponding to positions 181 and 182, 182 and 183, or 183 and 184 (according to the numbering of SEQ ID NO: 39).

[0171] Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% of the sequence of SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37. An isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a polypeptide of the present invention, the polypeptide comprising at least one amino acid residue at one or more amino acid positions selected from the group consisting of 401, 403, 405, 411, 413, 415, 424, 426, 428, 430, 432, 454, 455, 477, 479, and 481 (according to the numbering of SEQ ID NO:39) as present in SEQ ID NO:39.

[0172] Preferably, the isolated, synthetic, or recombinant polypeptide with alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:54, and the isolated, synthetic, or recombinant polypeptide with alpha-amylase activity comprises an amino acid residue at one or more amino acid positions selected from the group consisting of 401, 403, 405, 411, 413, 415, 424, 426, 428, 430, 432, 454, 455, 477, 479 and 481 (according to the numbering of SEQ ID NO:39) as present in SEQ ID NO:39.

[0173] Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37. An isolated, synthetic, or recombinant polypeptide comprising an amino acid sequence having the same identity as SEQ ID NO:39 and having alpha-amylase activity comprises an amino acid residue at at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, or at least thirteen, or all, of the amino acid positions selected from the group consisting of 401, 403, 405, 411, 413, 415, 424, 426, 428, 430, 432, 454, 455, 477, 479, and 481 (according to the numbering of SEQ ID NO:39) as present in SEQ ID NO:39.

[0174] Preferably, the isolated, synthetic, or recombinant polypeptide with alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:54, and the isolated, synthetic, or recombinant polypeptide with alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:39. The amino acid residues may be present at at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, or at least thirteen, or all, of the amino acid positions selected from the group consisting of 401, 403, 405, 411, 413, 415, 424, 426, 428, 430, 432, 454, 455, 477, 479, and 481 (according to the numbering of SEQ ID NO: 39).

[0175] Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% of the sequence of SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37. Isolated, synthetic, or recombinant polypeptides comprising an amino acid sequence with at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity and having alpha-amylase activity comprise an amino acid residue at one or more amino acid positions selected from the group consisting of 309, 313, 347, 348, 350, 351, 354, 355, 358, 359, 388, 389, 392, and 396 (according to the numbering of SEQ ID NO:39) as present in SEQ ID NO:40.

[0176] Preferably, the isolated, synthetic, or recombinant polypeptide with alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:54, and the isolated, synthetic, or recombinant polypeptide with alpha-amylase activity comprises an amino acid residue at one or more amino acid positions selected from the group consisting of 309, 313, 347, 348, 350, 351, 354, 355, 358, 359, 388, 389, 392, and 396 (according to the numbering of SEQ ID NO:39) as present in SEQ ID NO:40.

[0177] Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity has a sequence similar to that of SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37. An isolated, synthetic, or recombinant polypeptide comprising an amino acid sequence with 0% sequence identity and having alpha-amylase activity comprises an amino acid residue at at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, or all amino acid residues selected from the group consisting of 309, 313, 347, 348, 350, 351, 354, 355, 358, 359, 388, 389, 392, and 396 (according to the numbering of SEQ ID NO:39) as present in SEQ ID NO:40.

[0178] Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, or at least 96% affinity to SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37. An isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprising an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a polypeptide of the present invention comprises one or more deletions of amino acids corresponding to positions 181, 182, 183, and 184 according to the numbering of SEQ ID NO:39, preferably two or more deletions of amino acids corresponding to positions 181, 182, 183, and 184, preferably deletions of amino acids corresponding to positions 181 and 182, 182 and 183, or 183 and 184 (according to the numbering of SEQ ID NO:39).

[0179] Preferably, the isolated, synthetic, or recombinant polypeptide with alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, and the isolated, synthetic, or recombinant polypeptide with alpha-amylase activity comprises a deletion of one or more amino acids corresponding to positions 182 and 183, preferably a deletion of both amino acids corresponding to positions 182 and 183 (according to the numbering of SEQ ID NO:39).

[0180] Preferably, the isolated, synthetic, or recombinant polypeptide with alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:54, and the isolated, synthetic, or recombinant polypeptide with alpha-amylase activity comprises a deletion of one or more amino acids corresponding to positions 181, 182, 183 and 184 according to the numbering of SEQ ID NO:39, preferably a deletion of two or more amino acids corresponding to positions 181, 182, 183 and 184, preferably a deletion of amino acids corresponding to positions 181 and 182, 182 and 183, or 183 and 184 (according to the numbering of SEQ ID NO:39).

[0181] Preferably, the isolated, synthetic, or recombinant polypeptide with alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 54, and the isolated, synthetic, or recombinant polypeptide with alpha-amylase activity comprises a deletion of one or more amino acids corresponding to positions 182 and 183, preferably a deletion of both amino acids corresponding to positions 182 and 183 (according to the numbering of SEQ ID NO: 39).

[0182] Preferably, the isolated, synthetic, or recombinant polypeptide with alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 54, and the isolated, synthetic, or recombinant polypeptide with alpha-amylase activity comprises a deletion of both amino acids corresponding to positions 182 and 183 (according to the numbering of SEQ ID NO: 39).

[0183] Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity has a sequence similar to that of SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110% or at least 111% of that of SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37. An isolated, synthetic, or recombinant polypeptide comprising an amino acid sequence having 7%, at least 98%, at least 99%, or 100% sequence identity and having alpha-amylase activity comprises a substitution at one or more positions selected from the group consisting of 9, 130, 195, 206, 244, 202, 179, 181, 186, and 190 according to the numbering of SEQ ID NO:39, preferably one or more substitutions selected from the group consisting of M9L, E130V, N195F, I206L, S244Q, M202L, K179L, R181E, G186E / N / Q / S, and E190P according to the numbering of SEQ ID NO:39.

[0184] Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:54, and the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises one or more positions selected from the group consisting of 9, 130, 195, 206, 244, 202, 179, 181, 186, and 190 according to the numbering of SEQ ID NO:39, preferably or more substitutions at one or more positions selected from the group consisting of 9, 179, 186, 195, and 206, more preferably one or more positions selected from the group consisting of 179, 195, and 206, preferably one or more substitutions selected from the group consisting of M9L, E130V, N195F, I206L, S244Q, M202L, K179L, R181E, G186E / N / Q / S, and E190P, according to the numbering of SEQ ID NO: 39, preferably one or more substitutions selected from the group consisting of M9L, K179L, G186E / N / Q / S, N195F, and I206L, more preferably one or more substitutions selected from the group consisting of K179L, G186E, N195F, and I206L.

[0185] Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, and is an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity. The polypeptide comprises substitutions at at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or all amino acid residue positions selected from the group consisting of 9, 130, 195, 206, 244, 202, 179, 181, 186, and 190 according to the numbering of SEQ ID NO:39, preferably at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or all amino acid substitutions selected from the group consisting of M9L, E130V, N195F, I206L, S244Q, M202L, K179L, R181E, G186E / N / Q / S, and E190P according to the numbering of SEQ ID NO:39.

[0186] Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:54, and the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises at least one, at least two, or at least three amino acids selected from the group consisting of 9, 130, 195, 206, 244, 202, 179, 181, 186, and 190 according to the numbering of SEQ ID NO:39. , at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or all amino acid residue positions, preferably at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or all amino acid substitutions selected from the group consisting of M9L, E130V, N195F, I206L, S244Q, M202L, K179L, R181E, G186E / N / Q / S, and E190P according to the numbering of SEQ ID NO:39.

[0187] Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity has a sequence similar to that of SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, An isolated, synthetic, or recombinant polypeptide comprising an amino acid sequence having at least 97%, at least 98%, at least 99%, or 100% sequence identity and having alpha-amylase activity comprises an increase in expression, activity, thermostability, stability, laundry performance, specific activity, substrate specificity, pH-dependent activity, pH-dependent stability, oxidative stability, Ca2+ dependence, or any combination thereof, compared to the amylase set forth in SEQ ID NO:39 or SEQ ID NO:40; preferably, the amylase has increased thermostability compared to the amylase set forth in SEQ ID NO:39 or SEQ ID NO:40.

[0188] Preferably, the isolated, synthetic, or recombinant polypeptide with alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:54, and the isolated, synthetic, or recombinant polypeptide with alpha-amylase activity comprises an increase in expression, activity, thermostability, stability, laundry performance, specific activity, substrate specificity, pH-dependent activity, pH-dependent stability, oxidative stability, Ca2+ dependence, or any combination thereof, compared to the amylase set forth in SEQ ID NO:39 or SEQ ID NO:40, and preferably the amylase has increased thermostability compared to the amylase set forth in SEQ ID NO:39 or SEQ ID NO:40.

[0189] Preferably, the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:54, and the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprises increased thermostability compared to the amylase set forth in SEQ ID NO:39 or SEQ ID NO:40.

[0190] Preferably, the present invention provides an alpha-amylase comprising A and B domains and a C domain, wherein the amino acid sequences of the A and B domains are at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:42, and the amino acid sequence of the C domain is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:44. The present invention relates to an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity, comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37.

[0191] Preferably, the present invention provides a method for the preparation of a polypeptide comprising a polypeptide comprising an amino acid sequence of the A and B domains that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:42, and a polypeptide comprising an amino acid sequence of the C domain that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% or 100% identical to the amino acid sequence of SEQ ID NO:44. 54, wherein the A and B domains and the C domain are 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:54, wherein the A and B domains and the C domain are 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:54, and the C domain is 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:54.

[0192] The present invention also provides (a) a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:55, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38, wherein the nucleic acid encodes a polypeptide having amylase activity; (b) a nucleic acid sequence encoding a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, wherein the polypeptide has amylase activity, or any polypeptide described herein having amylase activity, preferably a nucleic acid sequence encoding a polypeptide having alpha-amylase activity comprising A and B domains and a C domain, wherein the amino acid sequences of the A and B domains are at least 75% identical to the amino acid sequence of SEQ ID NO:42 and the amino acid sequence of the C domain is at least 75% identical to the amino acid sequence of SEQ ID NO:44; (c) under high stringency conditions; (i) a sequence encoding SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37; or (ii) a polynucleotide set forth in SEQ ID NO:55, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38 a polynucleotide that hybridizes to the complement of; (d) a fragment of (a), (b), or (c), wherein the fragment encodes a polypeptide having amylase activity; or (e) a nucleic acid sequence that is completely complementary to any of (a) to (d) The present invention relates to an isolated, synthetic, or recombinant nucleic acid comprising:

[0193] Preferably, the isolated, synthetic, or recombinant nucleic acid is (a) a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:55, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38, wherein the nucleic acid encodes a polypeptide having amylase activity; (b) a nucleic acid sequence encoding a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, wherein the polypeptide has amylase activity; (c) a fragment of (a) or (b), wherein the fragment encodes a polypeptide having amylase activity; or (d) a nucleic acid sequence that is completely complementary to any of (a) to (c) Includes:

[0194] Further preferred herein are (a) a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:55, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38, wherein the nucleic acid encodes a polypeptide having amylase activity; (b) a nucleic acid sequence encoding a polypeptide having at least 90% sequence identity to SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, wherein the polypeptide has amylase activity; (c) a fragment of (a) or (b), wherein the fragment encodes a polypeptide having amylase activity; or (d) a nucleic acid sequence that is completely complementary to any of (a) to (c) is an isolated, synthetic, or recombinant nucleic acid comprising:

[0195] Further preferred herein are (a) a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 55, wherein the nucleic acid encodes a polypeptide having amylase activity; (b) a nucleic acid sequence encoding a polypeptide having at least 80% sequence identity to SEQ ID NO: 54, wherein the polypeptide has amylase activity; (c) a fragment of (a) or (b), wherein the fragment encodes a polypeptide having amylase activity; or (d) a nucleic acid sequence that is completely complementary to any of (a) to (c) is an isolated, synthetic, or recombinant nucleic acid comprising:

[0196] Preferably, the isolated, synthetic, or recombinant nucleic acid is (a) a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:55, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38, wherein the nucleic acid encodes a polypeptide having amylase activity; (b) a nucleic acid sequence encoding a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, wherein the polypeptide has amylase activity. Includes:

[0197] Preferably, the isolated, synthetic, or recombinant nucleic acid is (a) a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 55, wherein the nucleic acid encodes a polypeptide having amylase activity; (b) a nucleic acid sequence encoding a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 54, wherein the polypeptide has amylase activity. Includes:

[0198] Further preferred herein are amylases comprising an amino acid sequence that is at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37.

[0199] Further preferred herein are polypeptides (i.e., amylases) having amylase activity comprising an amino acid sequence that is at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of SEQ ID NO:54.

[0200] Further preferred herein are amylases comprising an amino acid sequence that is at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37.

[0201] Further preferred herein are amylases comprising an amino acid sequence that is at least 99% or 100% identical to the full-length amino acid sequence of SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37.

[0202] Further preferred herein are amylases comprising an amino acid sequence that is at least 99.5% identical to the full-length amino acid sequence of SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37.

[0203] Further preferred herein are amylases comprising an amino acid sequence that is 100% identical to the full-length amino acid sequence of SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37.

[0204] Further preferred herein are amylases comprising an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of SEQ ID NO:54, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37.

[0205] Further preferred herein are amylases comprising an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of SEQ ID NO:54.

[0206] Further preferred herein are amylases comprising an amino acid sequence that is at least at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of SEQ ID NO:54.

[0207] Further preferred herein are amylases that include one or more amino acid residue insertions, deletions, substitutions, or any combination thereof, relative to the amino acid sequence of SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37.

[0208] Further preferred herein are amylases comprising one or more amino acid residue substitutions relative to the amino acid sequence of SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, preferably at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, preferably positions 1 to 25, more preferably positions 1 to 10.

[0209] Further preferred herein are amylases encoded by a polynucleotide having a nucleic acid sequence wherein the amino acid sequence is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length polynucleotide sequence of SEQ ID NO:55, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38.

[0210] Further preferred herein are amylases encoded by a polynucleotide having a nucleic acid sequence whose amino acid sequence is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length polynucleotide sequence of SEQ ID NO:55.

[0211] Further preferred herein are those having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, or at least 86% similar to the full-length amino acid sequence of SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:35, or SEQ ID NO:37. An amylase comprising an amino acid sequence that is at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical, and that has increased expression, activity, thermostability, stability, laundry performance, and any combination thereof.

[0212] Further preferred herein is an amylase comprising an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:35, or SEQ ID NO:37, wherein the amylase has increased thermostability.

[0213] Further preferred herein are amylases comprising an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of SEQ ID NO: 54, wherein the amylase has increased thermostability.

[0214] Further preferred herein are those having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119 ...20%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 129%, at least 129%, at least 129%, at least 129%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 139% identical to the full-length amino acid sequence of SEQ ID NO:54, SEQ an amylase comprising an amino acid sequence that is at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the amino acid sequence of the amylase of the present invention, wherein the increased thermostability is after heat challenge at a temperature of 70°C to 100°C, preferably the increased thermostability is after heat challenge at a temperature of 70°C to 90°C, more preferably 75°C to 85°C, and most preferably 80°C.

[0215] Further preferred herein are amylases comprising an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of SEQ ID NO: 54, wherein the increased thermostability is after heat challenge at a temperature of 70°C to 100°C, preferably the increased thermostability is after heat challenge at a temperature of 70°C to 90°C, more preferably 75°C to 85°C, and most preferably 80°C.

[0216] Further preferred herein are amylases comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of SEQ ID NO: 54, wherein the amylase exhibits increased thermostability after heat challenge at 80°C.

[0217] In another aspect, the present invention relates to a composition comprising a polypeptide described herein. The composition may comprise a combination of the polypeptide and another enzyme. The enzyme combination may be from the same class, e.g., a composition comprising a first amylase and a second amylase. The enzyme combination may be from different classes of enzymes, e.g., a composition comprising a lipase and an amylase. The enzyme combination may be a composition comprising at least one amylase of the present invention and one or more second enzymes. In one embodiment, the composition comprises one second enzyme, two second enzymes, three second enzymes, four second enzymes, or more than four second enzymes. In some embodiments, the second enzyme is selected from the group consisting of a second amylase, a lipase, a protease, a cellulase, a laccase, a pectinase, and a nuclease, or any combination thereof.

[0218] The composition comprises an amylase comprising an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37. In another embodiment, the composition further comprises a second enzyme selected from the group consisting of a second amylase, a lipase, a protease, a cellulase, a laccase, a mannase, a pectinase, a xylanase, a nuclease, and any combination thereof. In a preferred embodiment, the composition further comprises a second enzyme, wherein the second enzyme is a different amylase. In another preferred embodiment, the composition further comprises a second enzyme, wherein the second enzyme is a protease.

[0219] Preferably, the composition comprises an amylase comprising an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of SEQ ID NO: 54. In another embodiment, the composition further comprises a second enzyme selected from the group consisting of a second amylase, a lipase, a protease, a cellulase, a laccase, a mannanase, a pectinase, a xylanase, a nuclease, and any combination thereof. In a preferred embodiment, the composition further comprises a second enzyme, wherein the second enzyme is a different amylase. In another preferred embodiment, the composition further comprises a second enzyme, wherein the second enzyme is a protease.

[0220] Additional enzymes suitable for hybrid or composition of the invention are further described below. In one embodiment, suitable enzymes include enzyme variants having enzymatic activity that is at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical when compared to the full-length polypeptide sequences of the parent enzymes disclosed below.

[0221] amylase Alpha-amylase (EC 3.2.1.1) enzymes are capable of carrying out the endohydrolysis of (1->4)-alpha-D-glucosidic bonds in polysaccharides containing three or more (1->4)-alpha-linked D-glucose units. Amylase enzymes act in a random manner on starch, glycogen, and related polysaccharides and oligosaccharides; the reducing group is released in the alpha configuration. Other examples of amylase enzymes include beta-amylase (EC 3.2.1.2), glucan 1,4-alpha-maltotetrahydrolase (EC 3.2.1.60), isoamylase (EC 3.2.1.68), glucan 1,4-alpha-maltohexaosidase (EC 3.2.1.98), and glucan 1,4-alpha-maltohydrolase (EC 3.2.1.133).

[0222] The amylases described below can be used as parent amylases to create variant amylases by introducing one or more amino acid substitutions and / or deletions as described herein.

[0223] Many amylase enzymes are described in patents and published patent applications, including, but not limited to, WO 2002 / 068589, WO 2002 / 068597, WO 2003 / 083054, WO 2004 / 091544, and WO 2008 / 080093.

[0224] The amylase is known to be derived from Bacillus licheniformis and has SEQ ID NO: 2 as described in WO 95 / 10603. Suitable variants are those that are at least 90% identical to SEQ ID NO: 2 as described in WO 95 / 10603 and / or contain one or more substitutions at the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243, 264, 304, 305, 391, 408, and 444, and have amylolytic activity. Such variants are described in SEQ ID NO: 4 of WO 94 / 02597, WO 94 / 018314, WO 97 / 043424 and WO 99 / 019467.

[0225] The amylase is known to be derived from B. stearothermophilus having SEQ ID NO:6 as described in WO 02 / 10355, or is at least 90% identical thereto, optionally with a C-terminal truncation across the wild-type sequence and having amylolytic activity. Suitable variants of SEQ ID NO:6 include those at least 90% identical thereto and / or those further comprising a deletion at positions 181 and / or 182 and / or a substitution at position 193.

[0226] The amylase is known to be derived from Bacillus sp. 707 having SEQ ID NO: 6 as disclosed in WO 99 / 19467, or an amylase that is at least 90% identical thereto that has amylolytic activity.

[0227] The amylase is an amylase known from Bacillus halmapalus having SEQ ID NO: 2 or SEQ ID NO: 7 described in WO 96 / 23872, also described as SP-722, or is at least 90% identical to one of the sequences having amylolytic activity.

[0228] The amylase is known to be derived from Bacillus sp. DSM12649 having SEQ ID NO: 4 as disclosed in WO 00 / 22103, or an amylase that is at least 90% identical thereto that has amylolytic activity.

[0229] The amylase is an amylase known from Bacillus strain TS-23 having SEQ ID NO: 2 as disclosed in WO 2009 / 061380 or an amylase that is at least 90% identical thereto that has amylolytic activity.

[0230] The amylase is an amylase known from Cytophaga sp. having SEQ ID NO: 1 as disclosed in WO 2013 / 184577 or at least 90% identical thereto that has amylolytic activity.

[0231] The amylase is an amylase known from Bacillus megaterium DSM90 having SEQ ID NO: 1 as disclosed in WO 2010 / 104675 or at least 90% identical thereto that has amylolytic activity.

[0232] The amylase is known to have amino acids 1 to 485 of SEQ ID NO: 2 as described in WO 00 / 60060 or is an amylase comprising an amino acid sequence that is at least 96% identical to amino acids 1 to 485 of SEQ ID NO: 2 that has amylolytic activity.

[0233] The amylase is also known to have SEQ ID NO: 12 as described in WO 2006 / 002643, or has at least 80% identity thereto, and has amylolytic activity. Suitable amylases include those having at least 80% identity compared to SEQ ID NO: 12 and / or containing substitutions at positions Y295F and M202LITV, and have amylolytic activity.

[0234] The amylase is also known to have SEQ ID NO: 6 as set forth in WO 2011 / 098531, or has at least 80% identity thereto, and is an amylase having amylolytic activity. Suitable amylases include those that have at least 80% identity to SEQ ID NO: 6 and / or contain substitutions at one or more positions selected from the group consisting of 193 [G,A,S,T or M], 195 [F,W,Y,L,I or V], 197 [F,W,Y,L,I or V], 198 [Q or N], 200 [F,W,Y,L,I or V], 203 [F,W,Y,L,I or V], 206 [F,W,Y,N,L,I,V,H,Q,D or E], 210 [F,W,Y,L,I or V], 212 [F,W,Y,L,I or V], 213 [G,A,S,T or M] and 243 [F,W,Y,L,I or V], and have amylolytic activity.

[0235] The amylase may also be an amylase known to have SEQ ID NO: 1 as set forth in WO 2013 / 001078 or having at least 85% identity thereto and having amylolytic activity. Suitable amylases include those having at least 85% identity compared to SEQ ID NO: 1 and / or containing alterations at two or more (several) positions corresponding to positions G304, W140, W189, D134, E260, F262, W284, W347, W439, W469, G476, and G477, and having amylolytic activity.

[0236] The amylase is an amylase that is known to have SEQ ID NO: 2 in WO 2013 / 001087 or has at least 85% identity thereto and has amylolytic activity. Suitable amylases include those that have amylolytic activity, have at least 85% identity compared to SEQ ID NO: 2 in WO 2013 / 001087, and / or include a deletion of positions 181+182, or 182+183, or 183+184 according to the numbering of SEQ ID NO. Suitable amylases include those which contain one or more modifications at any of the positions corresponding to W140, W159, W167, Q169, W189, E194, N260, F262, W284, F289, G304, G305, R320, W347, W439, W469, G476 and G477 and have amylolytic activity, have at least 85% identity to SEQ ID NO: 2 as set forth in WO 2013 / 001087, and / or contain a deletion of positions 181+182, or 182+183, or 183+184 according to the numbering of SEQ ID NO: 2.

[0237] Amylases also include hybrid α-amylases derived from the above amylases, for example as described in WO 2006 / 066594.

[0238] Commercially available amylase enzymes include Duramyl™, Termamyl™, Termamyl SC™, Termamyl Ultra™, Fungamyl™, Stainzyme™, Stainzyme Plus™, Natalase™, Liquozyme X, Supramyl™, Amplify™, Amplify Prime™, and BAN™ (from Novozymes A / S), as well as Rapidase™, Purastar™, Purastar OxAm™, Powerase™, Effectenz™ (M100, from DuPont), Preferenz™ (S1000, S110, and F1000; from DuPont), PrimaGreen™ (ALL; DuPont), Optisize™ (DuPont), and Kam™. (Kao) and Kemzyme™ (Biozym).

[0239] Lipase "Lipase," "lipolytic enzyme," and "lipid esterase" all refer to enzymes in EC class 3.1.1 ("carboxylic ester hydrolases"). Lipases (EC 3.1.1.3, triacylglycerol lipases) can hydrolyze triglycerides into more hydrophilic mono- and diglycerides, free fatty acids, and glycerol. Lipase enzymes typically also include enzymes that are active on substrates other than triglycerides or that cleave specific fatty acids, such as phospholipase A (EC 3.1.1.4), galactolipase (EC 3.1.1.26), cutinase (EC 3.1.1.74), and enzymes with sterol esterase activity (EC 3.1.1.13) and / or wax ester hydrolase activity (EC 3.1.1.50).

[0240] Many lipase enzymes are described in patents and published patent applications, including, but not limited to, WO2000032758, WO2003 / 089620, WO2005 / 032496, WO2005 / 086900, WO200600976, WO2006 / 031699, WO2008 / 036863, WO2011 / 046812, and WO2014059360.

[0241] Lipases are used in detergents and cleaning products to remove grease, fat, oil, and dairy stains. Commercially available lipases include, but are not limited to, Lipolase™, Lipex™, Lipolex™, and Lipoclean™ (Novozymes A / S), Lumafast (originally from Genencor), and Lipomax (Gist-Brocades / now DSM).

[0242] Methods for determining lipolytic activity are well known in the literature (see, for example, Gupta et al. (2003), Biotechnol. Appl. Biochem. 37, pp. 63-71). For example, lipase activity can be measured by hydrolyzing the ester bond in para-nitrophenyl palmitate substrate (pNP-Palmitate, C:16) to liberate pNP, which is yellow in color, and detecting it at 405 nm.

[0243] Proteases Enzymes that have proteolytic activity are called "proteases" or "peptidases." Proteases are active proteins that exhibit "protease activity" or "proteolytic activity."

[0244] Proteases are members of the class EC3.4. Proteases include aminopeptidases (EC 3.4.11), dipeptidases (EC 3.4.13), dipeptidyl-peptidases and tripeptidyl-peptidases (EC 3.4.14), peptidyl-dipeptidases (EC 3.4.15), serine-type carboxypeptidases (EC 3.4.16), metallocarboxypeptidases (EC 3.4.17), cysteine-type carboxypeptidases (EC 3.4.18), omega-peptidases (EC 3.4.19), serine endopeptidases (EC 3.4.21), cysteine ​​endopeptidases (EC 3.4.22), aspartic endopeptidases (EC 3.4.23), metallo-endopeptidases (EC 3.4.24), threonine endopeptidases (EC 3.4.25), and endopeptidases of unknown catalytic mechanism (EC 3.4.99).

[0245] Commercially available protease enzymes include, but are not limited to, Lavergy™ Pro (BASF); Alcalase®, Blaze®, Duralase™, Durazym™, Relase®, Relase® Ultra, Savinase®, Savinase® Ultra, Primase®, Polarzyme®, Kannase®, Liquanase®, Liquanase® Ultra, Ovozyme®, Coronase®, Coronase® Ultra, Neutrase®, Everlase®, and Esperase® (Novozymes A / S), under the trade names Maxatase®, Maxacal®, Maxapem®, Purafect®, Purafect® Prime, Purafect MA®, Purafect Ox®, Purafect These include those sold under the names OxP®, Puramax®, Properase®, FN2®, FN3®, FN4®, Excellase®, Eraser®, Ultimase®, Opticlean®, Effectenz®, Preferenz® and Optimase® (Danisco / DuPont), Axapem™ (Gist-Brocases NV), alkaline protease from Bacillus lentus, and KAP (Bacillus alkalophilus subtilisin) from Kao.

[0246] At least one protease can be selected from serine proteases (EC 3.4.21), which are characterized by having a serine in the catalytically active site and forming a covalent adduct with a substrate during catalysis. Serine proteases include chymotrypsin (e.g., EC 3.4.21.1), elastase (e.g., EC 3.4.21.36), elastase (e.g., EC 3.4.21.37 or EC 3.4.21.71), granzymes (e.g., EC 3.4.21.78 or EC 3.4.21.79), kallikreins (e.g., EC 3.4.21.34, EC 3.4.21.35, EC 3.4.21.118, or EC 3.4.21.119), plasmin (e.g., EC 3.4.21.7), trypsin (e.g., EC 3.4.21.4), thrombin (e.g., EC 3.4.21.5), and subtilisins (also known as subtilopeptidases, e.g., EC 3.4.21.62), the latter of which is also referred to hereinafter as "subtilisin."

[0247] cellulase "Cellulases," "cellulase enzymes," or "cellulolytic enzymes" are enzymes involved in the hydrolysis of cellulose. Three major types of cellulases are known: endo-ss-1,4-glucanases (endo-1,4-PD-glucan 4-glucanohydrolases, EC 3.2.1.4; hydrolyzes β-1,4-glucosidic bonds in cellulose), cellobiohydrolases (1,4-PD-glucan cellobiohydrolases, EC 3.2.1.91), and ss-glucosidases (EC 3.2.1.21).

[0248] Cellulase enzymes are described in patents and published patent applications such as, but not limited to, WO1997 / 025417, WO1998 / 024799, WO2003 / 068910, WO2005 / 003319, and WO2009020459.

[0249] Commercially available cellulase enzymes include Celluzyme™, Endolase™, Carezyme™, Cellusoft™, Renozyme™, Celluclean™ (from Novozymes A / S), Ecostone™, Biotouch™, Econase™, Ecopulp™ (from AB Enzymes Finland), Clazinase™, and Puradax HA™, Genencor detergent cellulase L, IndiAge™ Neutra (from Genencor International Inc. / DuPont), Revitalenz™ (2000, from DuPont), Primafast™ (DuPont), and KAC-500™ (from Kao Corporation).

[0250] The cellulases according to the present invention have "cellulolytic activity" or "cellulase activity." Assays for measuring cellulolytic activity are known to those skilled in the art. For example, cellulolytic activity can be determined by cellulase hydrolysis of carboxymethylcellulose to reducing carbohydrates and determining the reducing ability colorimetrically by the ferricyanide reaction according to Hoffman, WS, J. Biol. Chem. 120, 51 (1937).

[0251] Mannanase Mannanase (EC 3.2.1.78) enzymes hydrolyze internal β-1,4 beta-D-mannosidic bonds in mannans, galactomannans, and glucomannans. The "mannase" may be an alkaline mannanase of family 5 or 26. Because mannanase removes some of the dyes contained in hemicellulose, mannanase is a useful component of detergent and / or cleaning formulations. Inadequate removal of these types of dyes can result in, for example, graying of fabrics. The main components of hemicellulose are hetero-1,4-D-xylans and hetero-1,4-beta-mannans. Mannans are polysaccharides with a backbone of β-1,4-linked D-mannopyranosyl residues, which may contain galactose or acetyl substitutions, and may also contain glucose residues in the backbone. Mannanase enzymes are known to be wild-type from Bacillus or Humicola, particularly B. agaradhaerens, B. licheniformis, B. halodurans, B. clausii, or H. insolens. Suitable mannanases are described in WO 99 / 064619.

[0252] Commercially available mannanase enzymes include Mannaway® (Novozymes AIS).

[0253] Pectate lyase The enzyme pectate lyase (EC 4.2.2.2) excisively cleaves (1->4)-alpha-D-galacturonan to give oligosaccharides bearing a 4-deoxy-alpha-D-galact-4-enuronosyl group at their non-reducing ends.

[0254] Pectate lyase enzymes are described in patents and published patent applications, including, but not limited to, WO 2004 / 090099. Pectate lyases are known to be derived from Bacillus, particularly Bacillus licheniformis or Bacillus agaradoherens, or variants thereof, as described, for example, in U.S. Pat. No. 6,124,127, WO 99 / 027083, WO 99 / 027084, WO 2002 / 006442, WO 2002 / 092741, and WO 2003 / 095638.

[0255] Commercially available pectate lyase enzymes include Xpect™, Pectawash™ and Pectaway™ (Novozymes A / S); PrimaGreen™, EcoScour (DuPont).

[0256] nuclease Deoxyribonuclease I, or nuclease (EC 3.1.21.1), also known as DNase, performs endonucleolytic cleavage into 5'-phosphodinucleotide and 5'-phosphooligonucleotide end products.

[0257] Nuclease enzymes are described in patents and published patent applications such as, but not limited to, US3451935, GB1300596, DE10304331, WO2015155350, WO2015155351, WO2015166075, WO2015181287, and WO2015181286.

[0258] In one aspect of the present invention, at least one amylase variant of the present invention is provided together with at least one protease. In one embodiment, the amylase variant of the present invention is stable in the presence of at least one protease. In one embodiment, the amylase variant of the present invention has increased protease stability compared to the corresponding parent amylase. In one embodiment, the at least one protease is selected from subtilisin 309 disclosed as sequence a) in Table I of WO 89 / 06279, or a variant thereof at least 80% identical thereto and having proteolytic activity. In one embodiment, the amylase variant of the present invention has increased protease stability in the presence of said subtilisin 309 or a variant thereof at least 80% identical thereto, compared to an amylase according to SEQ ID NO: 1.

[0259] The protease can itself be stabilized by a protease stabilizer, or the protease can be destabilized. In one embodiment, the amylase variant of the invention has increased protease stability in the presence of destabilized subtilisin 309 or a destabilized variant thereof that is at least 80% identical thereto, when compared to an amylase according to SEQ ID NO: 1.

[0260] How to make: In another embodiment, the invention relates to a method of making a variant polypeptide described herein, comprising providing a nucleic acid sequence encoding a polypeptide described herein, transforming the nucleic acid sequence into a host cell, culturing the host cell to produce the variant polypeptide, and, optionally, purifying the variant polypeptide from the host cell.

[0261] A polynucleotide encoding a polypeptide can be "expressed." The term "expression" or "gene expression" refers to the transcription of a specific gene or genes or a specific nucleic acid construct. The term "expression" or "gene expression" refers to the transcription of a gene or genes or gene constructs into structural RNA (e.g., rRNA, tRNA) or mRNA, which may or may not then be translated into protein. This process involves transcription of DNA and processing of the resulting mRNA product.

[0262] Industrial production of enzymes is usually carried out by using an expression system. "Expression system" refers to a host microorganism, expression host, host cell, production organism, or production strain, and these terms can be used interchangeably. In one embodiment, the expression host is selected from the group consisting of a bacterial expression system, a yeast expression system, a fungal expression system, and a synthetic expression system. The expression host may be a wild-type cell or a recombinant cell. "Wild-type cell" herein refers to a cell prior to a specific modification. The term "recombinant cell" (also referred to herein as "genetically modified cell") refers to a cell that has been genetically altered, modified, or engineered to exhibit an altered, modified, or different genotype compared to the wild-type cell from which it was derived. A "recombinant cell" may contain an exogenous polynucleotide encoding a specific protein or enzyme and thus express said protein or enzyme.

[0263] Thus, in one embodiment, the present invention is directed to a genetic construct comprising a polynucleotide encoding an amylase described herein.

[0264] In one embodiment, the present invention is directed to an expression vector comprising a polynucleotide encoding an amylase described herein.

[0265] In one embodiment, the present invention is directed to a host cell comprising a polynucleotide encoding an amylase described herein.

[0266] In yet another embodiment, the present invention provides a method of expressing a polynucleotide, comprising: (a) providing a host cell comprising a heterologous nucleic acid construct comprising a polynucleotide encoding an amylase described herein by introducing into the host cell a nucleic acid construct comprising a polynucleotide encoding an amylase described herein; (b) culturing the recombinant host cell of step (a) under conditions conducive to expression of the polynucleotide; and (c) optionally recovering the protein of interest encoded by the polynucleotide; The present invention is directed to a method, including:

[0267] Examples of expression systems include, but are not limited to, Aspergillus niger, Aspergillus oryzae, Hansenula polymorpha, Thermomyces lanuginosus, Fusarium oxysporum, Fusarium heterosporum, Escherichia coli, Bacillus, preferably Bacillus pumilus, Bacillus subtilis, or Bacillus licheniformis, Pseudomonas, preferably Pseudomonas fluorescens, Examples of suitable expression systems include Pichia fluorescens, Pichia pastoris (also known as Komagataella phaffii), Myceliopthora thermophile (C1), Thermothelomyces thermophila, Schizosaccharomyces pombe, Trichoderma, preferably Trichoderma reesei, and Saccharomyces, preferably Saccharomyces cerevisiae. Variant polypeptides can be produced using the expression systems listed above.

[0268] In one embodiment, the bacterial expression system is selected from E. coli, Bacillus, Pseudomonas, and Streptomyces. In one embodiment, the yeast expression system is selected from Candida, Pichia, Saccharomyces, and / or Schizosaccharomyces. In one embodiment, the fungal expression system is selected from Penicillium, Aspergillus, Fusarium, Myceliopthora, Rhizomucor, Rhizopus, Thermomyces, and Trichoderma.

[0269] The term "heterologous" (or exogenous or foreign or recombinant) in the context of polynucleotides and polypeptides refers to (a) not native to the host cell; (b) is native to the host cell but contains structural modifications, e.g., deletions, substitutions, and / or insertions, as a result of manipulation of the host cell's DNA by recombinant DNA techniques to alter the native sequence; or (c) native to the host cell, but expression is quantitatively altered or expression is directed from a different genomic location than in the native host cell, e.g., a stronger promoter, as a result of manipulation of the host cell's DNA by recombinant DNA techniques; is defined herein.

[0270] With respect to two or more polynucleotide sequences or two or more amino acid sequences, the term "heterologous" is used to characterize that the two or more polynucleotide sequences or two or more amino acid sequences do not naturally occur in specific combination with each other.

[0271] As used herein, a "genetic construct" or "gene cassette" is a DNA molecule comprised of at least one sequence of interest to be expressed operably linked to one or more control sequences (at least a promoter) as described herein. Typically, an expression cassette contains three elements: a promoter sequence, an open reading frame, and a 3' untranslated region, which in eukaryotes usually contains a polyadenylation site. Additional regulatory elements include transcriptional as well as translational enhancers. Intron sequences can also be added to the 5' untranslated region (UTR) or within the coding sequence to increase the amount of mature message that accumulates in the cytosol. An expression cassette can be part of a vector or can be integrated into the genome of a host cell and replicated along with the genome of the host cell. An expression cassette can usually increase or decrease expression.

[0272] As used herein, the term "vector" includes any type of construct suitable for carrying an exogenous polynucleotide sequence for transfer into another cell or for stable or transient expression in a given cell. As used herein, the term "vector" encompasses any type of cloning vehicle, such as, but not limited to, a plasmid, a phagemid, a viral vector (e.g., a phage), a bacteriophage, a baculovirus, a cosmid, a fosmid, an artificial chromosome, or any other vector specific to a particular host of interest. Low-copy and high-copy vectors are also included. The exogenous polynucleotide sequence typically includes a coding sequence, which may be referred to herein as a "gene of interest." The gene of interest may include introns and exons, depending on the origin or target type of host cell.

[0273] The vectors used herein may provide segments for the transcription and translation of foreign polynucleotides upon transformation into host cells or host cell organelles. Such additional segments may include regulatory nucleotide sequences, one or more origins of replication required for its maintenance and / or replication in a particular cell type, one or more selectable markers, a polyadenylation signal, a suitable site for insertion of foreign coding sequences such as a multiple cloning site, and the like. One example is when the vector needs to be maintained in bacterial cells as an episomal genetic element (e.g., a plasmid or cosmid molecule). Non-limiting examples of suitable origins of replication include the f1-ori and colE1.

[0274] A vector can replicate without integrating into the host cell's genome, for example as a plasmid in a bacterial host cell, or it can integrate some or all of its DNA into the host cell's genome, thus bringing about the replication and expression of that DNA.

[0275] Foreign nucleic acids can be introduced into a vector by cloning, which may mean that cleavage of the vector (e.g., within the multiple cloning site) and foreign polynucleotide by suitable means and methods (e.g., restriction enzymes) can create compatible structures within the individual nucleic acids that allow controlled fusion of the foreign nucleic acid with the vector.

[0276] Once introduced into a vector, the foreign nucleic acid containing the coding sequence may be suitable for introduction (transformation, transduction, transfection, etc.) into a host cell or host cell organelle. Cloning vectors suitable for expression of foreign polynucleotide sequences in the host cell or host cell organelle can be selected.

[0277] The terms "introduction" or "transformation" referred to herein encompass the transfer of an exogenous polynucleotide into a host cell, regardless of the method used for transfer. That is, the term "transformation" as used herein is independent of vectors, shuttle systems, or host cells, and it encompasses not only the polynucleotide transfer methods of transformation known in the art (e.g., Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), but also any additional types of polynucleotide transfer methods, such as, but not limited to, transduction or transfection. Plant tissue that allows for subsequent clonal propagation, whether by organogenesis or embryogenesis, can be transformed with a gene construct and whole plants can be regenerated therefrom. The particular tissue selected will vary depending on the clonal propagation system available and best suited to the particular species being transformed. In one embodiment of the present invention, the vector is used for transformation of a host cell.

[0278] Polynucleotides can be transiently or stably introduced into host cells and maintained non-integrated, for example, as a plasmid. "Stable transformation" can also mean that the transformed cell or cellular organelle passes on the nucleic acid containing the exogenous coding sequence to subsequent generations of cells or cellular organelles. Typically, stable transformation results from the integration of the nucleic acid containing the exogenous coding sequence into a chromosome or as an episome (a separate piece of nuclear DNA).

[0279] "Transient transformation" may mean that a cell or cell organelle, once transformed, expresses an exogenous nucleic acid sequence for a specific period of time, often within one generation. Typically, transient transformation results from a nucleic acid containing the exogenous nucleic acid sequence that is not integrated into a chromosome or as an episome.

[0280] Alternatively, it may be integrated into the host genome. The resulting transformed plant cells can then be used to regenerate transformed plants in a manner known to those skilled in the art.

[0281] Recombinant cells may exhibit "increased" or "decreased" expression when compared to the corresponding wild-type cells.

[0282] As used herein, the terms "increased expression," "enhanced expression," or "overexpression" refer to any form of expression that is additional to the original wild-type expression level (which may also be absent or unmeasurable expression). Reference herein to "increased expression," "enhanced expression," or "overexpression" is taken to mean, insofar as it relates to an increase in gene expression and / or polypeptide, increased polypeptide levels and / or increased polypeptide activity compared to a control organism. The increase in expression may be, in order of decreasing priority, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or 100% or more compared to that of a control organism.

[0283] Methods for increasing the expression of genes or gene products have been well documented in the art, and include, for example, overexpression driven by an appropriate promoter, the use of transcriptional or translational enhancers. An isolated nucleic acid that acts as a promoter or enhancer element can be introduced into an appropriate position (typically upstream) of a non-heterologous polynucleotide to increase the expression of a nucleic acid encoding a polypeptide of interest. For example, endogenous promoters can be altered in vivo by mutation, deletion, and / or substitution (see U.S. Patent No. 5,565,350 to Kmiec et al.; WO 93 / 22443 to Zarling et al.), or an isolated promoter can be introduced into an organism with an appropriate orientation and distance from the gene of the present invention to control gene expression.

[0284] Intron sequences can also be added to the 5' untranslated region (UTR) or the coding sequence of a partial coding sequence to increase the amount of mature message that accumulates in the cytosol. The inclusion of splicable introns in transcription units in expression constructs has been shown to increase gene expression by up to 1000-fold at both the mRNA and protein levels (Buchman and Berg (1988) Mol. Cell Biol. 8: 4395-4405; Callis et al. (1987) Genes Dev 1:1183-1200). Such intron enhancement of gene expression is typically greatest when placed near the 5' end of the transcription unit.

[0285] To obtain increased or overexpression of a polypeptide, the nucleic acid encoding the polypeptide is most commonly overexpressed in the sense orientation with a polyadenylation signal. Introns or other enhancing elements can be used in addition to a promoter suitable for driving expression with the intended expression pattern.

[0286] Enzymes are generally produced commercially by using recombinant cells that express the desired enzyme by cultivation under conditions suitable for expression of the desired enzyme.

[0287] Culturing is typically carried out in a suitable nutrient medium to grow the recombinant cells (a process that can be called fermentation) and express the desired protein. At the end of fermentation, the fermentation broth is collected and, if it contains a liquid fraction and a solid fraction, may be further processed.

[0288] The enzyme of interest can be further purified from the fermentation broth. The term "purification" or "purifying" refers to the process of separating at least one component, such as a protein of interest, from at least another component, such as particulate matter in the fermentation broth, and transferring it to a different compartment or phase that does not necessarily need to be separated by a physical barrier. An example of such a different compartment is two compartments separated by a filter membrane or cloth, i.e., a filtrate and a retentate; an example of such a different phase is a precipitate and a supernatant, or a cake and a filtrate, respectively. The solution obtained after purifying the enzyme of interest from the fermentation broth is referred to herein as a "purified enzyme solution."

[0289] The desired enzyme may be secreted (into the liquid fraction of the fermentation broth) or may not be secreted from the host cells (and thus contained within the cells of the fermentation broth). Accordingly, the desired enzyme can be recovered from the liquid fraction of the fermentation broth or from the cell lysate. The recovery of the desired enzyme uses methods known to those skilled in the art. Suitable methods for recovering proteins or enzymes from the fermentation broth include, but are not limited to, collection, centrifugation, filtration, extraction, and sedimentation. If the desired enzyme precipitates or crystallizes in the fermentation broth or is at least partially bound to particulate matter in the fermentation broth, additional processing steps may be required to release the enzyme from the biomass or solubilize the enzyme crystals and precipitates. US Pat. No. 6,316,240 B1 describes a method for recovering enzymes that precipitate and / or crystallize during fermentation from the fermentation broth. If the desired enzyme is contained within the cells of the fermentation broth, release of the enzyme from the cells may be required. Release from the cells can be achieved, for example, but not limited to, by cell lysis using techniques known to those skilled in the art.

[0290] The purified enzyme solution may be further processed to form an "enzyme preparation." An "enzyme preparation" refers to any non-complex preparation containing a small number of components, the components serving to stabilize the enzyme contained in the enzyme preparation and / or the enzyme preparation itself. The term "enzyme stability" relates to the retention of enzyme activity as a function of time during storage or manipulation. The term "enzyme preparation stability" relates to the maintenance of the physical appearance of the enzyme preparation during storage or manipulation, as well as the avoidance of microbial contamination during storage or manipulation.

[0291] An "enzyme formulation" is a composition that is intended to be formulated into a complex formulation that can be determined for its own end use. An "enzyme formulation" according to the present invention is not a complex formulation containing several components that are formulated in the complex formulation so that each component individually performs a specific function in the end application. A complex formulation may be, but is not limited to, a detergent formulation in which the individual detergent components are formulated in amounts that are effective in the cleaning performance of the detergent formulation.

[0292] In one aspect of the invention, at least one amylase variant of the invention is comprised in an enzyme formulation.

[0293] The enzyme preparation may be solid or liquid. The enzyme preparation can be obtained by using techniques known in the art. For example, but not limited to, the solid enzyme preparation can be obtained by extrusion or granulation. Suitable extrusion and granulation techniques are known in the art and are described, for example, in WO9419444A1 and WO9743482A1.

[0294] "Liquid" in the context of an enzyme formulation refers to physical appearance at 20°C and 101.3 kPa.

[0295] The liquid enzyme formulation may contain an amount of enzyme in the range of 0.1 to 40% by weight, or 0.5 to 30% by weight, or 1 to 25% by weight, or 3 to 10% by weight, all based on the total weight of the enzyme formulation.

[0296] A liquid enzyme formulation may contain more than one type of enzyme. In one embodiment, the enzyme formulation comprises one or more amylases according to the present invention. In one embodiment, the enzyme formulation comprises one or more amylases according to the present invention and at least one additional enzyme selected from the group consisting of a second amylase, a lipase, a protease, a cellulase, a laccase, a pectinase, a nuclease, and any combination thereof.

[0297] Aqueous enzyme formulations of the present invention may contain water in an amount greater than about 50% by weight, greater than about 60% by weight, greater than about 70% by weight, or greater than about 80% by weight, all based on the total weight of the enzyme formulation.

[0298] The liquid enzyme formulations of the present invention may contain residual components such as salts from the fermentation medium, cell debris from the production host cells, and metabolites produced by the production host cells during fermentation.

[0299] In one embodiment, the remaining components may be present in the liquid enzyme formulation in an amount of less than 30 wt.%, less than 20 wt.%, less than 10 wt.%, or less than 5 wt.%, based on the total weight of the aqueous enzyme formulation. In one embodiment, the enzyme formulation, particularly the liquid enzyme formulation, contains, in addition to one or more enzymes, one or more additional components selected from the group consisting of solvents, salts, pH adjusters, preservatives, stabilizers, chelating agents, and thickeners. The preservative in the liquid enzyme formulation may be sorbitol, benzoate, Proxel, or any combination thereof. The stabilizer in the liquid enzyme formulation may be MPG, glycerol, acetate, or any combination thereof. The chelating agent in the liquid enzyme formulation may be citrate.

[0300] In one embodiment, the enzyme preparation comprises at least one polypeptide variant of the present invention and at least one preservative. Non-limiting examples of suitable preservatives include (quaternary) ammonium compounds, isothiazolinones, organic acids, and formaldehyde-releasing agents. Non-limiting examples of suitable (quaternary) ammonium compounds include benzalkonium chloride, polyhexamethylene biguanide (PHMB), didecyldimethylammonium chloride (DDAC), and N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine (diamine). Non-limiting examples of suitable isothiazolinones include 1,2-benzisothiazolin-3-one (BIT), 2-methyl-2H-isothiazol-3-one (MIT), 5-chloro-2-methyl-2H-isothiazol-3-one (CIT), 2-octyl-2H-isothiazol-3-one (OIT), and 2-butyl-benzo[d]isothiazol-3-one (BBIT). Non-limiting examples of suitable organic acids include benzoic acid, sorbic acid, L-(+)-lactic acid, formic acid, and salicylic acid. Non-limiting examples of suitable formaldehyde-releasing agents include N,N'-methylenebismorpholine (MBM), 2,2',2''-(hexahydro-1,3,5-triazine-1,3,5-triyl)triethanol (HHT), (ethylenedioxy)dimethanol, α,α',α''-trimethyl-1,3,5-triazine-1,3,5(2H,4H,6H)-triethanol (HPT), 3,3'-methylenebis[5-methyloxazolidine] (MBO), and cis-1-(3-chloroallyl)-3,5,7-triaza-1-azoniadamantane chloride (CTAC).

[0301] Further useful preservatives include halogen-releasing compounds such as iodopropynyl butylcarbamate (IPBC), dichloro-dimethyl-hydantoin (DCDMH), bromo-chloro-dimethyl-hydantoin (BCDMH), and dibromo-dimethyl-hydantoin (DBDMH); bromo-nitro compounds such as bronopol (2-bromo-2-nitropropane-1,3-diol), 2,2-dibromo-2-cyanoacetamide (DBNPA); aldehydes such as glutaraldehyde; phenoxyethanol; biphenyl-2-ol; and zinc or sodium pyrithione.

[0302] In one embodiment, the enzyme formulation comprises at least one polypeptide variant of the present invention and at least one enzyme stabilizer. The enzyme stabilizer is selected from substances capable of reducing the loss of enzyme activity of at least one enzyme contained in the liquid enzyme formulation during storage. In this context, a reduction in enzyme activity loss may mean that the loss of enzyme activity is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% compared to the initial enzyme activity before storage. Preferred stabilizers are selected from the group consisting of salts (e.g., CaCl), propanediol, polyethylene glycol, MPG, glycerol, acetate, or any combination thereof.

[0303] Enzyme uses In another embodiment, the polypeptide variants described herein can be used in food products, for example, the enzymes can be additives for baking. The enzymes can be used in feed, for example, as animal feed additives. The enzymes can be used in the starch processing industry, for example, amylases are used in the conversion of starch to ethanol or sugars (high fructose corn syrup) and oil, dry distiller grains, etc. The polypeptide variants can be used in pulp and paper processing, for example, enzymes can be used to improve paper strength. The enzymes can be used for mining and oil well servicing, for example, cellulases can be used to break down guar during oil well fracturing. In one embodiment, the polypeptide variants described herein are used in detergent or cleaning formulations.

[0304] In one embodiment, the invention relates to a method for preparing a dough or a baked product prepared from the dough, comprising adding one of the variant polypeptides with amylase activity described herein to the dough and baking it. In one embodiment, the invention relates to a method for using the variant polypeptides with amylase activity described herein to process starch. In one embodiment, the invention relates to a method for using the variant polypeptides with amylase activity described herein to clean or wash textiles, hard surfaces, or dishware. In one embodiment, the invention relates to a method for using the variant polypeptides with amylase activity described herein to make ethanol. In one embodiment, the invention relates to a method for using the variant polypeptides with amylase activity described herein to process pulp or paper. In one embodiment, the invention relates to a method for using the variant polypeptides with amylase activity described herein to feed animals.

[0305] In one embodiment, the amylases of the invention are used in detergent or cleaning formulations.

[0306] "Detergent formulation" or "cleaning formulation" means a composition designated for cleaning soiled materials. Cleaning includes laundry and hard surface cleaning. Soiled materials according to the present invention include textiles and / or hard surfaces.

[0307] The term "laundry" refers to the process of treating textiles with a solution containing the detergent composition of the present invention, both in relation to domestic and industrial laundering. The laundering process can be carried out by using technical devices such as domestic or industrial washing machines. Alternatively, the laundering process can be carried out by hand.

[0308] The term "textile" means any textile material, including yarns (threads made from natural or synthetic fibers used for knitting or weaving), yarn intermediates, fibers, nonwoven materials, natural materials, synthetic materials, and fabrics (textiles made by weaving, knitting, or felting fibers) made from these materials, such as garments (any clothing made from fabrics), cloth, and other items.

[0309] The term "fabric" includes natural fibers, synthetic fibers, and mixtures thereof. Examples of natural fibers are those of plant (such as flax, jute, and cotton) or animal (e.g., silk, wool, angora, mohair, cashmere) origin, which contain proteins such as collagen, keratin, and fibroin. Examples of fibers of synthetic origin are polyurethane fibers such as Spandex® or Lycra®, polyester fibers, polyolefins such as Elastofine, or polyamide fibers such as nylon. The fibers may be single fibers or part of a textile product such as knitwear, woven fabric, or nonwoven fabric.

[0310] The term "hard surface cleaning" is defined herein as cleaning of hard surfaces, which may include any hard surface in the home, such as floors, furniture, walls, sanitary ware, glass, metal surfaces including cutlery or tableware.

[0311] The term "dishwashing" refers to all forms of dishwashing, for example by hand or in an automatic dishwasher, including but not limited to cleaning all forms of crockery such as plates, cups, glasses, bowls, etc., all forms of cutlery such as spoons, knives, forks and serving utensils, as well as ceramic, plastics such as melamine, metal, porcelain, glass and acrylic.

[0312] The detergent formulations of the present invention comprise one or more detergent ingredients. The ingredients selected will depend on the desired cleaning application and / or the physical form of the detergent composition.

[0313] The term "detergent ingredient" is defined herein to mean any type of ingredient suitable for detergent compositions, such as surfactants, building agents, polymers, bleaching systems, etc. Any ingredient known in the art, recognizing its known properties, is a suitable detergent ingredient according to the present invention. In one embodiment, detergent ingredient refers to an ingredient that, when present in an effective amount, provides washing or cleaning performance or aids in processing efficiency (maintains physical properties during processing, storage, and use; e.g., rheology modifiers, hydrotropes, drying agents).

[0314] Typically, detergent compositions are complex formulations of three or more detergent ingredients.

[0315] A detergent ingredient may have more than one function in the final application of the detergent formulation, and therefore any detergent ingredient described herein in the context of a particular function may have another function in the final application of the detergent formulation. The function of a particular detergent ingredient in the final application of the detergent formulation depends on its amount in the detergent formulation, i.e., the effective amount of the detergent ingredient.

[0316] The term "effective amount" includes the amount of a particular ingredient that provides effective stain removal and effective cleaning conditions (e.g., pH, amount of foaming), the amount of a particular ingredient that effectively provides a visual benefit (e.g., optical brightening, dye transfer inhibition), and the amount of a particular ingredient that effectively aids processing (maintains physical properties during processing, storage, and use; e.g., rheology modifiers, hydrotropes, desiccants).

[0317] In one embodiment, the detergent formulation is a formulation of more than two detergent ingredients, where at least one ingredient is effective in stain removal, at least one ingredient is effective in providing visually clean conditions, and at least one ingredient is effective in maintaining the physical properties of the detergent.

[0318] The cleaning performance is increased under relevant cleaning conditions. The term "relevant cleaning conditions" as used herein refers to the conditions, in particular the cleaning temperature, time, cleaning mechanism, soapy water concentration, detergent type and water hardness, actually used in a washing machine, automatic dishwasher or hand cleaning process.

[0319] Individual detergent ingredients and their use in detergent compositions are known to those skilled in the art. Suitable detergent ingredients include, inter alia, surfactants, extenders, polymers, alkalis, bleaching systems, optical brighteners, soap solution inhibitors and stabilizers, hydrotropes, and preservatives. Further examples are described, for example, in "Complete Technology Book on Detergents with Formulations (Detergent Cake, Dishwashing Detergents, Liquid & Paste Detergents, Enzyme Detergents, Cleaning Powder & Spray Dried Washing Powder)", Engineers India Research Institute (EIRI), 6th Edition (2015). Another reference for those skilled in the art may be "Detergent Formulations Encyclopedia", Solverchem Publications, 2016.

[0320] The detergent ingredients vary in type and / or amount in the detergent formulation depending on the desired application, such as washing white textiles, colored textiles, and wool. The ingredients selected further depend on the physical form of the detergent formulation (liquid, solid, gel, etc., provided as a pouch or tablet). For example, the ingredients selected for a laundry formulation further depend on regional practices and geographic characteristics such as the average hardness of the water, which are themselves related to aspects such as the wash temperature used, the washing machine mechanism (vertical axis vs. horizontal axis machines), the amount of water consumed per wash cycle, etc.

[0321] For example, a low detergent concentration system includes laundry formulations in which less than about 800 ppm of detergent ingredients are present in the wash water, a medium detergent concentration system includes laundry formulations in which from about 800 ppm to about 2,000 ppm of detergent ingredients are present in the wash water, and a high detergent concentration system includes laundry formulations in which about 2,000 ppm of detergent ingredients are present in the wash water.

[0322] The numerical ranges recited for individual detergent ingredients provide the amounts to be included in the detergent composition. Such ranges should be understood to be inclusive of the numbers defining the range, including each integer within the stated range.

[0323] Unless otherwise stated, "% by weight" or "% w / w" means that it is of the total detergent composition, in which case "% by weight" or "% w / w" is calculated as follows: the concentration of that substance as the weight of that substance divided by the total weight of the composition and multiplied by 100.

[0324] The detergent formulations of the present invention may contain one or more surfactants. "Surfactant" (used interchangeably herein with "surface active agent") means an organic chemical that, when added to a liquid, changes the properties of that liquid at an interface. According to their ionic charge, surfactants are called nonionic, anionic, cationic, or amphoteric.

[0325] Non-limiting examples of surfactants are disclosed in McCutcheon's 2016 Detergents and Emulsifiers, and McCutcheon's 2016 Functional Materials, both North American and International Edition, MC Publishing Co, 2016. Further useful examples are disclosed in earlier editions of the same publications that are known to those skilled in the art.

[0326] Nonionic surfactants refer to surfactants that contain functional groups that are neither positively nor negatively charged (i.e., ionic). In contrast to anionic and cationic surfactants, nonionic surfactants do not ionize in solution.

[0327] Preferred Methods and Uses: Preferred herein is a method for producing an amylase comprising A and B domains and a C domain, wherein the amino acid sequences of the A and B domains are at least 75% identical to the amino acid sequence of SEQ ID NO: 42 and the amino acid sequence of the C domain is at least 75% identical to the amino acid sequence of SEQ ID NO: 44, the method comprising providing a nucleic acid sequence encoding the amylase; transforming the nucleic acid sequence into an expression host; culturing the expression host to produce the amylase; and, if necessary, purifying the amylase.

[0328] Preferred herein are SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37 and at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at 7%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical, the method comprising providing a nucleic acid sequence comprising SEQ ID NO:55, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38; transforming the nucleic acid sequence into an expression host, culturing the expression host to produce the amylase, and, if necessary, purifying the amylase.

[0329] Preferred herein is a method for making an amylase comprising an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of SEQ ID NO: 54, the method comprising: providing a nucleic acid sequence comprising SEQ ID NO: 55; transforming the nucleic acid sequence into an expression host; culturing the expression host to produce the amylase; and, if necessary, purifying the amylase.

[0330] A method for producing an amylase, wherein the expression host is selected from the group consisting of a bacterial expression system, a yeast expression system, a fungal expression system, and a synthetic expression system.

[0331] A method for producing amylase, wherein the bacterial expression system is selected from Escherichia coli, Bacillus, Pseudomonas, and Streptomyces, the yeast expression system is selected from Candida, Pichia, Saccharomyces, and Schizosaccharomyces, or the fungal expression system is selected from Penicillium, Aspergillus, Fusarium, Mycelioptora, Thermoteromyces, Rhizomucor, Rhizopus, Thermomyces, and Trichoderma, preferably Bacillus.

[0332] Preferred herein are those having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, or at least the full-length amino acid sequence of SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:35, or SEQ ID NO:37. 100% identical to an amylase comprising an amino acid sequence at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to an amylase comprising an amylase having ...

[0333] Preferred herein is a method for producing an amylase comprising an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of SEQ ID NO:54, wherein the expression host is preferably a Bacillus host cell selected from Bacillus pumilus, Bacillus subtilis, and Bacillus licheniformis, and is most preferably Bacillus licheniformis.

[0334] Preferred herein is a method of use of a C domain of a first amylase, said C domain having an amino acid sequence at least 75% identical to the amino acid sequence of SEQ ID NO: 44, for improving one or more properties selected from the group consisting of stability, pH profile, expression, activity, thermostability, specific activity, substrate specificity, pH-dependent activity, pH-dependent stability, oxidative stability, Ca2+ dependence, performance in laundry, processing starch, cleaning textiles, cleaning hard surfaces, cleaning dishes, making ethanol, processing pulp or paper, and feeding animals, of a second alpha-amylase having A and B domains having at least 75% identity to the amino acid sequence of SEQ ID NO: 42, wherein said use comprises replacing the C domain of the second alpha-amylase with the C domain of the first alpha-amylase.

[0335] Preferred herein is a method of preparing a dough or a baked product prepared from the dough, comprising adding an amylase comprising an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37 to a dough and baking it.

[0336] Preferred herein is a method comprising adding an amylase comprising an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of SEQ ID NO: 54 to dough and baking it.

[0337] Preferred herein are those having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, or at least 91% similar to the full-length amino acid sequence of SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:35, or SEQ ID NO:37. A method of using an amylase comprising an amino acid sequence that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the amylase for processing starch, cleaning or washing textiles, hard surfaces, or tableware, making ethanol, processing pulp or paper, or feeding animals, preferably for cleaning or washing textiles, hard surfaces, or tableware.

[0338] Preferred herein are methods of using an amylase comprising an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of SEQ ID NO: 54 for processing starch, cleaning or washing textiles, hard surfaces, or tableware, making ethanol, processing pulp or paper, or feeding animals.

[0339] Preferred herein is a method of using an amylase comprising an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, for cleaning or washing textiles, hard surfaces, or tableware.

[0340] Preferred herein is a method of using an amylase comprising an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of SEQ ID NO:54, for cleaning or washing textiles, hard surfaces, or tableware.

[0341] Preferred herein is a method of using an amylase comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of SEQ ID NO: 54 for cleaning or washing textiles, hard surfaces, or tableware.

[0342] Preferred herein is a method of using an amylase comprising an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, for cleaning or washing textiles.

[0343] Preferred herein is a method of using an amylase comprising an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of SEQ ID NO:54 for cleaning or washing textiles.

[0344] Preferred herein is a method of using an amylase comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full-length amino acid sequence of SEQ ID NO: 54 for cleaning or washing textiles. (Example) [Example]

[0345] Generation of amylase expression constructs Genes encoding amino acid sequences were codon-optimized to match the natural codon abundance of B. subtilis and physically synthesized by an external commercial DNA supplier. Upon receipt, these genes were cloned by standard restriction-ligation protocols into a Gram-positive expression vector featuring a promoter known to provide expression in Bacillaceae organisms, a secretory signal peptide, and a region encoding a ribosome binding site (e.g., the promoter driving expression of the B. subtilis amyE gene, the signal peptide of the B. subtilis YdjM enzyme, and a consensus Shine-Dalgarno sequence). This vector also contained an antibiotic-based selection marker and an origin of replication.

[0346] After the reaction, the plasmid assembly mixture was transformed into Bacillus subtilis PY79 KO-7S (Bacillus Genetic Stock Center, BGSCID:1S145; Zeigler DR) derivative Bs#056, which contained the DNA methyltransferase gene described for Bacillus subtilis Bs#053 in WO2019016051, according to the method of Spizizen (Anagnostopoulos, C. and Spizizen, J. (1961). J. Bacteriol. 81, 741-746). Successful transformants were selected by plating on LB agar plates supplemented with 20 μg / ml kanamycin sulfate and incubating overnight at 37°C. After overnight selection, individual colonies were obtained and grown overnight at 37°C with shaking at 250 rpm in rich medium (e.g., LB broth) containing 20 μg / ml kanamycin sulfate. The following morning, cells were pelleted by centrifugation, and plasmid DNA was isolated by alkaline lysis using a Macherey-Nagel NucleoSpin kit. The isolated DNA could then be transformed into electrocompetent B. licheniformis cells.

[0347] Preparation of electrocompetent Bacillus licheniformis cells and DNA transformation were performed essentially as described by Brigidi et al. (Brigidi, P., Mateuzzi, D. (1991). Biotechnol. Techniques 5, 5) with the following modifications: for DNA transformation, cells were plated on selective LB agar plates (20 μl / ml kanamycin sulfate) and incubated overnight at 37°C, then recovered in 1 ml of LBSPG buffer and incubated at 37°C for 60 min (Vehmaanpera J., 1989, FEMS Microbio. Lett., 61: 165-170). [Example]

[0348] Amylase expression and protein quantification A single colony of the expression strain was inoculated into 60 μL of rich medium (e.g., LB broth) supplemented with 20 μg / mL kanamycin sulfate in a 96-well plate (GE Life Sciences part 28403943). The culture was grown at 37°C for 16 hours, after which 15 μL of the culture was used to inoculate 600 μL of defined glucose-mineral medium and 20 μg / mL kanamycin sulfate in a 96-well plate. The culture was grown at 37°C for 48 hours, after which the supernatant was harvested by sedimenting the cells using centrifugation and removing the residual culture liquid. The expression level (concentration in mg / mL) of the variant polypeptide hybrid with alpha-amylase activity was identified by LabChip (LC) analysis, and the corresponding expression plasmid was sequenced for verification. [Example]

[0349] Red starch and residual activity assay The amount of starch hydrolysis for the alpha-amylase-containing variant polypeptides was measured using the red starch method described by Megazyme, "Assay of Alpha-Amylase using Red Starch," with the following modifications. Ten microliters of 1.33% red starch prepared in 50 mM HEPES, pH 8.0 buffer, was reacted with 10 μL of enzyme diluted in 50 mM HEPES, pH 8.0 buffer at 25°C. The reaction was terminated after 10 minutes by the addition of 50 μL of 200-proof ethanol. After vigorous mixing, the reaction was allowed to equilibrate at room temperature for 10 minutes and then centrifuged at 1,200 x g for 10 minutes. A 40 μL aliquot of the reaction mixture was transferred, and the absorbance of the solution was read at 510 nm in a BioTek plate reader. Residual activity was calculated by comparing the activity of each enzyme measured using the red starch assay before and after heat challenge. Heat challenge was performed on enzyme diluted in 50 mM HEPES, pH 8.0 buffer by first heating the sample to 80°C for 15 min, cooling to 4°C for 10 min, holding at 24°C for 5 min, and then testing using the red starch assay at 25°C.

[0350] Each enzyme / temperature pair was performed in technical duplicates (triplicates at 25°C, or n=5), and two independent experiments were performed on different days. For the residual activity plots, the reference to "100% activity" was the highest activity measurement across all temperatures. Results for an 80°C heat challenge are shown in Figure 1. It is clearly visible that replacing the C domain of SEQ ID NO:41 with the C domain of SEQ ID NO:40, resulting in an amylase with SEQ ID NO:54 (with additional mutations at positions 430 and 454), greatly increases thermostability. Use of a closely related C domain (such as one closely related to the C domain of SEQ ID NO:40) results in a hybrid that is more stable than SEQ ID NO:41, which is highlighted by the results obtained with SEQ ID NO:9. [Example]

[0351] Enzyme performance in laundry applications (stain removal) The hybrid amylases were measured for their washing performance on cotton soiled with starch (stain types EMPA161 and CS28) purchased from Swissatest Testmaterilien AG and CFT (Center for Testmaterials BV). The amylase according to SEQ ID NO: 39 is used as a reference for other amylases that are hybrid molecules. SEQ ID NO: 54 is, for example, a hybrid of SEQ ID NO: 42 and SEQ ID NO: 44, containing a double deletion at positions 183 and 184 and additional mutations at positions 430 and 454. The other selected amylases (SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 17 and SEQ ID NO: 27) are also hybrids with significant changes in the C domain relative to SEQ ID NO: 54.

[0352] Amylase was dosed at 0.05, 0.1, 0.2, or 0.4 ppm in 2.5 mM hard water (14°C dH, Deutsche Harte) yielding a pH of 8.0–8.5 + 3.3 g / L of detergent ES1 (Maranil DBS / LC LAS 5.5% w / w, Edenor palm fatty acid C12-C18 palm fatty acid 2.4% w / w, Lutensol AO7 AEO 5.4% w / w, Texapon N70 FAEO 5.4% w / w, 1,2 propylene glycol 6.0% w / w, ethanol 2.0% w / w, KOH 2.2% w / w) + 3% sodium citrate. Washing was performed at 40°C for 30 minutes using a Launder-O-meter (SDS Atlas). After removing the wash solution, the fabrics were rinsed three times with water. The samples were dried overnight at room temperature. Cleaning performance was measured using digital image analysis of the cleaned stains using a spectrophotometer (ELREPHPO, Datacolor) and the average L * AB intensities are presented as the average of four data points (0.05, 0.1, 0.2, or 0.4 ppm) after normalization to the detergent base without amylase.

[0353] The cleaning performance on the test stains CS28 and EMPA161 is shown as ddE to indicate the amylase specific effect (dE amy - dE detergent base).

[0354] [Table 1] The results provided in the table above show that the hybrid amylase enzyme has improved performance (stain removal) when compared to the parent enzyme SEQ ID NO:39. [Example]

[0355] Heat challenge stability test The hybrid enzymes of the invention can be stabilized by further changes in the amino acid sequence, which was tested by storing the variants at elevated temperatures and measuring the residual activity after a certain period of time.

[0356] Therefore, the enzyme was diluted to approximately 20 μg / mL in 0.1 M Hepes pH 8.0 and loaded on a PCR machine at 92°C for 10 min, followed by chilling at 4°C. Unloaded controls were kept at 4°C. To measure residual activity, the loaded enzyme and controls were diluted 10-fold in 1% red starch prepared in 0.1 M Hepes pH 8.0 and 0.05% Tween 20 according to the manufacturer's instructions (Megazyme) and incubated at room temperature for 10 min. The reaction was quenched with two volumes of ice-cold ethanol, incubated for 10 min, and then spun down. The supernatant was transferred to a new plate and the absorbance was read at 510 nm. The improvement factor (IF) was calculated as the ratio between a specific variant and the parent SEQ ID NO: 54.

[0357] The numbering according to SEQ ID NO:39 is used to introduce mutations into SEQ ID NO:54.

[0358] [Table 2] [Example]

[0359] Preservation of hybrid variants in liquid laundry detergent The stabilization of the hybrid enzymes of the present invention by further changes in the amino acid sequence was further tested by storing the variants in liquid detergent and measuring the residual activity after a certain time.

[0360] Storage in liquid laundry detergent was performed by incubating amylase in ES1-C detergent solution (Maranil DBS / LC LAS 5.5% w / w, Edenor C12-C18 coconut fatty acids 2.4% w / w, Lutensol AO7 AEO 5.5% w / w, Texapon N70 FAEO 5.5% w / w, 1,2 propylene glycol 6.0% w / w, ethanol 2.0% w / w, KOH 2.2% w / w, and sodium citrate 3%, pH 8) containing 1% Bacillus lentus alkaline protease (BLAP) with the R101E mutation and storing at 37°C for 7 days. Samples were taken on days 0 and 7 and diluted in 50 mM MOPS, pH 7, and then analyzed using the Infinity Amylase Reagent at room temperature. Activity is calculated as a 5-point MaxV gradient at 405 nm over 5 minutes. The improvement factor (IF) relative to the reference hybrid amylase, SEQ ID NO: 54, is calculated by taking the percent remaining activity after storage of each variant and dividing it by the percent remaining activity after storage of SEQ ID NO: 54. Mutations are introduced into SEQ ID NO: 54 using the numbering according to SEQ ID NO: 39.

[0361] [Table 3] [Example]

[0362] Microscale cleaning studies on hybrid variants The hybrids of the present invention can be made more efficient in cleaning performance by small changes in the amino acid sequence, which was tested by microscale washing tests.

[0363] Using the variant, an old corn starch stain (CS-126) was washed in 3.3 g / L of ES1 C (5.5% w / w Lutensit A-LBS LAS, 2.4% w / w Edenor C12-C18 palm fatty acids, 5.5% w / w Lutensol AO7 AEO, 5.5% w / w Texapon N70 FAEO, 6.0% w / w 1,2 propylene glycol, 2.0% w / w ethanol, 2.2% w / w KOH, and 3% sodium citrate, pH 8) containing 2.78 mM hard water (15.5° dH, Deutsche Harte) with 0.02, 0.05, and 0.1 ppm amylase added to the wash solution for 60 min at room temperature, then rinsed under running water for 5 min, dried, and measured for reflected light intensity as the average of RGB values. A ratio of the performance of a particular variant to that of SEQ ID NO: 54 was generated, with numbers greater than 1 indicating better performance.

[0364] The numbering according to SEQ ID NO:39 is used to introduce mutations into SEQ ID NO:54.

[0365] [Table 4]

Claims

1. An isolated, synthetic, or recombinant polypeptide having alpha-amylase activity and stability under thermal stress, comprising A and B domains and a C domain, in that order, wherein the amino acid sequences of the A and B domains are at least 95% identical to the amino acid sequence of SEQ ID NO: 42 and the amino acid sequence of the C domain is at least 95% identical to the amino acid sequence of SEQ ID NO:

44.

2. 2. The polypeptide of claim 1, having alpha-amylase activity, comprising A and B domains and a C domain, wherein the amino acid sequences of the A and B domains are at least 95% identical to the amino acid sequence of SEQ ID NO: 42, and the amino acid sequence of the C domain is at least 95% identical to the amino acid sequence of SEQ ID NO:

44.

3. 3. The polypeptide of claim 1 or 2, which comprises substitutions, deletions, and / or insertions at one or more positions.

4. 4. The polypeptide of any one of claims 1 to 3, comprising the sequence TQXDYLDHPDVIGWTREGDXXHXXSGLAXLMSDGPXGXKWMXVGKNNAGEXWXDITGNQTNTVTINXDGXGQFXVXXGSXSIYXQX, where X can be any amino acid.

5. 5. The polypeptide of any one of claims 1 to 4, having one or more amino acid residues selected from the group consisting of: 402R,H; 419S,G,D; 420V,I; 422,A,V; 423N,D,G,K; 428T,A; 435G,R,E; 437S,A; 441N,E,D; 444K,E; 450V,I; 452Y,H,R; 466K,R; 469W,S; 473H,Q,R; 475S,N; 476G,E; 479V,A; 483V,I; and 485R,Q,K according to the numbering of SEQ ID NO:

39.

6. 6. The polypeptide of any one of claims 1 to 5, comprising substitutions at one or more positions selected from the group consisting of I430M and M454I according to the numbering of SEQ ID NO:

39.

7. 7. The polypeptide of any one of claims 1 to 6, comprising an amino acid residue at one or more amino acid positions selected from the group consisting of 401, 403, 405, 411, 413, 415, 424, 426, 428, 432, 455, 477, 479 and 481 (according to the numbering of SEQ ID NO: 39) present in SEQ ID NO:

39.

8. 8. The polypeptide of any one of claims 1 to 7, comprising an amino acid residue at one or more amino acid positions selected from the group consisting of 309, 313, 347, 348, 350, 351, 354, 355, 358, 359, 388, 389, 392 and 396 (according to the numbering of SEQ ID NO: 39) present in SEQ ID NO:

40.

9. 9. The polypeptide of any one of claims 1 to 8, comprising a deletion of amino acids corresponding to positions 181 and 182, 182 and 183, or 183 and 184 according to the numbering of SEQ ID NO: 39 (according to the numbering of SEQ ID NO: 39).

10. 10. The polypeptide of any one of claims 1 to 9, comprising one or more substitutions selected from the group consisting of M9L, E130V, N195F, I206L, S244Q, M202L, K179L, R181E, G186E / N / Q / S and E190P according to the numbering of SEQ ID NO:

39.

11. below: (a) an amino acid sequence having at least 95% sequence identity to SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37; (b) an amino acid sequence encoded by a polynucleotide having at least 95% sequence identity to SEQ ID NO:55, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38; (c) under high stringency conditions; (i) a sequence encoding SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37; or (ii) a polynucleotide set forth in SEQ ID NO:55, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38 an amino acid sequence encoded by a polynucleotide that hybridizes to the complement of or (d) a fragment of (a), (b), or (c) having amylase activity. The polypeptide according to any one of claims 1 to 10, comprising:

12. 12. The polypeptide of any one of claims 1 to 11, wherein the amylase has increased thermostability compared to the amylase shown in SEQ ID NO: 39 or SEQ ID NO:

40.

13. An isolated, synthetic, or recombinant nucleic acid comprising: (a) a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:55, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38, wherein the nucleic acid encodes a polypeptide having amylase activity; (b) a nucleic acid sequence encoding a polypeptide having at least 95% sequence identity to SEQ ID NO:54, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, or SEQ ID NO:37, wherein the polypeptide has amylase activity; or (e) a nucleic acid sequence that is completely complementary to any of (a) to (b) An isolated, synthetic, or recombinant nucleic acid comprising:

14. A nucleic acid construct comprising the nucleic acid of claim 13.

15. 15. An expression vector comprising the nucleic acid of claim 13 or the nucleic acid construct of claim 14.

16. A host cell comprising the nucleic acid of claim 13, the nucleic acid construct of claim 14, or the expression vector of claim 15.

17. 13. A composition comprising an isolated, synthetic or recombinant polypeptide having alpha-amylase activity according to any one of claims 1 to 12 and having stability under thermal stress.

18. 18. The composition of claim 17, further comprising at least one second enzyme selected from the group consisting of a second amylase, a lipase, a protease, a cellulase, a laccase, a mannase, a pectinase, a xylanase, and a nuclease.

19. 13. A method for producing an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity and stability under heat stress according to any one of claims 1 to 12, comprising providing a nucleic acid encoding the polypeptide, transforming the nucleic acid sequence into an expression host, culturing the expression host to produce the polypeptide, and optionally purifying the polypeptide.

20. 13. A method for preparing a starch dough or a baked product prepared from a starch dough, the method comprising adding an isolated, synthetic or recombinant polypeptide having alpha-amylase activity and stability under heat stress according to any one of claims 1 to 12 to a dough and baking it.

21. 13. Use of an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity and stability under heat stress according to any one of claims 1 to 12 for cleaning or washing textiles, hard surfaces, or tableware, for processing starch, for making ethanol, for processing pulp or paper, or for feeding animals.

22. 22. A method of using an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity and stability under thermal stress, as described in claim 21, for cleaning or washing textiles, hard surfaces, or tableware.

23. A method for producing an isolated, synthetic, or recombinant polypeptide having alpha-amylase activity and stability under thermal stress, comprising the step of producing a hybrid from at least two different amylases, wherein the hybrid comprises A and B domains and a C domain, and wherein the amino acid sequences of the A and B domains are at least 95% identical to the amino acid sequence of SEQ ID NO: 42 and the amino acid sequence of the C domain is at least 95% identical to the amino acid sequence of SEQ ID NO:

44.

24. 1. A method of using the C domain of a first amylase, comprising replacing the C domain of a second alpha-amylase with the C domain of a first alpha-amylase, the second alpha-amylase having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 44, having alpha-amylase activity and stability under heat stress, wherein the C domain has A and B domains at least 95% identical to the amino acid sequence of SEQ ID NO: 42 for improving one or more properties selected from the group consisting of stability, pH profile, expression, activity, thermostability, specific activity, substrate specificity, pH-dependent activity, pH-dependent stability, oxidative stability, Ca2+ dependence, performance in laundry, processing starch, cleaning textiles, cleaning hard surfaces, cleaning dishware, making ethanol, processing pulp or paper, and feeding animals.

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