Alpha-amylase variants with improved activity

Abstract

The present disclosure concerns an alpha-amylase variant having at least 70% identity with the amino acid sequence of SEQ ID NO: 2, and comprising an amino acid substitution at any one of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71, 79, 80, 84, 86, 91, 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141, 143, 148, 151, 152, 155, 157, 158, 168, 174, 178, 181, 5 183, 185, 204, 207, 208, 209, 211, 219, 230, 233, 242, 246, 252, 261, 268, 273, 290, 301, 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442 as well as fusion polypeptides comprising same. The alpha-amylase variants or the fusion polypeptides can be used to hydrolyze starch.

Description

[0001] ALPHA-AMYLASE VARIANTS WITH IMPROVED ACTIVITY

[0002] CROSS-REFERENCE TO RELATED APPLICATION(S) AND DOCUMENT(S)

[0003] This application claims priority from U.S. provisional patent application 63 / 740,120 filed on December 30, 2024 and herewith incorporated in its entirety. The present application comprises a sequence listing in HTML format also incorporated herein in its entirety.

[0004] TECHNOLOGICAL FIELD

[0005] The present disclosure concerns alpha-amylase variants exhibiting increased extracellular activity when expressed in yeast. The recombinant yeast host cells of the present disclosure expressing the alpha-amylase variants of the present exhibit improved activity on raw starch.

[0006] BACKGROUND

[0007] Amylases are among the oldest and most intensively studied commercial enzymes and are estimated to make up 25% of the global enzyme market. Alpha-amylase (EC 3.2.1.1) is an endo-acting glycoside hydrolase active on starch, glycogen, and related oligosaccharides, and is used in a variety of applications including food, fuel, beverage, detergents, textiles, and pulp / paper. In such a diverse field, it is in the best interest of enzyme suppliers to continuously improve and expand their alpha-amylase products to provide the highest value in applications. Conventional approaches for improving enzyme activity include enzyme discovery, directed evolution, and rational design enzyme engineering strategies. Fungal alpha-amylase variants with improved secreted activity would be desirable.

[0008] SUMMARY

[0009] The present disclosure concerns alpha-amylase variants with increased activity, particularly extracellular activity, and recombinant yeast host cells expressing the alpha-amylase variants such that starch hydrolysis is improved in the presence of the recombinant yeast host cells compared to a reference strain (lacking the alpha-amylase variant).

[0010] According to a first aspect, the present disclosure provides an alpha-amylase variant (i) having at least 70% identity with the amino acid sequence of SEQ ID NO: 2, and (ii) comprising an amino acid substitution at any one of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In some embodiments, the alpha-amylase variant comprises an amino acid substitution at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348,357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In additional embodiments, the alpha-amylase variant comprises an amino acid substitution at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variant comprises an amino acid substitution at any one of positions 71 , 80, 84, 91 , 93, 96, 117, 119, 125, 133, 141 , 158, 174, 209, 211 , 252, 268, 336, 348, 357, 380, 383, 393, and / or 402. In some embodiments, the alpha-amylase variant comprises an amino acid substitution at any one of positions 43, 71 , 79, 80, 93, 103, 106, 117, 124, 143, 178,181 , 204, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 343, 347, 369, 372, 373, 385, 386, 393, and / or 396. In some embodiments, the alpha-amylase variant comprises an amino acid substitution at any one of positions 133, 207, and / or 423. In some embodiments, the alpha-amylase variant comprises at least one of the following amino acid substitutions: K43Q / A / R / E, R71G / N / D / A, S79Q / K / T / R / N, N80D / S / H, T84I / V / L, S91A / E / P, E93D / S / Q, G96S / D / N / A, G103E / S / A / N / Q, I106M / F / V / L, V117M / A / T, N119P / S / A / Q, E124D / S / T, M125N / T / K / S, G133A / D / E / Q / S, G141D / E / T / Q / K, N143D / S / Q / K, D158G / S / E, K174Q / N / Y / R, K178S / R / Q / A, M181V / I / L, D204G / E / P / K, W207R / S / Q / K / A / E, S209A / T / G, S211V / A / I / F, S219D / N / H / E / T, G233D / S / P / A, F242Y / W / K / D, N252G / S / D / Q, V268F / Y / M / L / I, T273A / V / L / I / S, T301A / S / V / G, A319G / S / E, Q327A / E / G / N, R336G / N / S / K, M343L / V / I, F347I / L / V / T, Q348A / S / K / T, H357A / V / N, F369Y / H / L / M, S372D / N / E / G / A, D373T / N / G / S, Q380A / E / G / D, A383D / K / R / N / S, M385I / V / L / F / A, A386V / I / T / S, K393S / Q / R / A, Q396S / T / N / G, F402N / S / D / G / T, and G423S / N / K / A / E / F. In some embodiments, the alphaamylase variant comprises at least one of the following amino acid substitutions: K43Q, R71G, S79Q, N80D, T84I, S91A, E93D, G96S, G103E, I106M, V117M, N119P, E124D, M125N, G132S, G133A, G141D, N143D, D158G, K174Q, K178S, M181V, D204G, W207R, S209A, S211V, S219D, G233D, F242Y, N252G, V268F, T273A, T301A, A319G, Q327A, R336G, M343L, F347I, Q348A, H357A, F369Y, S372D, D373T, Q380A, A383D, M385I, A386V, K393S, Q396S, F402N, and G423S. In some embodiments, the alpha-amylase variant further comprises at least one amino acid deletion at position 355.

[0011] According to a second aspect, the present disclosure provides a fusion polypeptide having alpha-amylase activity, the fusion polypeptide comprising (i) the alpha-amylase variant as described herein, and (ii) a carbohydrate-binding module. In some embodiments, the carbohydrate-binding module has at least 70% identity with the amino acid sequence of SEQ ID NO: 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 164 or 168. In some embodiments, the fusion polypeptide has at least 70% identity with the amino acid sequence of SEQ ID NO: 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 166 or 170. In some embodiments, the fusion polypeptide has the following formula:

[0012] NH2-AA-L-(CBM)n-COOH (la), orNH2-AA-(L-CBM)n-COOH (lb),

[0013] wherein: NH2is the amino terminus of the fusion polypeptide;

[0014] AA is the alpha-amylase moiety;

[0015] L is an optional amino acid linker;

[0016] CBM is the carbohydrate binding moiety;

[0017] n is an integral number between 1 and 5, and

[0018] COOH is the carboxy terminus of the fusion polypeptide.

[0019] In some embodiments, the fusion polypeptide has the following formula:

[0020] NH2-(CBM)n-L-AA-COOH (Ila), or

[0021] NH2-(CBM-L)n-AA-COOH (lib),

[0022] wherein: NH2is the amino terminus of the fusion polypeptide;

[0023] CBM is the carbohydrate binding moiety;

[0024] n is an integral number between 1 and 5;

[0025] L is an optional amino acid linker;

[0026] AA is the alpha-amylase moiety; and

[0027] COOH is the carboxy terminus of the fusion polypeptide.

[0028] In additional embodiments, the fusion polypeptide has the following formula:

[0029] NH2-(CBM)n-L1-AA-L2-(CBM)m-COOH (Illa),

[0030] NH2-(CBM-L1)n-AA-L2-(CBM)m-COOH (lllb),

[0031] NH2-(CBM)n-L1-AA-(L2-CBM)m-COOH (lllc),

[0032] NH2-(CBM-L1)n-AA-(L2-CBM)m-COOH (Hid),

[0033] wherein: NH2is the amino terminus of the fusion polypeptide;

[0034] CBM is the carbohydrate binding moiety;

[0035] n and m are integral numbers independently selected between 1 and 5;

[0036] L1 and L2 are optional amino acid linkers;

[0037] AA is the alpha-amylase moiety; and

[0038] COOH is the carboxy terminus of the fusion polypeptide.

[0039] According to a third aspect, the present disclosure provides heterologous nucleic acid molecule comprising an open reading frame encoding the alpha-amylase variant described herein orthe fusion polypeptide described herein. In additional embodiments, the heterologous nucleic acid molecule comprises at least one promoter operably associated with the open reading frame. In some embodiments, the open reading frame encoding the alpha-amylase variant further encodes a signal sequence, orthe fusion polypeptide further comprises a signal sequence.According to a fourth aspect, the present disclosure provides a vector comprising the heterologous nucleic acid molecule described herein.

[0040] According to a fifth aspect, the present disclosure provides an expression cassette comprising the heterologous nucleic acid molecule described herein.

[0041] According to a sixth aspect, the present disclosure provides a recombinant microbial host cell expressing the alpha-amylase variant described herein or the fusion polypeptide described herein. In some embodiments, the recombinant microbial host cell comprises the heterologous nucleic acid molecule described herein, the vector described herein orthe expression cassette described herein. In some embodiments, the recombinant microbial host cell is a yeast. In some embodiments, the yeast is from Saccharomyces sp. In some embodiments, the Saccharomyces sp is from Saccharomyces cerevisiae. In other embodiments, the yeast is from Komagataella sp. In another embodiment, the Komagataella sp is from Komagataella phaffii. According to a seventh aspect, the present disclosure provides a method for making the alphaamylase variant described herein or the fusion polypeptide described herein, wherein the method comprises (i) expressing the heterologous nucleic acid molecule described herein, the vector described herein, or the expression cassette described herein in the recombinant microbial host cell described herein. In some embodiments, the alpha-amylase variant is an intracellular polypeptide or a secreted polypeptide. In some embodiments, the secreted polypeptide is in a free form or is associated to the surface of the recombinant microbial host cell. In some embodiments, the polypeptide associated to the surface of the recombinant yeast host cell is a tethered polypeptide. In additional embodiments, the method comprises, after step (i), (ii) substantially separating the alpha-amylase variant or the fusion polypeptide from the recombinant microbial host cell. In additional embodiments, the method comprises, after step (i) or (ii), (iii) drying the alpha-amylase variant orthe fusion polypeptide.

[0042] According to an eighth aspect, the present disclosure provides a composition comprising the alpha-amylase variant described herein or the fusion polypeptide described herein, and a carrier. In some embodiments, the alpha-amylase variant described herein is obtainable or obtained by the method described herein. In additional embodiments, the composition further comprises another enzyme.

[0043] In the context of the present disclosure, “a” refers to one or more such that “a” refers to a minimum of one.

[0044] DETAILED DESCRIPTION OF THE DRAWINGS

[0045] Having thus generally described the nature of the invention, reference will now be made to the accompanying drawings, showing by way of illustration, a preferred embodiment thereof, and in which:Figure 1 provides the metabolites measured at the end of fermentations conducted in a corn mash in the presence of a commercial alpha-amylase (AtAA), an alpha-amylase variant (CfAA_30) or an alpha-amylase variant fusion protein (CfAA_30 / HgCBM). Results are shown in function of alpha-amylase enzyme used for the yield in ethanol (in g / L, bars), the generation of glycerol (in g / L, •) and the remaining glucose levels (in g / L, ♦).

[0046] Figure 2 provides the metabolites measured at the end of fermentations conducted in a corn mash in the presence of a commercial glucoamylase (ReGA) alone or in combination with a commercial alpha-amylase (AtAA), an alpha-amylase variant (CfAA_30) or an alpha-amylase variant fusion protein (CfAA_30 / HgCBM). Results are shown in function of enzyme(s) used for the yield in ethanol (in g / L, bars), the generation of glycerol (in g / L, •) and the remaining glucose levels (in g / L, ♦).

[0047] DETAILLED DESCRIPTION

[0048] The present disclosure provides variant polypeptides having alpha-amylase activity which belong to Enzyme Commission # 3.2.1.1. The variant polypeptides are derived from a naturally occurring alpha-amylase from Cordyceps farinosa which corresponds to the GenBank accession number 6SAU_A or to Uniprot accession number A0A5S8WFA8 (which corresponds to the amino acid sequence of SEQ ID NO: 2). The alpha-amylase from Cordyceps farinosa having the GenBank accession number 6SAU_A or the Uniprot accession number A0A5S8WFA8 (which corresponds to the amino acid sequence of SEQ ID NO: 2) can be referred to as the parental alpha-amylase or the wildtype (WT) alpha-amylase. In the context of the present disclosure, polypeptides are considered being “derived from a naturally occurring alpha-amylase from Cordyceps farinosa" when they can be obtained from modifying (adding, deleting, and / or substituting) at least one amino acid residue which is present in the amino acid sequence of SEQ ID NO: 2. In the context of the present disclosure, polypeptides derived from the naturally occurring alpha-amylase from C. farinosa can be referred to as alpha-amylase variants.

[0049] In some embodiments, the alpha-amylase variants exhibit at least 70% identity to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants exhibit at least 70% identity or more to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants exhibit at least 75% identity or more to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alphaamylase variants exhibit at least 80% identity or more to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants exhibit at least 81% identity or more to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants exhibit at least 82% identity or more to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants exhibit at least83% identity or more to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants exhibit at least 84% identity or more to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants exhibit at least 85% identity or more to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants exhibit at least 86% identity or more to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alphaamylase variants exhibit at least 87% identity or more to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants exhibit at least 88% identity or more to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants exhibit at least 89% identity or more to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants exhibit at least 90% identity or more to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants exhibit at least 91% identity or more to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants exhibit at least 92% identity or more to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants exhibit at least 93% identity or more to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alphaamylase variants exhibit at least 94% identity or more to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants exhibit at least 95% identity or more to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants exhibit at least 96% identity or more to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants exhibit at least 97% identity or more to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants exhibit at least 98% identity or more to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants exhibit at least 99% identity or more to the amino acid sequence of SEQ ID NO: 2. The alphaamylase variants of the present disclosure have less than 100% identity with respect to the amino acid sequence of SEQ ID NO: 2 because they include a modification with respect to the amino acid sequence of SEQ ID NO: 2. The at least one amino acid modification can refer to an amino acid residue that has been added, substituted, or deleted. The term “% identity”, as known in the art, is a relationship between two or more polypeptide sequences, as determined by comparing the sequences. The level of identity can be determined conventionally using known computer programs. Identity can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology(von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, NY (1991). Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.) or Geneious Prime. Multiple alignments of the sequences disclosed herein were performed using the Clustal Omega alignment method, which employs a Hidden Markov Model-based algorithm, using default parameters (including a cluster size = 100).

[0050] As used in the context of the present disclosure, a “fragment” is a type of variant which includes at least one deleted amino acid residues when compared to the amino acid sequence of SEQ ID NO: 2. In some embodiments, fragments can refer to the mature form of a secreted alphaamylase fragment (from which the signal sequence has been cleaved). In additional embodiments, the fragment can be, for example, a truncation of one or more, two or more, three or more, four, five or more, six or more, seven or more, eight or more, nine or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more at the amino terminus of the amino acid sequence of SEQ ID NO: 2. In additional embodiments, the fragment can be, for example, a truncation of one or more, two or more, three or more, four, five or more, six or more, seven or more, eight or more, nine or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more at the carboxyl terminus of the amino acid sequence of SEQ ID NO: 2. Alternatively, or in combination, the fragment can be generated from removing one or more internal amino acid residues to the amino acid sequence of SEQ ID NO: 2. In an embodiment, the polypeptide fragment can have at least 100, 150, 200, 250, 300, 350, 400, 450, or more consecutive amino acid residues of the amino acid sequence of SEQ ID NO: 2. The present disclosure also provides variants of alphaamylase variants including at least one amino acid substitution at any one of positions 8, 43, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 148, 155, 158, 174, 178, 181 , 204, 207, 208, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402, 423, and / or 442 (with respect to the amino acid sequence of SEQ ID NO: 2). Such variants include the at least one amino acid substitution at any one of positions 8, 43, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 148, 155, 158, 174, 178, 181 , 204, 207, 208, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402, 423, and / or 442 (with respect to the amino acid sequence of SEQ ID NO: 2) and, in some embodiments, additional modifications.Variants (including fragments) exhibit more biological activity when compared to the biological activity of the parental alpha-amylase. In some embodiments, the variants can also have at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000%, 3000%, 4000%, 5000%, 6000% or more biological activity when compared to the biological activity of the parental alpha-amylase. The alpha-amylase variants described herein may be (i) one in which one or more of the amino acid residues are substituted with a conservative or non-conservative (preferably a conservative) amino acid residue and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide for purification of the polypeptide. Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics, e.g., substitutions of an amino acid by another one belonging to same category determined by its side chain: within amino acids presenting hydrophobic side chain (alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan); within amino acids presenting positively charged side chain (arginine, histidine, or lysine); negatively charged side chain (aspartic acid or glutamic acid) and polar-uncharged side chain (serine, threonine, asparagine, or glutamine); or substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Other conservative amino acid substitutions are known in the art and are included herein. Non-conservative substitutions, such as replacing a basic amino acid with a hydrophobic one, are also well-known in the art.

[0051] An alpha-amylase variant can also be a conservative variant or an allelic variant. As used herein, a conservative variant refers to alterations in the amino acid sequence that do not adversely affect the biological function(s) of the polypeptide. A substitution, insertion, or deletion is said to adversely affect the polypeptide when the altered sequence prevents or disrupts a biological function associated with the polypeptide. For example, the overall charge, structure, or hydrophobic-hydrophilic properties of the polypeptide can be altered without adversely affecting a biological activity. Accordingly, the amino acid sequence can be altered, for example to render the polypeptide more hydrophobic or hydrophilic, without adversely affecting the biological activities of the polypeptide.

[0052] In some embodiments, the alpha-amylase variants of the present disclosure exhibit more activity when they are secreted from a recombinant yeast host cell than the parental alphaamylase. The parental alpha-amylase is of fungal origin. As shown in the Examples below, when the parental alpha-amylase was expressed in a recombinant yeast, its activity was verylow. Surprisingly, the alpha-amylase variants exhibited a higher activity than the parental alphaamylase when expressed in a recombinant yeast host cell.

[0053] It is well known in the art how to determine if a polypeptide exhibits alpha-amylase activity. For example, in a first step, the polypeptide suspected of having alpha-amylase activity can be placed in the presence of a polysaccharide known to be acted upon by the enzyme (starch for example). If the tested polypeptide exhibits alpha-amylase activity, it will generate dextrins, maltose and / or other smaller polysaccharides having a reduced degree of polymerization compared to the starting polysaccharide. In an embodiment, the alpha-amylase variant exhibits an increase of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100-fold or more in activity when compared to the activity of the parental alpha-amylase (when both enzymes are expressed independently in a recombinant yeast host cell and the activity is measured under similar conditions). In some embodiments, the alphaamylase variant exhibits an increase of at least 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100-fold or more in activity when compared to the activity of the parental alpha-amylase (when both enzymes are expressed independently in a recombinant yeast host cell and the activity is measured under similar conditions), the alphaamylase variant exhibits an increase of at least 80, 85, 90, 95, 100-fold or more in activity when compared to the activity of the parental alpha-amylase (when both enzymes are expressed independently in a recombinant yeast host cell and the activity is measured under similar conditions).

[0054] Alpha-amylase variants

[0055] As indicated herein, the alpha-amylase variants of the present disclosure are derived from a naturally occurring alpha-amylase from Cordyceps farinosa having the GenBank accession number 6SAU_A or the Uniprot accession number A0A5S8WFA8 (which corresponds to the amino acid sequence of SEQ ID NO: 2). In the context of the present disclosure, the numbering of the amino acid residues which are modified in the alpha-amylase variants are provided with respect to the full length of the amino acid sequence of SEQ ID NO: 2. The alpha-amylase variants of the present disclosure have at least 70% identity and less than 100% identity to the amino acid sequence of SEQ ID NO: 2. As such, the alpha-amylase variants include one or more amino acid modification with respect to the amino acid sequence of SEQ ID NO: 2. The amino modification(s) can include at least one amino acid addition, at least one amino acid deletion and / or at least one amino acid substitution. In some specific embodiments, the alphaamylase variants include amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least two, three, four, five, six, seven, eight, nine, 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least three, four, five, six, seven, eight, nine, 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least four, five, six, seven, eight, nine, 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least five, six, seven, eight, nine, 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least six, seven, eight, nine, 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least seven, eight, nine, 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least eight, nine, 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least nine, 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alphaamylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alphaamylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alphaamylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alphaamylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alphaamylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alphaamylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least44, 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alphaamylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 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 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alphaamylase variants include at least 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87 or more amino acid substitutions with respect tothe amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alphaamylase variants include at least 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alphaamylase variants include at least 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 79, 80, 81 , 82, 83, 84, 85, 86, 87 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alphaamylase variants include at least 80, 81 , 82, 83, 84, 85, 86, 87 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 81 , 82, 83, 84, 85, 86, 87 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 82, 83, 84, 85, 86, 87 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 83, 84, 85, 86, 87 or more aminoacid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 84, 85, 86, 87 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 85, 86, 87 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 86, 87 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2. In some additional embodiments, the alpha-amylase variants include at least 87 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2.

[0056] In some further embodiments, the alpha-amylase variants include at least one amino acid substitution at any one of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In additional embodiments, the alpha-amylase variants include at least two amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In additional embodiments, the alpha-amylase variants include at least three amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least four amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least five amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369,372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least six amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least seven amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least eight amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least nine amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 10 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 11 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 12 amino acid substitutions at anycombinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 13 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 14 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 15 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 16 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 17 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 18 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183,185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 19 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 20 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 21 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 22 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 23 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 24 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416,422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 25 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 26 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 27 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 28 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 29 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 30 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 31 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84,86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 32 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 33 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 34 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 35 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 36 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 37 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290,301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 38 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 39 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 40 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 41 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 42 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 43 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylasevariants include at least 44 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 45 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 46 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 47 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 48 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 49 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In further embodiments, the alpha-amylase variants include at least 50 amino acid substitutions at any combinations of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148,151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In yet additional embodiments, the alpha-amylase variants include at least 51 amino acid substitutions at positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In yet additional embodiments, the alpha-amylase variants include at least 52 amino acid substitutions at positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In yet additional embodiments, the alpha-amylase variants include at least 53 amino acid substitutions at positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In yet additional embodiments, the alpha-amylase variants include at least 54 amino acid substitutions at positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In yet additional embodiments, the alpha-amylase variants include at least 55 amino acid substitutions at positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In yet additional embodiments, the alpha-amylase variants include at least 55 amino acid substitutions at positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In yetadditional embodiments, the alpha-amylase variants include at least 57 amino acid substitutions at positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In yet additional embodiments, the alpha-amylase variants include at least 58 amino acid substitutions at positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In yet additional embodiments, the alpha-amylase variants include at least 59 amino acid substitutions at positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In yet additional embodiments, the alpha-amylase variants include at least 60 amino acid substitutions at positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In yet additional embodiments, the alpha-amylase variants include at least 61 amino acid substitutions at positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In yet additional embodiments, the alpha-amylase variants include at least 62 amino acid substitutions at positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In yet additional embodiments, the alpha-amylase variants include at least 63 amino acid substitutions at positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174,178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In yet additional embodiments, the alpha-amylase variants include at least 64 amino acid substitutions at positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In yet additional embodiments, the alpha-amylase variants include at least 65 amino acid substitutions at positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In yet additional embodiments, the alpha-amylase variants include at least 66 amino acid substitutions at positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In yet additional embodiments, the alpha-amylase variants include at least 67 amino acid substitutions at positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In yet additional embodiments, the alpha-amylase variants include at least 68 amino acid substitutions at positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In yet additional embodiments, the alpha-amylase variants include at least 69 amino acid substitutions at positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In yet additional embodiments,the alpha-amylase variants include at least 70 amino acid substitutions at positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In yet additional embodiments, the alpha-amylase variants include at least 71 amino acid substitutions at positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In yet additional embodiments, the alpha-amylase variants include at least 72 amino acid substitutions at positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In yet additional embodiments, the alpha-amylase variants include at least 73 amino acid substitutions at positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In yet additional embodiments, the alpha-amylase variants include at least 74 amino acid substitutions at positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In yet additional embodiments, the alpha-amylase variants include at least 75 amino acid substitutions at positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In yet additional embodiments, the alpha-amylase variants include at least 76 amino acid substitutions at positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208,209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In yet additional embodiments, the alpha-amylase variants include at least 77 amino acid substitutions at positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In yet additional embodiments, the alpha-amylase variants include at least 78 amino acid substitutions at positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In yet additional embodiments, the alpha-amylase variants include at least 79 amino acid substitutions at positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In yet additional embodiments, the alpha-amylase variants include at least 80 amino acid substitutions at positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In yet additional embodiments, the alpha-amylase variants include at least 81 amino acid substitutions at positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In yet additional embodiments, the alpha-amylase variants include at least 82 amino acid substitutions at positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442.In yet additional embodiments, the alpha-amylase variants include at least 83 amino acid substitutions at positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In yet additional embodiments, the alpha-amylase variants include at least 84 amino acid substitutions at positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In yet additional embodiments, the alpha-amylase variants include at least 85 amino acid substitutions at positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In yet additional embodiments, the alpha-amylase variants include at least 86 amino acid substitutions at positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In yet additional embodiments, the alpha-amylase variants include acid substitutions at positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442.

[0057] In some embodiments, the alpha-amylase variants include at least one amino acid substitution at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In additional embodiments, the alpha-amylase variants include at least two amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. Inadditional embodiments, the alpha-amylase variants include at least three amino acid substitutions at any combinations of positions 8, 43, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 148, 155, 158, 174, 178, 181 , 204, 207, 208, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402, 423, and / or 442. In further embodiments, the alpha-amylase variants include at least four amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least five amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least six amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least seven amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least eight amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least nine amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least 10 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least 11 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variantsinclude at least 12 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least 13 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least 14 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least 15 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least 16 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least 17 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least 18 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least 19 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least 20 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least 21 amino acid substitutions at any combinations of positions 43, 71 , 79, 80,84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least 22 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least 23 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least 24 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least 25 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least 26 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least 27 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least 28 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least 29 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least 30 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209,211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least 31 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least 32 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least 33 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least 34 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least 35 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least 36 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least 37 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least 38 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least 39 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380,383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least 40 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least 41 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least 42 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least 43 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least 44 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least 45 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least 46 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least 47 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variants include at least 48 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In further embodiments, the alpha-amylase variantsinclude at least 49 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 84. 91 . 93. 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In yet additional embodiments, the alpha-amylase variants include amino acid substitutions at positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and 423.

[0058] In some embodiments, the alpha-amylase variants include at least one amino acid substitution at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least two amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least three amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least four amino acid substitutions at any one of positions 43. 71 . 79. 80. 84. 91. 93. 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least five amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least six amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least seven amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least eight amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include atleast nine amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least 10 amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least 11 amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least 12 amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least 13 amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least 14 amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alphaamylase variants include at least 15 amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least 16 amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least 17 amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least 10 amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least 18 amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242,252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least 19 amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least 20 amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least 21 amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least 22 amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least 23 amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least 24 amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least 25 amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least 26 amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least 27 amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least 28 amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least 29amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least 30 amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least 31 amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least 32 amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least 33 amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least 34 amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least 35 amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least 36 amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least 37 amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least 38 amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least 39 amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347,348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least 40 amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least 41 amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants include at least 42 amino acid substitutions at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423. In some embodiments, the alpha-amylase variants comprises amino acid substitutions at positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and 423.

[0059] In some embodiments, the alpha-amylase variants include at least one amino acid substitution at any one of positions 71 , 80, 84, 91 , 93, 96, 117, 119, 125, 133, 141 , 158, 174, 209, 211 , 252, 268, 336, 348, 357, 380, 383, 393, and / or 402. In additional embodiments, the alphaamylase variants include at least two amino acid substitutions at any combinations of positions 71 , 80, 84, 91 , 93, 96, 117, 119, 125, 133, 141 , 158, 174, 209, 211 , 252, 268, 336, 348, 357, 380, 383, 393, and / or 402. In additional embodiments, the alpha-amylase variants include at least three amino acid substitutions at any combinations of positions 71 , 80, 84, 91 , 93, 96, 117, 119, 125, 133, 141 , 158, 174, 209, 211 , 252, 268, 336, 348, 357, 380, 383, 393, and / or 402. In additional embodiments, the alpha-amylase variants include at least four amino acid substitutions at any combinations of positions 71 , 80, 84, 91 , 93, 96, 117, 119, 125, 133, 141 , 158, 174, 209, 211 , 252, 268, 336, 348, 357, 380, 383, 393, and / or 402. In additional embodiments, the alpha-amylase variants include at least five amino acid substitutions at any combinations of positions 71 , 80, 84, 91 , 93, 96, 117, 119, 125, 133, 141 , 158, 174, 209, 211 , 252, 268, 336, 348, 357, 380, 383, 393, and / or 402. In additional embodiments, the alphaamylase variants include at least six amino acid substitutions at any combinations of positions 71 , 80, 84, 91 , 93, 96, 117, 119, 125, 133, 141 , 158, 174, 209, 211 , 252, 268, 336, 348, 357, 380, 383, 393, and / or 402. In additional embodiments, the alpha-amylase variants include at least seven amino acid substitutions at any combinations of positions 71 , 80, 84, 91 , 93, 96, 117, 119, 125, 133, 141 , 158, 174, 209, 211 , 252, 268, 336, 348, 357, 380, 383, 393, and / or 402. In additional embodiments, the alpha-amylase variants include at least eight amino acid substitutions at any combinations of positions 71 , 80, 84, 91 , 93, 96, 117, 119, 125, 133, 141 , 158, 174, 209, 211 , 252, 268, 336, 348, 357, 380, 383, 393, and / or 402. In additionalembodiments, the alpha-amylase variants include at least nine amino acid substitutions at any combinations of positions 71, 80, 84, 91, 93, 96, 117, 119, 125, 133, 141, 158, 174, 209, 211, 252, 268, 336, 348, 357, 380, 383, 393, and / or 402. In additional embodiments, the alphaamylase variants include at least 10 amino acid substitutions at any combinations of positions 71, 80, 84, 91, 93, 96, 117, 119, 125, 133, 141, 158, 174, 209, 211, 252, 268, 336, 348, 357, 380, 383, 393, and / or 402. In additional embodiments, the alpha-amylase variants include at least 11 amino acid substitutions at any combinations of positions 71 , 80, 84, 91 , 93, 96, 117, 119, 125, 133, 141, 158, 174, 209, 211 , 252, 268, 336, 348, 357, 380, 383, 393, and / or402. In additional embodiments, the alpha-amylase variants include at least 12 amino acid substitutions at any combinations of positions 71 , 80, 84, 91 , 93, 96, 117, 119, 125, 133, 141 , 158, 174, 209, 211, 252, 268, 336, 348, 357, 380, 383, 393, and / or 402. In additional embodiments, the alpha-amylase variants include at least 13 amino acid substitutions at any combinations of positions 71, 80, 84, 91, 93, 96, 117, 119, 125, 133, 141, 158, 174, 209, 211, 252, 268, 336, 348, 357, 380, 383, 393, and / or 402. In additional embodiments, the alphaamylase variants include at least 14 amino acid substitutions at any combinations of positions 71, 80, 84, 91, 93, 96, 117, 119, 125, 133, 141, 158, 174, 209, 211, 252, 268, 336, 348, 357, 380, 383, 393, and / or 402. In additional embodiments, the alpha-amylase variants include at least 15 amino acid substitutions at any combinations of positions 71, 80, 84, 91, 93, 96, 117, 119, 125, 133, 141, 158, 174, 209, 211 , 252, 268, 336, 348, 357, 380, 383, 393, and / or402. In additional embodiments, the alpha-amylase variants include at least 16 amino acid substitutions at any combinations of positions 71, 80, 84, 91, 93, 96, 117, 119, 125, 133, 141, 158, 174, 209, 211, 252, 268, 336, 348, 357, 380, 383, 393, and / or 402. In additional embodiments, the alpha-amylase variants include at least 17 amino acid substitutions at any combinations of positions 71, 80, 84, 91, 93, 96, 117, 119, 125, 133, 141, 158, 174, 209, 211, 252, 268, 336, 348, 357, 380, 383, 393, and / or 402. In additional embodiments, the alphaamylase variants include at least 18 amino acid substitutions at any combinations of positions 71, 80, 84, 91, 93, 96, 117, 119, 125, 133, 141, 158, 174, 209, 211, 252, 268, 336, 348, 357, 380, 383, 393, and / or 402. In additional embodiments, the alpha-amylase variants include at least 19 amino acid substitutions at any combinations of positions 71 , 80, 84, 91 , 93, 96, 117, 119, 125, 133, 141, 158, 174, 209, 211, 252, 268, 336, 348, 357, 380, 383, 393, and / or 402. In additional embodiments, the alpha-amylase variants include at least 20 amino acid substitutions at any combinations of positions 71 , 80, 84, 91 , 93, 96, 117, 119, 125, 133, 141 , 158, 174, 209, 211, 252, 268, 336, 348, 357, 380, 383, 393, and / or 402. In additional embodiments, the alpha-amylase variants include at least 21 amino acid substitutions at any combinations of positions 71, 80, 84, 91, 93, 96, 117, 119, 125, 133, 141, 158, 174, 209, 211, 252, 268, 336, 348, 357, 380, 383, 393, and / or 402. In additional embodiments, the alphaamylase variants include at least 22 amino acid substitutions at any combinations of positions71 , 80, 84, 91 , 93, 96, 117, 119, 125, 133, 141 , 158, 174, 209, 211 , 252, 268, 336, 348, 357, 380, 383, 393, and / or 402. In additional embodiments, the alpha-amylase variants include at least 23 amino acid substitutions at any combinations of positions 71 , 80, 84, 91 , 93, 96, 117, 119, 125, 133, 141 , 158, 174, 209, 211 , 252, 268, 336, 348, 357, 380, 383, 393, and / or402. In additional embodiments, the alpha-amylase variants include amino acid substitutions at positions 71 , 80, 84, 91 , 93, 96, 117, 119, 125, 133, 141 , 158, 174, 209, 211 , 252, 268, 336, 348, 357, 380, 383, 393, and 402.

[0060] In further embodiments, the alpha-amylase variants include at least one amino acid substitution at any one of positions 43, 71 , 79, 80, 93, 103, 106, 117, 124, 143, 178,181 , 204, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 343, 347, 369, 372, 373, 385, 386, 393, and / or 396. In additional embodiments, the alpha-amylase variants include at least two amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 93, 103, 106, 117, 124, 143, 178,181 , 204, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 343, 347, 369, 372, 373, 385, 386, 393, and / or 396. In additional embodiments, the alpha-amylase variants include at least three amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 93, 103, 106, 117, 124, 143, 178,181 , 204, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 343, 347, 369, 372, 373, 385, 386, 393, and / or 396. In further embodiments, the alpha-amylase variants include at least four amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 93, 103, 106, 117, 124, 143, 178,181 , 204, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 343, 347, 369, 372, 373, 385, 386, 393, and / or 396. In further embodiments, the alpha-amylase variants include at least five amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 93, 103, 106, 117, 124, 143, 178,181 , 204, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 343, 347, 369, 372, 373, 385, 386, 393, and / or 396. In further embodiments, the alpha-amylase variants include at least six amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 93, 103, 106, 117, 124, 143, 178,181 , 204, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 343, 347, 369, 372, 373, 385, 386, 393, and / or 396. In further embodiments, the alpha-amylase variants include at least seven amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 93, 103, 106, 117, 124, 143, 178,181 , 204, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 343, 347, 369, 372, 373, 385, 386, 393, and / or 396. In further embodiments, the alpha-amylase variants include at least eight amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 93, 103, 106, 117, 124, 143, 178,181 , 204, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 343, 347, 369, 372, 373, 385, 386, 393, and / or 396. In further embodiments, the alpha-amylase variants include at least nine amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 93, 103, 106, 117, 124, 143, 178,181 , 204, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 343, 347, 369, 372, 373, 385, 386, 393, and / or 396. In further embodiments, the alphaamylase variants include at least 10 amino acid substitutions at any combinations of positions43, 71 , 79, 80, 93, 103, 106, 117, 124, 143, 178,181 , 204, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 343, 347, 369, 372, 373, 385, 386, 393, and / or 396. In further embodiments, the alpha-amylase variants include at least 11 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 93, 103, 106, 117, 124, 143, 178,181 , 204, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 343, 347, 369, 372, 373, 385, 386, 393, and / or 396. In further embodiments, the alpha-amylase variants include at least 12 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 93, 103, 106, 117, 124, 143, 178,181 , 204, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 343, 347, 369, 372, 373, 385, 386, 393, and / or 396. In further embodiments, the alpha-amylase variants include at least 13 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 93, 103, 106, 117, 124, 143, 178,181 , 204, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 343, 347, 369, 372, 373, 385, 386, 393, and / or 396. In further embodiments, the alpha-amylase variants include at least 14 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 93, 103, 106, 117, 124, 143, 178,181 , 204, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 343, 347, 369, 372, 373, 385, 386, 393, and / or 396. In further embodiments, the alphaamylase variants include at least 15 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 93, 103, 106, 117, 124, 143, 178,181 , 204, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 343, 347, 369, 372, 373, 385, 386, 393, and / or 396. In further embodiments, the alpha-amylase variants include at least 16 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 93, 103, 106, 117, 124, 143, 178,181 , 204, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 343, 347, 369, 372, 373, 385, 386, 393, and / or 396. In further embodiments, the alpha-amylase variants include at least 17 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 93, 103, 106, 117, 124, 143, 178,181 , 204, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 343, 347, 369, 372, 373, 385, 386, 393, and / or 396. In further embodiments, the alpha-amylase variants include at least 18 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 93, 103, 106, 117, 124, 143, 178,181 , 204, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 343, 347, 369, 372, 373, 385, 386, 393, and / or 396. In further embodiments, the alpha-amylase variants include at least 19 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 93, 103, 106, 117, 124, 143, 178,181 , 204, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 343, 347, 369, 372, 373, 385, 386, 393, and / or 396. In further embodiments, the alphaamylase variants include at least 20 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 93, 103, 106, 117, 124, 143, 178,181 , 204, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 343, 347, 369, 372, 373, 385, 386, 393, and / or 396. In further embodiments, the alpha-amylase variants include at least 21 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 93, 103, 106, 117, 124, 143, 178,181 , 204, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 343, 347, 369, 372, 373, 385, 386, 393, and / or396. In further embodiments, the alpha-amylase variants include at least 22 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 93, 103, 106, 117, 124, 143, 178,181 , 204, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 343, 347, 369, 372, 373, 385, 386, 393, and / or 396. In further embodiments, the alpha-amylase variants include at least 23 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 93, 103, 106, 117, 124, 143, 178,181 , 204, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 343, 347, 369, 372, 373, 385, 386, 393, and / or 396. In further embodiments, the alpha-amylase variants include at least 24 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 93, 103, 106, 117, 124, 143, 178,181 , 204, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 343, 347, 369, 372, 373, 385, 386, 393, and / or 396. In further embodiments, the alphaamylase variants include at least 25 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 93, 103, 106, 117, 124, 143, 178,181 , 204, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 343, 347, 369, 372, 373, 385, 386, 393, and / or 396. In further embodiments, the alpha-amylase variants include at least 26 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 93, 103, 106, 117, 124, 143, 178,181 , 204, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 343, 347, 369, 372, 373, 385, 386, 393, and / or 396. In further embodiments, the alpha-amylase variants include at least 27 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 93, 103, 106, 117, 124, 143, 178,181 , 204, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 343, 347, 369, 372, 373, 385, 386, 393, and / or 396. In further embodiments, the alpha-amylase variants include at least 28 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 93, 103, 106, 117, 124, 143, 178,181 , 204, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 343, 347, 369, 372, 373, 385, 386, 393, and / or 396. In further embodiments, the alpha-amylase variants include at least 29 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 93, 103, 106, 117, 124, 143, 178,181 , 204, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 343, 347, 369, 372, 373, 385, 386, 393, and / or 396. In further embodiments, the alphaamylase variants include at least 30 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 93, 103, 106, 117, 124, 143, 178,181 , 204, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 343, 347, 369, 372, 373, 385, 386, 393, and / or 396. In further embodiments, the alpha-amylase variants include at least 31 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 93, 103, 106, 117, 124, 143, 178,181 , 204, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 343, 347, 369, 372, 373, 385, 386, 393, and / or 396. In further embodiments, the alpha-amylase variants include at least 32 amino acid substitutions at any combinations of positions 43, 71 , 79, 80, 93, 103, 106, 117, 124, 143, 178,181 , 204, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 343, 347, 369, 372, 373, 385, 386, 393, and / or 396. In further embodiments, the alpha-amylase variants include amino acid substitutions at positions 43, 71 , 79, 80, 93, 103, 106, 117, 124, 143, 178,181 , 204, 209,211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 343, 347, 369, 372, 373, 385, 386, 393, and 396.

[0061] In some embodiments, the alpha-amylase variants include at least one amino acid substitution at any one of positions 133, 207, and / or 423. In additional embodiments, the alpha-amylase variants include at least two amino acid substitutions at any combinations of positions 133, 207, and / or 423. In additional embodiments, the alpha-amylase variants include the amino acid substitutions at positions 133, 207, and 423.

[0062] Embodiments of alpha-amylase variants comprising at least one amino acid substitution at any one of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442 are provided in Table 1. In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 4 or is a variant of the amino acid sequence of SEQ ID NO: 4 exhibiting alpha-amylase activity (and in some embodiments, a higher alpha-amylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 6 or is a variant of the amino acid sequence of SEQ ID NO: 6 exhibiting alpha-amylase activity (and in some embodiments, a higher alpha-amylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 8 or is a variant of the amino acid sequence of SEQ ID NO: 8 exhibiting alpha-amylase activity (and in some embodiments, a higher alpha-amylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 10 or is a variant of the amino acid sequence of SEQ ID NO: 10 exhibiting alpha-amylase activity (and in some embodiments, a higher alphaamylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 12 or is a variant of the amino acid sequence of SEQ ID NO: 12 exhibiting alpha-amylase activity (and in some embodiments, a higher alpha-amylase activity than the parental alphaamylase when expressed in a recombinant yeast host cell). In an embodiment, the alphaamylase variant has the amino acid sequence of SEQ ID NO: 14 or is a variant of the amino acid sequence of SEQ ID NO: 14 exhibiting alpha-amylase activity (and in some embodiments, a higher alpha-amylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 16 or is a variant of the amino acid sequence of SEQ ID NO: 16 exhibiting alpha-amylase activity (and in some embodiments, a higher alpha-amylase activitythan the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 18 or is a variant of the amino acid sequence of SEQ ID NO: 18 exhibiting alpha-amylase activity (and in some embodiments, a higher alpha-amylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 20 or is a variant of the amino acid sequence of SEQ ID NO: 20 exhibiting alpha-amylase activity (and in some embodiments, a higher alphaamylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 22 or is a variant of the amino acid sequence of SEQ ID NO: 22 exhibiting alpha-amylase activity (and in some embodiments, a higher alpha-amylase activity than the parental alphaamylase when expressed in a recombinant yeast host cell). In an embodiment, the alphaamylase variant has the amino acid sequence of SEQ ID NO: 24 or is a variant of the amino acid sequence of SEQ ID NO: 24 exhibiting alpha-amylase activity (and in some embodiments, a higher alpha-amylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 30 or is a variant of the amino acid sequence of SEQ ID NO: 30 exhibiting alpha-amylase activity (and in some embodiments, a higher alpha-amylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 32 or is a variant of the amino acid sequence of SEQ ID NO: 32 exhibiting alpha-amylase activity (and in some embodiments, a higher alpha-amylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 34 or is a variant of the amino acid sequence of SEQ ID NO: 34 exhibiting alpha-amylase activity (and in some embodiments, a higher alphaamylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 36 or is a variant of the amino acid sequence of SEQ ID NO: 36 exhibiting alpha-amylase activity (and in some embodiments, a higher alpha-amylase activity than the parental alphaamylase when expressed in a recombinant yeast host cell). In an embodiment, the alphaamylase variant has the amino acid sequence of SEQ ID NO: 38 or is a variant of the amino acid sequence of SEQ ID NO: 38 exhibiting alpha-amylase activity (and in some embodiments, a higher alpha-amylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 40 or is a variant of the amino acid sequence of SEQ ID NO: 40 exhibiting alpha-amylase activity (and in some embodiments, a higher alpha-amylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In anembodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 42 or is a variant of the amino acid sequence of SEQ ID NO: 42 exhibiting alpha-amylase activity (and in some embodiments, a higher alpha-amylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 44 or is a variant of the amino acid sequence of SEQ ID NO: 44 exhibiting alpha-amylase activity (and in some embodiments, a higher alphaamylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 46 or is a variant of the amino acid sequence of SEQ ID NO: 46 exhibiting alpha-amylase activity (and in some embodiments, a higher alpha-amylase activity than the parental alphaamylase when expressed in a recombinant yeast host cell). In an embodiment, the alphaamylase variant has the amino acid sequence of SEQ ID NO: 48 or is a variant of the amino acid sequence of SEQ ID NO: 48 exhibiting alpha-amylase activity (and in some embodiments, a higher alpha-amylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 50 or is a variant of the amino acid sequence of SEQ ID NO: 50 exhibiting alpha-amylase activity (and in some embodiments, a higher alpha-amylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 52 or is a variant of the amino acid sequence of SEQ ID NO: 52 exhibiting alpha-amylase activity (and in some embodiments, a higher alpha-amylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 54 or is a variant of the amino acid sequence of SEQ ID NO: 54 exhibiting alpha-amylase activity (and in some embodiments, a higher alphaamylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 56 or is a variant of the amino acid sequence of SEQ ID NO: 56 exhibiting alpha-amylase activity (and in some embodiments, a higher alpha-amylase activity than the parental alphaamylase when expressed in a recombinant yeast host cell). In an embodiment, the alphaamylase variant has the amino acid sequence of SEQ ID NO: 106 or is a variant of the amino acid sequence of SEQ ID NO: 106 exhibiting alpha-amylase activity (and in some embodiments, a higher alpha-amylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 108 or is a variant of the amino acid sequence of SEQ ID NO: 108 exhibiting alpha-amylase activity (and in some embodiments, a higher alphaamylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ IDNO: 110 or is a variant of the amino acid sequence of SEQ ID NO: 110 exhibiting alphaamylase activity (and in some embodiments, a higher alpha-amylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 112 or is a variant of the amino acid sequence of SEQ ID NO: 112 exhibiting alpha-amylase activity (and in some embodiments, a higher alpha-amylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 114 or is a variant of the amino acid sequence of SEQ ID NO: 114 exhibiting alpha-amylase activity (and in some embodiments, a higher alphaamylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 116 or is a variant of the amino acid sequence of SEQ ID NO: 116 exhibiting alphaamylase activity (and in some embodiments, a higher alpha-amylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 118 or is a variant of the amino acid sequence of SEQ ID NO: 118 exhibiting alpha-amylase activity (and in some embodiments, a higher alpha-amylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 120 or is a variant of the amino acid sequence of SEQ ID NO: 120 exhibiting alpha-amylase activity (and in some embodiments, a higher alphaamylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 122 or is a variant of the amino acid sequence of SEQ ID NO: 122 exhibiting alphaamylase activity (and in some embodiments, a higher alpha-amylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 124 or is a variant of the amino acid sequence of SEQ ID NO: 124 exhibiting alpha-amylase activity (and in some embodiments, a higher alpha-amylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 126 or is a variant of the amino acid sequence of SEQ ID NO: 126 exhibiting alpha-amylase activity (and in some embodiments, a higher alphaamylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 128 or is a variant of the amino acid sequence of SEQ ID NO: 128 exhibiting alphaamylase activity (and in some embodiments, a higher alpha-amylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 130 or is a variant of theamino acid sequence of SEQ ID NO: 130 exhibiting alpha-amylase activity (and in some embodiments, a higher alpha-amylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 132 or is a variant of the amino acid sequence of SEQ ID NO: 132 exhibiting alpha-amylase activity (and in some embodiments, a higher alphaamylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 134 or is a variant of the amino acid sequence of SEQ ID NO: 134 exhibiting alphaamylase activity (and in some embodiments, a higher alpha-amylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 136 or is a variant of the amino acid sequence of SEQ ID NO: 136 exhibiting alpha-amylase activity (and in some embodiments, a higher alpha-amylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 138 or is a variant of the amino acid sequence of SEQ ID NO: 138 exhibiting alpha-amylase activity (and in some embodiments, a higher alphaamylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 140 or is a variant of the amino acid sequence of SEQ ID NO: 140 exhibiting alphaamylase activity (and in some embodiments, a higher alpha-amylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 142 or is a variant of the amino acid sequence of SEQ ID NO: 142 exhibiting alpha-amylase activity (and in some embodiments, a higher alpha-amylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 144 or is a variant of the amino acid sequence of SEQ ID NO: 144 exhibiting alpha-amylase activity (and in some embodiments, a higher alphaamylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 146 or is a variant of the amino acid sequence of SEQ ID NO: 146 exhibiting alphaamylase activity (and in some embodiments, a higher alpha-amylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 148 or is a variant of the amino acid sequence of SEQ ID NO: 148 exhibiting alpha-amylase activity (and in some embodiments, a higher alpha-amylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 150 or is a variant of the amino acid sequence ofSEQ ID NO: 150 exhibiting alpha-amylase activity (and in some embodiments, a higher alphaamylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 152 or is a variant of the amino acid sequence of SEQ ID NO: 152 exhibiting alphaamylase activity (and in some embodiments, a higher alpha-amylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 154 or is a variant of the amino acid sequence of SEQ ID NO: 154 exhibiting alpha-amylase activity (and in some embodiments, a higher alpha-amylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 156 or is a variant of the amino acid sequence of SEQ ID NO: 156 exhibiting alpha-amylase activity (and in some embodiments, a higher alphaamylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 158 or is a variant of the amino acid sequence of SEQ ID NO: 158 exhibiting alphaamylase activity (and in some embodiments, a higher alpha-amylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 160 or is a variant of the amino acid sequence of SEQ ID NO: 160 exhibiting alpha-amylase activity (and in some embodiments, a higher alpha-amylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell). In an embodiment, the alpha-amylase variant has the amino acid sequence of SEQ ID NO: 162 or is a variant of the amino acid sequence of SEQ ID NO: 162 exhibiting alpha-amylase activity (and in some embodiments, a higher alphaamylase activity than the parental alpha-amylase when expressed in a recombinant yeast host cell).

[0063] The present disclosure provides variants of alpha-amylase variants including at least one amino acid substitution at any one of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442. In some embodiments, such variants include at least one amino acid substitution at any one of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408,415, 416, 422, 423, 426, and / or 442 as well as at least one other modifications (addition, deletion and / or substitutions) at other positions.

[0064] In some embodiments, the alpha-amylase variant comprises, at position 3, a naturally occurring amino acid residue that is different from T. For example, the alpha-amylase variant can include, at position 3, one of the following amino acid residues: A (T3A), C (T3C), D (T3D), E (T3E), F (T3F), G (T3G), H (T3H), I (T3I), K (T3K), L (T3L), M (T3M), N (T3N), P (T3P), Q (T3Q), R (T3R), S (T3S), V (T3V), W (T3W), or Y (T3Y). In a further example, the alphaamylase variant can include, at position 3, the following amino acid residue: S (T3S). Embodiments of an alpha-amylase variant having T3S have the amino acid sequence of SEQ ID NO: 160 or 162 as well as variants thereof.

[0065] In some embodiments, the alpha-amylase variant comprises, at position 4, a naturally occurring amino acid residue that is different from N. For example, the alpha-amylase variant can comprise, at position 4, on of the following amino acid residue: A (N4A), C (N4C), D (N4D), E (N4E), F (N4F), G (N4G), H (N4H), I (N4I), K (N4K), L (N4L), M (N4M), P (N4P), Q (N4Q), R (N4R), S (N4S), T (N4T), V (N4V), W (N4W), or Y (N4Y). In a further example, the alphaamylase variant can include, at position 4, the following amino acid residue: D (N4D). Embodiments of alpha-amylase variants having N4D include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID 160 or 162 as well as variants thereof. In some embodiments, the alpha-amylase variant comprises, at position 5, a naturally occurring amino acid residue that is different from S. For example, the alpha-amylase variant can comprise, at position 5, one of the following amino acid residues: A (S5A), C (S5C), D M (S5M), N (S5N), P

[0066]

[0067] another example, the alpha-amylase variant can include, at position 5, the following amino acid residue: D (S5D). An embodiment of an alpha-amylase variant having S5D has the amino acid sequence of SEQ ID NO: 158, 160 or 162 as well as variants thereof. In some embodiments, the alpha-amylase variant comprises, at position 8, a naturally occurring amino acid residue that is different from S. For example, the alpha-amylase variant can comprise, at position 8, one of the following amino acid residues: ,K (S8K), L (S8L), M

[0068]

[0069] or Y (S8Y). In another example, the alpha-amylase variant can include, at position 8, the following amino acid residue: G (S8G). An embodiment of an alpha-amylase variant having S8G has the amino acid sequence of SEQ ID NO: 16 as well as variants thereof.

[0070] In some embodiments, the alpha-amylase variant comprises, at position 18, a naturally occurring amino acid residue that is different from G. For example, the alpha-amylase variant can include, at position 18, one of the following amino acid residues: A (G18A), C (G18C), D (G18D), E (G18E), F (G18F), H (G18H), I (G18I), K (G18K), L (G18L), M (G18M), N (G18N),P (G18P), Q (G18Q), R (G18R), S (G18S), T (G18T), V (G18V), W (G18W), orY (G18Y). In a further example, the alpha-amylase variant can include, at position 18, the following amino acid residue: W (G18W). An embodiment of alpha-amylase variants having G18W includes, but is not limited to, a polypeptide having the amino acid sequence of SEQ ID NO: 140 as well as variants thereof.

[0071] In some embodiments, the alpha-amylase variant comprises, at position 24, a naturally occurring amino acid residue that is different from S. For example, the alpha-amylase variant can comprise, at position 24, one of the following amino acid residues: A (S24A), C (S24C), D 24M), N

[0072]

[0073] another example, the alpha-amylase variant can include, at position 24, the following amino acid residue: G (S24G). An embodiment of an alpha-amylase variant having S24G has the amino acid sequence of SEQ ID NO: 160 or 162 as well as variants thereof.

[0074] In some embodiments, the alpha-amylase variant comprises, at position 26, a naturally occurring amino acid residue that is different from N. For example, the alpha-amylase variant can comprise, at position 26, on of the following amino acid residue: A (N26A), C (N26C), D M), P

[0075]

[0076] . In a further example, the alpha-amylase variant can include, at position 26, the following amino acid residue: D (N26D). Embodiments of alpha-amylase variants having N26D include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID 160 or 162 as well as variants thereof.

[0077] In some embodiments, the alpha-amylase variant comprises, at position 33 a naturally occurring amino acid residue that is different from Q. For example, the alpha-amylase variant can include, at position 33, one of the following amino acid residues: A (Q33A), C (Q33C), D

[0078]

[0079] further example, the alpha-amylase variant can include, at position 33, the following amino acid residue: E (Q33E). Embodiments of alpha-amylase variants having Q33E include, but are not limited to, the polypeptides having the amino acid sequence of SEQ ID NO: 144 or 146 as well as variants thereof.

[0080] In some embodiments, the alpha-amylase variant comprises, at position 43, a naturally occurring amino acid residue that is different from K. For example, the alpha-amylase variant can comprise, at position 43, one of the following amino acid residues: A (K43A), C (K43C), D (K43D), E (K43E), F (K43F), G (K43G), H (K43H), I (K43I), L (K43L), M (K43M), N (K43N), P (K43P), Q (K43Q),R (K43R), S (K43S), T (K43T), V (K43V), W(K43W), orY (K43Y). In another example, the alpha-amylase variant can include, at position 43, one of the following amino acidresidues: Q (K43Q), A (K43A), E (K43E) or R (K43R). In a further example, the alpha-amylase variant can include, at position 43, the following amino acid residue: Q (K43Q). Embodiments of alpha-amylase variants having K43Q include, but are not limited to, the polypeptides having the amino acid sequence of SEQ ID NO: 32, 38, 40, 42, 46, 50, 54, 106, 108, 110, 112, 14, 116, 118, 120, 122, 124, 126, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, or 160 as well as variants thereof. In a further example, the alpha-amylase variant can include, at position 43, the following amino acid residue: E (K43E). An embodiment of an alpha-amylase variant having K43E includes, but is not limited to, the polypeptide having the amino acid sequence of SEQ ID NO: 162 as well as variants thereof. In still another embodiment, the alpha-amylase variant of the present disclosure excludes having a substitution at position 43 (e.g., it remains a Q).

[0081] In some embodiments, the alpha-amylase variant comprises, at position 70, a naturally occurring amino acid residue that is different from A. For example, the alpha-amylase variant can include, at position 70, one of the following amino acid residues: C (A70C), D (A70D), E (A70E), F (A70F), G (A70G), H (A70H), I (A70I), K (A70K), L (A70L), M (A70M), N (A70N), P (A70P), Q (A70Q), R (A70R), S (A70S), T (A70T), V (A70V), W(A70W), orY (A70Y). In another example, the alpha-amylase variant can include, at position 70, the following amino acid residue: D (A70D). Embodiments of an alpha-amylase variant including A70D is a polypeptide having the amino acid sequence of SEQ ID NO: 106, 114, 156, 158, 160, or 162 as well as variants thereof.

[0082] In some embodiments, the alpha-amylase variant comprises, at position 71 , a naturally occurring amino acid residue that is different from R. In an example, the alpha-amylase variant can comprise, at position 71 , one of the following amino acid residues: A (R71A), C (R71C), D

[0083]

[0084] another example, the alpha-amylase variant can include, at position 71 , one of the following amino acid residues: G (R71G), N (R71N), D (R71D), or A (R71A). In another example, the alpha-amylase variant can include, at position 71 , the following amino acid residue: G (R71G) or D(R71D). In a further example, the alpha-amylase variant can include, at position 71 , the following amino acid residue: G (R71G). Embodiments of alpha-amylase variants having R71G include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 30, 32, 34, 36, 38, 40, 42, 48, 50, 52, 54, 56, 106, 108, 110, 112, 114, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 150, 154, 156, 158, 160, or 162 as well as variants thereof. In a further example, the alpha-amylase variant can include, at position 71 , the following amino acid residue: D (R71D). Embodiments of alpha-amylase variants having R71D include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 148 or 152 as well as variants thereof. In still another embodiment, the alpha-amylasevariant of the present disclosure excludes having a substitution at position 71 (e.g., it remains a R).

[0085] In some embodiments, the alpha-amylase variant comprises, at position 79, a naturally occurring amino acid residue that is different from S. In an example, the alpha-amylase variant can comprise, at position 79, one of the following amino acid residues: A (S79A), C (S79C), D 79M), N

[0086]

[0087] another example, the alpha-amylase variant can include, at position 79, one of the following amino acid residues: Q (S79Q), K (S79K), T (S79T), R (S79R), or N (S79N). In a further example, the alpha-amylase variant can include, at position 79, the following amino acid residue: Q (S79Q). Embodiments of alpha-amylase variants having S79Q include, but are not limited to, the polypeptides having the amino acid sequence of SEQ ID NO: 32, 38, 40, 42, 46, 50, 54, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162 as well as variants thereof.

[0088] In some embodiments, the alpha-amylase variant comprises, at position 80, a naturally occurring amino acid residue that is different from N. For example, the alpha-amylase variant can comprise, at position 80, on of the following amino acid residue: A (N80A), C (N80C), D

[0089]

[0090] another example, the alpha-amylase variant can include, at position 80, one of the following amino acid residues: N (N80D), S (N80S) or H (N80H). In a further example, the alpha-amylase variant can include, at position 80, the following amino acid residue: D (N80D). In some embodiments, the alpha-amylase variants do not include an amino acid modification at position 80. Embodiments of alpha-amylase variants having N80D include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 30, 32, 34, 36, 38, 40, 42, 48, 50, 52, 54, 56, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162 as well as variants thereof. In some embodiments, the alpha-amylase variant comprises, at position 84, a naturally occurring amino acid residue that is different from T. For example, the alpha-amylase variant can include, at position 84, one of the following amino acid residues: A (T84A), C (T84C), D (T84D), E (T84E), F (T84F), G (T84G), H (T84H), I (T84I), K (T84K), L (T84L), M (T84M), N (T84N), P (T84P), Q (T84Q), R (T84R), S (T84S), V (T84V), W(T84W), orY (T84Y). In another example, the alpha-amylase variant can include, at position 84, one of the following amino acid residues: I (T84I), V (T84V), or L (T84L). In a further example, the alpha-amylase variant can include, at position 84, the following amino acid residue: I (T84I). Embodiments of alphaamylase variants having T84I include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 30, 36, 40, 42, 44, 46, 48, 52, 56, 106, 108, 110, 112, 114, 116,118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, or 160 as well as variants thereof.

[0091] In some embodiments, the alpha-amylase variant comprises, at position 86, a naturally occurring amino acid residue that is different from S. For example, the alpha-amylase variant can include, at position 86, one of the following amino acid residues: A (S86A), C (S86C), D 86M), N

[0092]

[0093] another example, the alpha-amylase variant can include, at position 86, the following amino acid residue: P (S86P). An embodiment of an alpha-amylase variants having S86P has the amino acid sequence of SEQ ID NO: 8 as well as variants thereof.

[0094] In some embodiments, the alpha-amylase variant comprises, at position 91 , a naturally occurring amino acid residue that is different from S. For example, the alpha-amylase variant can include, at position 91 , one of the following amino acid residues: A (S91A), C (S91C), D (S91M), N

[0095]

[0096] . In another example, the alpha-amylase variant can include, at position 91 , one of the following amino acid residues: A (S91 A), E (S91 E) or P (S91 P). In a further example, the alpha-amylase variant can include, at position 91 , the following amino acid residue: A (S91A). Embodiments of alphaamylase variants having S91A include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 30, 36, 40, 42, 44, 46, 48, 52, 56, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162 as well as variants thereof.

[0097] In some embodiments, the alpha-amylase variant comprises, at position 93, a naturally occurring amino acid residue that is different from E. For example, the alpha-amylase variant can include, at position 93, one of the following amino acid residues: A (E93A), C (E93C), D 93N), P

[0098]

[0099] another example, the alpha-amylase variant can include, at position 93, one of the following amino acid residues: D (E93D), S (E93S), or Q (E93Q). In a further example, the alpha-amylase variant can include, at position 93, the following amino acid residue: D (E93D). Embodiments of alphaamylase variants having E93D include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 30, 32, 34, 36, 38, 40, 42, 48, 50, 52, 54, 56, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162 as well as variants thereof.

[0100] In some embodiments, the alpha-amylase variant comprises, at position 96, a naturally occurring amino acid residue that is different from G. For example, the alpha-amylase variant can include, at position 96, one of the following amino acid residues: A (G96A), C (G96C), D

[0101]

[0102] another example, the alpha-amylase variant can include, at position 96, one of the following amino acid residues: S (G96S), D (G96D), N (G96N), or A (G96A). In a further example, the alpha-amylase variant can include, at position 96, the following amino acid residue: S (G96S). Embodiments of alpha-amylase variants having G96S include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 18, 30, 36, 40, 42, 48, 50, 52, 54, 56, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 150, 154, 156, 158, 160, or 162 as well as variants thereof. In a further example, the alpha-amylase variant can include, at position 96, the following amino acid residue: D (G96D). Embodiments of alpha-amylase variants having G96D include, but are not limited to, a polypeptide having the amino acid sequence of SEQ ID NO: 20, 146, 148, or 152 as well as variants thereof. In a further example, the alpha-amylase variant can include, at position 96, the following amino acid residue: E (G96E). An embodiment of an alpha-amylase variant having G96E has the amino acid sequence of SEQ ID NO: 22. In a further example, the alphaamylase variant can include, at position 96, the following amino acid residue: A (G96A). An embodiment of an alpha-amylase variant having G96A has the amino acid sequence of SEQ ID NO: 24 as well as variants thereof. In still another embodiment, the alpha-amylase variant of the present disclosure excludes having a substitution at position 96 (e.g., it remains a G). In some embodiments, the alpha-amylase variant comprises, at position 103, a naturally occurring amino acid residue that is different from G. For example, the alpha-amylase variant can include, at position 103, one of the following amino acid residues: A (G103A), C (G103C),

[0103]

[0104] (G103W), orY (G103Y). In another example, the alpha-amylase variant can include, at position 103, one of the following amino acid residues: E (G103E), S (G103S), A (G103A), N (G103N), or Q (G103Q). In a further example, the alpha-amylase variant can include, at position 103, the following amino acid residue: E (G103E). Embodiments of alpha-amylase variants having G103E include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 32, 38, 40, 42, 46, 50, 54, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162 as well as variants thereof.

[0105] In some embodiments, the alpha-amylase variant comprises, at position 105, a naturally occurring amino acid residue that is different from G. For example, the alpha-amylase variant can include, at position 105, one of the following amino acid residues: A (G105A), C (G105C),

[0106]

[0107] (G105W), or Y (G105Y). In a further example, the alpha-amylase variant can include, at position 105, the following amino acid residue: D (G105D). Embodiments of alpha-amylase variants having G105D includes, but is not limited to, a polypeptide having the amino acid sequence of SEQ ID NO: 158, 160, or 162 as well as variants thereof.

[0108] In some embodiments, the alpha-amylase variant comprises, at position 106, a naturally occurring amino acid residue that is different from I. For example, the alpha-amylase variant can include, at position 106, one of the following amino acid residues: A (1106A), C (I106C), D (I106D), E (I106E), F (I106F), G (I106G), H (I106H), K (I106K), L (I106L), M (I106M), N (I106N), P (I106P), Q (I106Q), R (I106R), S (I106S), T (I106T), V (1106V), W (I106W), orY (l106Y). In another example, the alpha-amylase variant can include, at position 106, one of the following amino acid residues: M (I106M), F (I106F), V (1106V), or L (I106L). In a further example, the alpha-amylase variant can include, at position 106, the following amino acid residue: M (I106M). Embodiments of alpha-amylase variants having I106M include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 32, 38, 40, 42, 46, 50, 54, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162 as well as variants thereof.

[0109] In some embodiments, the alpha-amylase variant comprises, at position 117, a naturally occurring amino acid residue that is different from V. For example, the alpha-amylase variant can include, at position 117, one of the following amino acid residues: A (V117A), C (V117C),

[0110]

[0111] or Y (V117Y). In another example, the alpha-amylase variant can include, at position 117, one of the following amino acid residues: M (V117M), A(V117A), orT (V117T). In a further example, the alpha-amylase variant can include, at position 117, the following amino acid residue: M (V117M). Embodiments of alpha-amylase variants having V117M include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 30, 32, 34, 36, 38, 40, 42, 48, 50, 52, 54, 56, 106, 108, 110, 112, 114, 122, 124, 126, 128, 130, 132, 134, 136, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162 as well as variants thereof. In an embodiment, the alpha-amylase variant of the present disclosure excludes having F at position 117. In still another embodiment, the alpha-amylase variant of the present disclosure excludes having a substitution at position 117 (e.g., it remains a V).

[0112] In some embodiments, the alpha-amylase variant comprises, at position 119, a naturally occurring amino acid residue that is different from N. For example, the alpha-amylase variant can include, at position 119, one of the following amino acid residues: A (N119A), C (N119C),

[0113]

[0114] orY (N119Y). In another example, the alpha-amylase variant can include, at position 119, oneof the following amino acid residues: P (N119P), S (N119S), A (N119A), or Q (N119Q). In a further example, the alpha-amylase variant can include, at position 119, the following amino acid residue: P (N119P). Embodiments of alpha-amylase variants having N119P include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 30, 36, 40, 42, 44, 46, 48, 52, 56, 106, 108, 110, 112, 114, 116, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162 as well as variants thereof. In an embodiment, the alpha-amylase variant of the present disclosure excludes having Y at position 119. In still another embodiment, the alpha-amylase variant of the present disclosure excludes having a substitution at position 119 (e.g., it remains a N).

[0115] In some embodiments, the alpha-amylase variant comprises, at position 123, a naturally occurring amino acid residue that is different from N. For example, the alpha-amylase variant can include, at position 123, one of the following amino acid residues: A (N123A), C (N123C),

[0116]

[0117] (N123W), or Y (N123Y). In a further example, the alpha-amylase variant can include, at position 123, the following amino acid residue: W (N123W). An embodiment of alpha-amylase variant having N123W includes, but is not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 138 as well as variants thereof. In an embodiment, the alpha-amylase variants of the present disclosure exclude having Y at position 123.

[0118] In some embodiments, the alpha-amylase variant comprises, at position 124, a naturally occurring amino acid residue that is different from E. For example, the alpha-amylase variant can include, at position 124, one of the following amino acid residues: A (E124A), C (E124C),

[0119]

[0120] or Y (E124Y). In another example, the alpha-amylase variant can include, at position 124, one ofthe following amino acid residues: D (E124D), S (E124S), orT (E124T). In a further example, the alpha-amylase variant can include, at position 124, the following amino acid residue: D (E124D). Embodiments of alpha-amylase variants having E124D include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 32, 38, 40, 42, 46, 50, 54, 106, 108, 110, 112, 114, 116,118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162 as well as variants thereof. In an embodiment, the alpha-amylase variant ofthe present disclosure excludes having Y at position 124.

[0121] In some embodiments, the alpha-amylase variant comprises, at position 125, a naturally occurring amino acid residue that is different from M. For example, the alpha-amylase variant can include, at position 125, one ofthe following amino acid residues: A (M125A), C (M125C), D (M125D), E (M125E), F (M125F), G (M125G), H (M125H), I (M125I), K (M125K), L (M125L),N (M125N), P (M125P), Q (M125Q), R (M125R), S (M125S), T (M125T), V (M125V), W (M125W), or Y (M125Y). In another example, the alpha-amylase variant can include, at position 125, one of the following amino acid residues: N (M125N), T (M125T), K (M125K), F (M125F), orS (M125S). In a further example, the alpha-amylase variant can include, at position 125, the following amino acid residue: N (M125N). Embodiments of alpha-amylase variants having M125N include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 30, 36, 40, 42, 48, 50, 52, 56, 106, 108, 110, 112, 114, 116,118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162 as well as variants thereof. In an embodiment, the alpha-amylase variants of the present disclosure exclude having S at position 125.

[0122] In some embodiments, the alpha-amylase variant comprises, at position 132 a naturally occurring amino acid residue that is different from Q. For example, the alpha-amylase variant can include, at position 132, one of the following amino acid residues: A (Q132A), C (Q132C),

[0123]

[0124] (Q132W), or Y (Q132Y). In a further example, the alpha-amylase variant can include, at position 132, the following amino acid residue: D (Q132D). An embodiment of an alphaamylase variant having Q132D includes, but is not limited to, the polypeptide having the amino acid sequence of SEQ ID NO: 162 as well as variants thereof.

[0125] In some embodiments, the alpha-amylase variant comprises, at position 133, a naturally occurring amino acid residue that is different from G. For example, the alpha-amylase variant can include, at position 133, one of the following amino acid residues: A (G133A), C (G133C),

[0126]

[0127] (G133W), orY (G133Y). In another example, the alpha-amylase variant can include, at position 133, one of the following amino acid residues: A (G133A), D (G133D), E (G133E), Q (G133Q), or S (G133S). In another example, the alpha-amylase variant can include, at position 133, the following amino acid residue: S (G133S). Embodiments of alpha-amylase variants having G133S include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 6, 12, 14, 38, 40, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 150, 154, 156, 158, 160, or 162 as well as variants thereof. In another example, the alpha-amylase variant can include, at position 133, the following amino acid residue: A (G133A). Embodiments of alpha-amylase variants having G133A include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 30, 36, 42, 48, 50, 52, or 56 as well as variants thereof. In another example, the alpha-amylase variant can include, at position 133, the following amino acid residue: E (G133E). Embodiments of alphaamylase variants having G133E include, but are not limited to, polypeptides having the aminoacid sequence of SEQ ID NO: 148, or 152 as well as variants thereof. In an embodiment, the alpha-amylase variant of the present disclosure excludes having Q at position 133. In still another embodiment, the alpha-amylase variant of the present disclosure excludes having a substitution at position 133 (e.g., it remains a G).

[0128] In some embodiments, the alpha-amylase variant comprises, at position 141, a naturally occurring amino acid residue that is different from G. For example, the alpha-amylase variant can include, at position 141 , one of the following amino acid residues: A (G141A), C (G141C),

[0129]

[0130] (G141 W), orY(G141Y). In another example, the alpha-amylase variant can include, at position 141, one ofthe following amino acid residues: D (G141D), E (G141E), T (G141T), Q (G141Q), or K (G141K). In a further example, the alpha-amylase variant can include, at position 141 , the following amino acid residue: D (G141D). Embodiments of alpha-amylase variants having G141D include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 30, 36, 40, 42, 44, 46, 48, 52, or 56 as well as variants thereof. In an embodiment, the alpha-amylase variant of the present disclosure excludes having L at position 141. In still another embodiment, the alpha-amylase variant of the present disclosure excludes having a substitution at position 141 (e.g., it remains a G).

[0131] In some embodiments, the alpha-amylase variant comprises, at position 143, a naturally occurring amino acid residue that is different from N. For example, the alpha-amylase variant can include, at position 143, one ofthe following amino acid residues: A (N143A), C (N143C),

[0132]

[0133] (N143W), orY (N143Y). In another example, the alpha-amylase variant can include, at position 143, one of the following amino acid residues: D (N143D), S (N143S), Q (N143Q), or K (N143K). In a further example, the alpha-amylase variant can include, at position 143, the following amino acid residue: D (N143D). Embodiments of alpha-amylase variants having N143D) include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 32, 38, 40, 42, 46, 50, 54, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162 as well as variants thereof.

[0134] In some embodiments, the alpha-amylase variant comprises, at position 148, a naturally occurring amino acid residue that is different from H. For example, the alpha-amylase variant can include, at position 148, one ofthe following amino acid residues: A (H148A), C (H148C),

[0135]

[0136] (H148W), orY (H148Y). In an example, the alpha-amylase variant can include at position 148S (H148S), N (H148N) or D (H148D). In another example, the alpha-amylase variant can include, at position 148, the following amino acid residue: S (H148S). Embodiments of alphaamylase variants having H148S include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 48, or 50 as well as variants thereof. In another example, the alpha-amylase variant can include, at position 148, the following amino acid residue: N (H148N). Embodiments of alpha-amylase variants having H148N include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 144, or 146 as well as variants thereof. In another example, the alpha-amylase variant can include, at position 148, the following amino acid residue: D (H148D). Embodiments of alpha-amylase variants having H148D include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 148, or 152 as well as variants thereof.

[0137] In some embodiments, the alpha-amylase variant comprises, at position 151 , a naturally occurring amino acid residue that is different from E. For example, the alpha-amylase variant can include, at position 151 , one of the following amino acid residues: A (E151A), C (E151C),

[0138]

[0139] or Y (E151Y). In another example, the alpha-amylase variant can include, at position 151 , one of the following amino acid residues: Y (E151 Y), or K (E151 K). In a further example, the alphaamylase variant can include, at position 151 , the following amino acid residue: Y (E151Y). An embodiment of an alpha-amylase variant having E151Y includes, but is not limited to, a polypeptide having the amino acid sequence of SEQ ID NO: 128 as well as variants thereof. In a further example, the alpha-amylase variant can include, at position 151 , the following amino acid residue: K (E151K). An embodiment of an alpha-amylase variant having E151K includes, but is not limited to, a polypeptide having the amino acid sequence of SEQ ID NO: 124 as well as variants thereof.

[0140] In some embodiments, the alpha-amylase variant comprises, at position 152, a naturally occurring amino acid residue that is different from T. For example, the alpha-amylase variant can include, at position 152, one of the following amino acid residues: A (T152A), C (T152C),

[0141]

[0142] orY (T152Y). In a further example, the alpha-amylase variant can include, at position 152, the following amino acid residue: N (T152N). Embodiments of an alpha-amylase variant having T152N have the amino acid sequence of SEQ ID NO: 144, or 146 as well as variants thereof. In some embodiments, the alpha-amylase variant comprises, at position 155, a naturally occurring amino acid residue that is different from V. For example, the alpha-amylase variant can include, at position 155, one of the following amino acid residues: A (V155A), C (V155C), D (V155D), E (V155E), F (V155F), G (V155G), H (V155H), I (V155I), K (V155K), L (V155L), M(V155M), N (V155N), P (V155P), Q (V155Q), R (V155R), S (V155S), T (V155T), W (V155W), or Y (V155Y). In another example, the alpha-amylase variant can include, at position 155, the following amino acid residue: I (V155I). Embodiments of alpha-amylase variants having V155I include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 52, or 54 as well as variants thereof.

[0143] In some embodiments, the alpha-amylase variant comprises, at position 157, a naturally occurring amino acid residue that is different from S. For example, the alpha-amylase variant can comprise, at position 157, one of the following amino acid residues: A (S157A), C (S157C),

[0144]

[0145] or Y (S157Y). In another example, the alpha-amylase variant can include, at position 157, the following amino acid residue: D (S157D). An embodiment of an alpha-amylase variant having S157D has the amino acid sequence of SEQ ID NO: 108, or 114 as well as variants thereof. In some embodiments, the alpha-amylase variant comprises, at position 158, a naturally occurring amino acid residue that is different from D. For example, the alpha-amylase variant can include, at position 158, one of the following amino acid residues: A (D158A), C (D158C),

[0146]

[0147] (D158W), orY (D158Y). In another example, the alpha-amylase variant can include, at position 158, one of the following amino acid residues: G (D158G), S (D158S), or E (D158E). In a further example, the alpha-amylase variant can include, at position 158, the following amino acid residue: G (D158G). Embodiments of alpha-amylase variants having D158G include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 30, 36, 40, 42, 44, 46, 52, 56, 106, 110, 112, 116, 118, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, or 158 as well as variants thereof. In a further example, the alpha-amylase variant can include, at position 158, the following amino acid residue: E (D158E). Embodiments of alpha-amylase variants having D158E include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 108, 114, 160, or 162 as well as variants thereof. In an embodiment, the alpha-amylase variants of the present disclosure exclude having T at position 158. In still another embodiment, the alpha-amylase variant of the present disclosure excludes having a substitution at position 158 (e.g., it remains a D).

[0148] In some embodiments, the alpha-amylase variant comprises, at position 168, a naturally occurring amino acid residue that is different from K. For example, the alpha-amylase variant can include, at position 168, one of the following amino acid residues: A (K168A), C (K168C),

[0149]

[0150] or Y (K168Y). In a further example, the alpha-amylase variant can include, at position 168, thefollowing amino acid residue: S (K168S), or D(K168D). In still a further embodiment, the alphaamylase variant can include, at position 168, the amino acid residue S (K168S). Embodiments of alpha-amylase variants having K168S include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 150, or 152, as well as variants thereof. In still a further embodiment, the alpha-amylase variant can include, at position 168, the amino acid residue D (K168D). Embodiments of alpha-amylase variants having K168D include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 156, 160, or 162 as well as variants thereof. In some embodiments, the alpha-amylase variant comprises, at position 174, a naturally occurring amino acid residue that is different from K. For example, the alpha-amylase variant can include, at position 174, one of the following amino acid residues:

[0151]

[0152] V (K174V), W (K174W), or Y (K174Y). In another example, the alpha-amylase variant can include, at position 174, one of the following amino acid residues: Q (K174Q), N (K174N), Y (K174Y), or R (K174R). In a further example, the alpha-amylase variant can include, at position 174, the following amino acid residue: Q (K174Q). Embodiments of alpha-amylase variants having K174Q include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 30, 36, 40, 42, 44, 46, 48, 52, 56, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162 as well as variants thereof.

[0153] In some embodiments, the alpha-amylase variant comprises, at position 178, a naturally occurring amino acid residue that is different from K. For example, the alpha-amylase variant can include, at position 178, one of the following amino acid residues: A (K178A), C (K178C),

[0154]

[0155] or Y (K178Y). In another example, the alpha-amylase variant can include, at position 178, one of the following amino acid residues: S (K178S), R (K178R), Q (K178Q), or A (K178A). In a further example, the alpha-amylase variant can include, at position 178, the following amino acid residue: S (K178S). Embodiments of alpha-amylase variants having K178S include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 32, 38, 40, 42, 46, 50, 54, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162 as well as variants thereof. In some embodiments, the alpha-amylase variant comprises, at position 181 , a naturally occurring amino acid residue that is different from M. For example, the alpha-amylase variant can include, at position 181 , one of the following amino acid residues: A (M181A), C (M181C),

[0156]

[0157] (M181W), orY (M181Y). For example, the alpha-amylase variant can include, at position 181 , one of the following amino acid residues: V (M181V), I (M181 I), or L (M181L). In a further example, the alpha-amylase variant can include, at position 181 , the following amino acid residue: V (M181V). Embodiments of alpha-amylase variants having M181V include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 32, 38, 40, 42, 46, 50, 54, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162 as well as variants thereof. In some embodiments, the alpha-amylase variant comprises, at position 183, a naturally occurring amino acid residue that is different from T. For example, the alpha-amylase variant can include, at position 183, one of the following amino acid residues: A (T183A), C (T183C),

[0158]

[0159] orY (T183Y). In a further example, the alpha-amylase variant can include, at position 183, the following amino acid residue: E (T183E). Embodiments of an alpha-amylase variant having T183E have the amino acid sequence of SEQ ID NO: 144, or 146 as well as variants thereof. In some embodiments, the alpha-amylase variant comprises, at position 185, a naturally occurring amino acid residue that is different from K. For example, the alpha-amylase variant can comprise, at position 185, one of the following amino acid residues: A (K185A), C (K185C),

[0160]

[0161] orY (K185Y). In a further example, the alpha-amylase variant can include, at position 185, the following amino acid residue: S (K185S). Embodiments of alpha-amylase variants having K185S include, but are not limited to, the polypeptides having the amino acid sequence of SEQ ID NO: 156, 158, 160, or 162 as well as variants thereof.

[0162] In some embodiments, the alpha-amylase variant comprises, at position 204, a naturally occurring amino acid residue that is different from D. For example, the alpha-amylase variant can include, at position 204, one of the following amino acid residues: A (D204A), C (D204C),

[0163]

[0164] (D204W), orY (D204Y). In another example, the alpha-amylase variant can include, at position 204, one of the following amino acid residues: G (D204G), E (D204E), P (D204P), or K (D204K). For example, the alpha-amylase variant can include, at position 204, the following amino acid residue: G (D204G). Embodiments of alpha-amylase variants having D204G include, but are not limited to, the polypeptides having the amino acid sequence of SEQ ID NO: 32, 38, 40, 42, 46, 50, 54, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162 as well as variants thereof.In some embodiments, the alpha-amylase variant comprises, at position 207, a naturally occurring amino acid residue that is different from W. For example, the alpha-amylase variant can include, at position 207, one of the following amino acid residues: A (W207A), C (W207C),

[0165]

[0166] (W207T), V (W207V), or Y (W207Y). In another example, the alpha-amylase variant can include, at position 207, one of the following amino acid residues: R (W207R), S (W207S), Q (W207Q), K (W207K), or A (W207A), or E (W207E). In a further example, the alpha-amylase variant can include, at position 207, the following amino acid residue: R (W207R). Embodiments of alpha-amylase variants having W207R include, but are not limited to, the polypeptides having the amino acid sequence of SEQ ID NO: 12, 14, 36, 38, 40, 106, 108, 112, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, or 154 as well as variants thereof. In a further example, the alpha-amylase variant can include, at position 207, the following amino acid residue: E (W207E). Embodiments of alphaamylase variants having W207E include, but are not limited to, the polypeptides having the amino acid sequence of SEQ ID NO: 110, 114, 156, 158, 160, or 162 as well as variants thereof. In still another embodiment, the alpha-amylase variant of the present disclosure excludes having a substitution at position 207 (e.g., it remains a W).

[0167] In some embodiments, the alpha-amylase variant comprises, at position 208, a naturally occurring amino acid residue that is different from A. For example, the alpha-amylase variant can include, at position 208, one of the following amino acid residues: C (A208C), D (A208D), E (A208E), F (A208F), G (A208G), H (A208H), I (A208I), K (A208K), L (A208L), M (A208M), N (A208N), P (A208P), Q (A208Q), R (A208R), S (A208S), T (A208T), V (A208V), W (A208W), orY (A208Y). In another example, the alpha-amylase variant can include, at position 208, the following amino acid residue: E (A208E). An embodiment of an alpha-amylase variant including A208E is a polypeptide having the amino acid sequence of SEQ ID NO: 56 as well as variants thereof.

[0168] In some embodiments, the alpha-amylase variant comprises, at position 209, a naturally occurring amino acid residue that is different from S. For example, the alpha-amylase variant can include, at position 209, one of the following amino acid residues: A (S209A), C (S209C), D (S209D), E (S209E), F (S209F), G (S209G), H (S209H), I (S209I), K (S209K), L (S209L), M (S209M), N (S209N), P (S209P), Q (S209Q), R (S209R), T (S209T), V (S209V), W (S209W), or Y (S209Y). In another example, the alpha-amylase variant can include, at position 209, one of the following amino acid residues: A (S209A), T (S209T), orG (S209G). In a further example, the alpha-amylase variant can include, at position 209, the following amino acid residue: A (S209A). Embodiments of alpha-amylase variants having S209A include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 30, 32, 34, 36, 38, 40, 42, 48,50, 52, 54, 56, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162 as well as variants thereof. In some embodiments, the alpha-amylase variant comprises, at position 211 , a naturally occurring amino acid residue that is different from S. For example, the alpha-amylase variant can include, at position 211 , one of the following amino acid residues: A (S211A), C (S211C),

[0169]

[0170] or Y (S211Y). In another example, the alpha-amylase variant can include, at position 211 , one of the following amino acid residues: V (S211 V), A (S211 A), I (S2111), or F (S211F). In a further example, the alpha-amylase variant can include, at position 211 , the following amino acid residue: V (S211V). Embodiments of alpha-amylase variants having S211V include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 30, 32, 34, 36, 38, 40, 42, 48, 50, 52, 54, 56, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162 as well as variants thereof.

[0171] In some embodiments, the alpha-amylase variant comprises, at position 219, a naturally occurring amino acid residue that is different from S. For example, the alpha-amylase variant can include, at position 219, one of the following amino acid residues: A (S219A), C (S219C),

[0172]

[0173] or Y (S219Y). In another example, the alpha-amylase variant can include, at position 219, one of the following amino acid residues: D (S219D), N (S219N), H (S219H), E (S219E), or T (S219T). In a further example, the alpha-amylase variant can include, at position 219, the following amino acid residue: D (S219D). Embodiments of alpha-amylase variants having S219D include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 32, 38, 40, 42, 46, 50, 54, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162 as well as variants thereof.

[0174] In some embodiments, the alpha-amylase variant comprises, at position 230, a naturally occurring amino acid residue that is different from K. For example, the alpha-amylase variant can comprise, at position 230, one of the following amino acid residues: A (K230A), C (K230C), D (K230D), E (K230E), F (K230F), G (K230G), H (K230H), I (K230I), L (K230L), M (K230M), N (K230N), P (K230P), Q (K230Q),R (K230R), S (K230S), T (K230T), V (K230V), W (K230W), or Y (K230Y). In a further example, the alpha-amylase variant can include, at position 230, the following amino acid residue: G (K230G). Embodiments of alpha-amylase variants having K230G include, but are not limited to, the polypeptides having the amino acid sequence of SEQ ID NO: 150, or 152 as well as variants thereof.In some embodiments, the alpha-amylase variant comprises, at position 233, a naturally occurring amino acid residue that is different from G. For example, the alpha-amylase variant can include, at position 233, one of the following amino acid residues: A (G233A), C (G233C),

[0175]

[0176] (G233W), orY (G233Y). In another example, the alpha-amylase variant can include, at position 233, one of the following amino acid residues: D (G233D), S (G233S), P (G233P), or A (G233A). In a further example, the alpha-amylase variant can include, at position 233, the following amino acid residue: D (G233D). Embodiments of alpha-amylases having G233D include, but are not limited to, the polypeptides having the amino acid sequence of SEQ ID NO: 32, 38, 40, 42, 46, 50, 54, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162 as well as variants thereof.

[0177] In some embodiments, the alpha-amylase variant comprises, at position 242 a naturally occurring amino acid residue that is different from F. For example, the alpha-amylase variant can include, at position 242, one of the following amino acid residues: A (F242A), C (F242C), D (F242D), E (F242E), G (F242G), H (F242H), I (F242I), K (F242K), L (F242L), M (F242M), N (F242N), P (F242P), Q (F242Q), R (F242R), S (F242S), T (F242T), V (F242V), W (F242W), or Y (F242Y). In another example, the alpha-amylase variant can include, at position 242, one of the following amino acid residues: Y (F242Y), W (F242W), K (F242K), or D (F242D). In a further example, the alpha-amylase variant can include, at position 242, the following amino acid residue: Y (F242Y). Embodiments of alpha-amylase variants having F242Y include, but are not limited to, the polypeptides having the amino acid sequence of SEQ ID NO: 32, 38, 40, 42, 50, 54, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162 as well as variants thereof. In some embodiments, the alpha-amylase variant comprises, at position 246, a naturally occurring amino acid residue that is different from R. In an example, the alpha-amylase variant can comprise, at position 246, one of the following amino acid residues: A (R246A), C (R246C),

[0178]

[0179] (R246W) orY (R246Y). In a further example, the alpha-amylase variant can include, at position 246, the following amino acid residue: D (R246D). Embodiments of alpha-amylase variants having R246D include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 158, 160, or 162 as well as variants thereof.

[0180] In some embodiments, the alpha-amylase variant comprises, at position 252 a naturally occurring amino acid residue that is different from N. For example, the alpha-amylase variant can include, at position 252, one of the following amino acid residues: A (N252A), C (N252C),

[0181]

[0182] (N252W), orY (N252Y). In another example, the alpha-amylase variant can include, at position 252, one of the following amino acid residues: G (N252G), S (N252S), D (N252D), or Q (N252Q). In a further example, the alpha-amylase variant can include, at position 252, the following amino acid residue: D (N252D). Embodiments of alpha-amylase variants having N252G include, but are not limited to, the polypeptide having the amino acid sequence of SEQ ID NO: 10 as well as variants thereof. In a further example, the alpha-amylase variant can include, at position 252, the following amino acid residue: G (N252G). Embodiments of alphaamylase variants having N252G include, but are not limited to, the polypeptides having the amino acid sequence of SEQ ID NO: 30, 32, 34, 36, 38, 40, 42, 48, 50, 52, 54, 56, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162 as well as variants thereof. In still another embodiment, the alpha-amylase variant of the present disclosure excludes having a substitution at position 252 (e.g., it remains a N).

[0183] In some embodiments, the alpha-amylase variant comprises, at position 261 , a naturally occurring amino acid residue that is different from H. For example, the alpha-amylase variant can include, at position 261 , one of the following amino acid residues: A (H261 A), C (H261C),

[0184]

[0185] (H261 W), orY (H261Y). In another example, the alpha-amylase variant can include, at position 261 , the following amino acid residue: L (H261 L). Embodiments of alpha-amylase variants having H261L include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 156, 158, 160, or 162 as well as variants thereof.

[0186] In some embodiments, the alpha-amylase variant comprises, at position 268 a naturally occurring amino acid residue that is different from V. For example, the alpha-amylase variant can include, at position 268, one of the following amino acid residues: A (V268A), C (V268C), D (V268D), E (V268E), F (V268F), G (V268G), H (V268H), I (V268I), K (V268K), L (V268L), M (V268M), N (V268N), P (V268P), Q (V268Q), R (V268R), S (V268S), T (V268T), W (V268W), orY (V268Y). In another example, the alpha-amylase variant can include, at position 268, one of the following amino acid residues: F (V268F), Y (V268Y), L (V268L), M (V268M), or I (V268I). In a further example, the alpha-amylase variant can include, at position 268, the following amino acid residue: F (V268F). Embodiments of alpha-amylase variants having V268F include, but are not limited to, the polypeptides having the amino acid sequence of SEQ ID NO: 30, 32, 34, 36, 38, 40, 42, 48, 50, 52, 54, 56, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162 as well as variants thereof.In some embodiments, the alpha-amylase variant comprises, at position 273 a naturally occurring amino acid residue that is different from T. For example, the alpha-amylase variant can include, at position 273, one of the following amino acid residues: A (T273A), C (T273C), D (T273D), E (T273E), F (T273F), G (T273G), H (T273H), I (T273I), K (T273K), L (T273L), M (T273M), N (T273N), P (T273P), Q (T273Q), R (T273R), S (T273S), V (T273V), W (T273W), or Y (T273Y). In another example, the alpha-amylase variant can include, at position 273, one of the following amino acid residues: A (T273A), V (T273V), L (T273L), I (T273I), or S (T273S). In a further example, the alpha-amylase variant can include, at position 273, the following amino acid residue: A (T273A). Embodiments of alpha-amylase variants having T273A include, but are not limited to, the polypeptide having the amino acid sequence of SEQ ID NO: 32, 38, 40, 42, 46, 50, 54, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162 as well as variants thereof.

[0187] In some embodiments, the alpha-amylase variant comprises, at position 301 a naturally occurring amino acid residue that is different from T. For example, the alpha-amylase variant can include, at position 301 , one of the following amino acid residues: A (T301A), C (T301C),

[0188]

[0189] or Y (T301 Y). In another example, the alpha-amylase variant can include, at position 301 , one of the following amino acid residues: A (T301A), S (T301S), V (T301V), or G (T301G). In a further example, the alpha-amylase variant can include, at position 301 , the following amino acid residue: A (T301 A). Embodiments of alpha-amylases having T301A include, but are not limited to, the polypeptides having the amino acid sequence of SEQ ID NO: 32, 38, 40, 42, 46, 50, 54, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162 as well as variants thereof. In some embodiments, the alpha-amylase variant comprises, at position 319 a naturally occurring amino acid residue that is different from A. For example, the alpha-amylase variant can include, at position 319, one of the following amino acid residues: C (A319C), D (A319D),

[0190]

[0191] or Y (A319Y). In another example, the alpha-amylase variant can include, at position 319, one of the following amino acid residues: G (A319G), S (A319S), or E (A319E). In a further example, the alpha-amylase variant can include, at position 319, the following amino acid residue: G (A319G). Embodiments of alpha-amylase variants having A319G include, but are not limited to, the polypeptides having the amino acid sequence of SEQ ID NO: 32, 38, 40, 42, 46, 50, 54, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162 as well as variants thereof.In some embodiments, the alpha-amylase variant comprises, at position 327 a naturally occurring amino acid residue that is different from Q. For example, the alpha-amylase variant can include, at position 327, one of the following amino acid residues: A (Q327A), C (Q327C), D (Q327D), E (Q327E), F (Q327F), G (Q327G), H (Q327H), I (Q327I), K (Q327K), L (Q327L), M (Q327M), N (Q327N), P (Q327P), R (Q327R), S (Q327S), T (Q327T), V (Q327V), W (Q327W), orY (Q327Y). In another example, the alpha-amylase variant can include, at position 327, one of the following amino acid residues: A (Q327A), E (Q327E), G (Q327G), or N (Q327N). In a further example, the alpha-amylase variant can include, at position 327, the following amino acid residue: A (Q327A). Embodiments of alpha-amylase variants having Q327A include, but are not limited to, the polypeptides having the amino acid sequence of SEQ ID NO: 32, 38, 40, 42, 46, 50, 54, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162 as well as variants thereof.

[0192] In some embodiments, the alpha-amylase variant comprises, at position 334 a naturally occurring amino acid residue that is different from R. For example, the alpha-amylase variant can include, at position 334, one of the following amino acid residues: A (R334A), C (R334C),

[0193]

[0194] (R334W), or Y (R334Y). In a further example, the alpha-amylase variant can include, at position 334, the following amino acid residue: T (R334T). An embodiment of an alpha-amylase variant having R334T include, but is not limited to, the polypeptide having the amino acid sequence of SEQ ID NO: 162 as well as variants thereof.

[0195] In some embodiments, the alpha-amylase variant comprises, at position 336 a naturally occurring amino acid residue that is different from R. For example, the alpha-amylase variant can include, at position 336, one of the following amino acid residues: A (R336A), C (R336C),

[0196]

[0197] (R336W), orY (R336Y). In another example, the alpha-amylase variant can include, at position 336, one of the following amino acid residues: G (R336G), N (R336N), S (R336S), or K (R336K). In a further example, the alpha-amylase variant can include, at position 336, the following amino acid residue: G (R336G). Embodiments of alpha-amylase variants having R336G include, but are not limited to, the polypeptides having the amino acid sequence of SEQ ID NO: 30, 36, 40, 42, 48, 50, 52, 54, 56, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162 as well as variants thereof.

[0198] In some embodiments, the alpha-amylase variant comprises, at position 338 a naturally occurring amino acid residue that is different from N. For example, the alpha-amylase variantcan include, at position 338, one of the following amino acid residues: A (N338A), C (N338C),

[0199]

[0200] (N338W), or Y (N338Y). In a further example, the alpha-amylase variant can include, at position 338, following amino acid residue: D (N338D). Embodiments of alpha-amylase variants having N338D include, but are not limited to, the polypeptides having the amino acid sequence of SEQ ID NO: 158, 160, or 162 as well as variants thereof.

[0201] In some embodiments, the alpha-amylase variant comprises, at position 343 a naturally occurring amino acid residue that is different from M. For example, the alpha-amylase variant can include, at position 343, one of the following amino acid residues: A (M343A), C (M343C),

[0202]

[0203] (M343W), or Y (M343Y). In another example, the alpha-amylase variant can include, at position 343, one of the following amino acid residues: L (M343L), V (M343V), or I (M343I). In a further example, the alpha-amylase variant can include, at position 343, following amino acid residue: L (M343L). Embodiments of alpha-amylase variants having M343L include, but are not limited to, the polypeptides having the amino acid sequence of SEQ ID NO: 32, 38, 40, 42, 46, 50, 54, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162 as well as variants thereof. In some embodiments, the alpha-amylase variant comprises, at position 345, a naturally occurring amino acid residue that is different from K. For example, the alpha-amylase variant can comprise, at position 345, one of the following amino acid residues: A (K345A), C (K345C), D (K345D), E (K345E), F (K345F), G (K345G), H (K345H), I (K345I), L (K345L), M (K345M), N (K345N), P (K345P), Q (K345Q),R (K345R), S (K345S), T (K345T), V (K345V), W (K345W), or Y (K345Y). In a further example, the alpha-amylase variant can include, at position 345, the following amino acid residue: Q (K345Q). Embodiments of alpha-amylase variants having K345Q include, but are not limited to, the polypeptides having the amino acid sequence of SEQ ID NO: 158, 160, or 162 as well as variants thereof.

[0204] In some embodiments, the alpha-amylase variant comprises, at position 347 a naturally occurring amino acid residue that is different from F. For example, the alpha-amylase variant can include, at position 347, one of the following amino acid residues: A (F347A), C (F347C), D (F347D), E (F347E), G (F347G), H (F347H), I (F347I), K (F347K), L (F347L), M (F347M), N (F347N), P (F347P), Q (F347Q), R (F347R), S (F347S), T (F347T), V (F347V), W (F347W), or Y (F347Y). For example, the alpha-amylase variant can include, at position 347, one of the following amino acid residues: I (F347I), L (F347L), V (F347V), or T (F347T). In a further example, the alpha-amylase variant can include, at position 347, the following amino acid residue: I (F347I). Embodiments of alpha-amylase variants having F347I include, but are notlimited to, the polypeptides having the amino acid sequence of SEQ ID NO: 32, 38, 40, 42, 46, 50, 54, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 148, 150, 152, 154, 156, 158, 160, or 162 as well as variants thereof. In a further example, the alpha-amylase variant can include, at position 347, the following amino acid residue: L (F347L). An embodiment of an alpha-amylase variant having F347L includes a polypeptide having the amino acid sequence of SEQ ID NO: 4, 144, or 146 as well as variants thereof. In still another embodiment, the alpha-amylase variant of the present disclosure excludes having a substitution at position 347 (e.g., it remains a F).

[0205] In some embodiments, the alpha-amylase variant comprises, at position 348 a naturally occurring amino acid residue that is different from Q. For example, the alpha-amylase variant can include, at position 348, one of the following amino acid residues: A (Q348A), C (Q348C), D (Q348D), E (Q348E), F (Q348F), G (Q348G), H (Q348H), I (Q348I), K (Q348K), L (Q348L), M (Q348M), N (Q348N), P (Q348P), R (Q348R), S (Q348S), T (Q348T), V (Q348V), W (Q348W), orY (Q348Y). In another example, the alpha-amylase variant can include, at position 348, one of the following amino acid residues: A (Q348A), S (Q348S), K (Q348K), orT (Q348T). In a further example, the alpha-amylase variant can include, at position 348, the following amino acid residue: A (Q348A). Embodiments of the alpha-amylase variants having Q348A include, but are not limited to, the polypeptides having the amino acid sequence of SEQ ID NO: 30, 36, 40, 42, 44, 46, 48, 52, 56, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162 as well as variants thereof.

[0206] In some embodiments, the alpha-amylase variant comprises, at position 357 a naturally occurring amino acid residue that is different from H. For example, the alpha-amylase variant can include, at position 357, one of the following amino acid residues: A (H357A), C (H357C),

[0207]

[0208] (H357W), orY (H357Y). For example, the alpha-amylase variant can include, at position 357, one of the following amino acid residues: A (H357A), V (H357V), or N (H357N). In a further example, the alpha-amylase variant can include, at position 357, the following amino acid residue: A (H357A). Embodiments of alpha-amylase variants having H357A include, but are not limited to, the polypeptides having the amino acid sequence of SEQ ID NO: 30, 36, 40, 42, 48, 50, 52, 54, 56, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162 as well as variants thereof.

[0209] In some embodiments, the alpha-amylase variant comprises, at position 369 a naturally occurring amino acid residue that is different from F. For example, the alpha-amylase variant can include, at position 369, one of the following amino acid residues: A (F369A), C (F369C),D (F369D), E (F369E), G (F369G), H (F369H), I (F369I), K (F369K), L (F369L), M (F369M), N (F369N), P (F369P), Q (F369Q), R (F369R), S (F369S), T (F369T), V (F369V), W (F369W), or Y (F369Y). In another example, the alpha-amylase variant can include, at position 369, one of the following amino acid residues: Y (F369Y), H (F369H), L (F369L), or M (F369M). In a further example, the alpha-amylase variant can include, at position 369, the following amino acid residue: Y (F369Y). Embodiments of alpha-amylase variants having F369Y include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 32, 38, 40, 42, 46, 50, 54, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162 as well as variants thereof. In some embodiments, the alpha-amylase variant comprises, at position 372 a naturally occurring amino acid residue that is different from S. For example, the alpha-amylase variant can include, at position 372, one of the following amino acid residues: A (S372A), C (S372C), D (S372D), E (S372E), F (S372F), G (S372G), H (S372H), I (S372I), K (S372K), L (S372L), M (S372M), N (S372N), P (S372P), Q (S372Q), R (S372R), T (S372T), V (S372V), W (S372W), or Y (S372Y). In another example, the alpha-amylase variant can include, at position 372, one of the following amino acid residues: D (S372D), N (S372N), E (S372E), G (S372G), or A (S372A). In a further example, the alpha-amylase variant can include, at position 372, the following amino acid residue: D (S372D). Embodiments of alpha-amylase variants having S372D include, but are not limited to, the polypeptides having the amino acid sequence of SEQ ID NO: 32, 38, 40, 42, 46, 50, 54, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162 as well as variants thereof.

[0210] In some embodiments, the alpha-amylase variant comprises, at position 373 a naturally occurring amino acid residue that is different from D. For example, the alpha-amylase variant can include, at position 373, one of the following amino acid residues: A (D373A), C (D373C),

[0211]

[0212] (D373W), orY (D373Y). In another example, the alpha-amylase variant can include, at position 373, one of the following amino acid residues: T (D373T), N (D373N), G (D373G), or S (D373S). In a further example, the alpha-amylase variant can include, at position 373, the following amino acid residue: T (D373T). Embodiments of alpha-amylase variants having D373T include, but are not limited to, the polypeptides having the amino acid sequence of SEQ ID NO: 32, 38, 40, 42, 46, 50, 54, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162 as well as variants thereof.

[0213] In some embodiments, the alpha-amylase variant comprises, at position 380 a naturally occurring amino acid residue that is different from Q. For example, the alpha-amylase variantcan include, at position 380, one of the following amino acid residues: A (Q380A), C (Q380C), D (Q380D), E (Q380E), F (Q380F), G (Q380G), H (Q380H), I (Q380I), K (Q380K), L (Q380L), M (Q380M), N (Q380N), P (Q380P), R (Q380R), S (Q380S), T (Q380T), V (Q380V), W (Q380W), orY (Q380Y). In another example, the alpha-amylase variant can include, at position 380, one of the following amino acid residues: A (Q380A), E (Q380E), G (Q380G), or D (Q380D). In a further example, the alpha-amylase variant can include, at position 380, the following amino acid residue: A (Q380A). Embodiments of alpha-amylase variants having Q380A including, but are not limited to, the polypeptides having the amino acid sequence of SEQ ID NO: 30, 36, 40, 42, 48, 50, 52, 54, or 56, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 150, 154, 156, 158, 160 or 162as well as variants thereof. In a further example, the alpha-amylase variant can include, at position 380, the following amino acid residue: E (Q380E). Embodiments of alpha-amylase variants having Q380E including, but are not limited to, the polypeptides having the amino acid sequence of SEQ ID NO: 148, or 152 as well as variants thereof. In still another embodiment, the alpha-amylase variant of the present disclosure excludes having a substitution at position 380 (e.g., it remains a Q). In some embodiments, the alpha-amylase variant comprises, at position 383 a naturally occurring amino acid residue that is different from A. For example, the alpha-amylase variant can include, at position 383, one of the following amino acid residues: C (A383C), D (A383D), E (A383E), F (A383F), G (A383G), H (A383H), I (A383I), K (A383K), L (A383L), M (A383M), N (A383N), P (A383P), Q (A383Q), R (A383R), S (A383S), T (A383T), V (A383V), W (A383W), or Y (A383Y). In another example, the alpha-amylase variant can include, at position 383, one of the following amino acid residues: D (A383D), K (A383K), R (A383R), N (A383N), or S (A383S). In a further example, the alpha-amylase variant can include, at position 383, the following amino acid residues: D (A383D). Embodiments of alphaamylase variants having A383D include, but are not limited to, the polypeptides having the amino acid sequence of SEQ ID NO: 30, 36, 40, 42, 44, 46, 48, 52, 56, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162 as well as variants thereof.

[0214] In some embodiments, the alpha-amylase variant comprises, at position 385 a naturally occurring amino acid residue that is different from M. For example, the alpha-amylase variant can include, at position 385, one of the following amino acid residues: A (M385A), C (M385C),

[0215]

[0216] (M385W), or Y (M385Y). In another example, the alpha-amylase variant can include, at position 385, one of the following amino acid residues: I (M385I), V (M385V), L (M385L), F (M385F), or A (M385A). In a further example, the alpha-amylase variant can include, at position 385, the following amino acid residue: I (M385I). Embodiments of alpha-amylase variantshaving M385I include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 32, 38, 40, 42, 46, 50, 54, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162 as well as variants thereof.

[0217] In some embodiments, the alpha-amylase variant comprises, at position 386 a naturally occurring amino acid residue that is different from A. For example, the alpha-amylase variant can include, at position 386, one of the following amino acid residues: C (A386C), D (A386D), E (A386E), F (A386F), G (A386G), H (A386H), I (A386I), K (A386K), L (A386L), M (A386M), N (A386N), P (A386P), Q (A386Q), R (A386R), S (A386S), T (A386T), V (A386V), W (A386W), or Y (A386Y). In another example, the alpha-amylase variant can include, at position 386, one of the following amino acid residues: V (A386V), I (A386I), T (A386T), or S (A386S). In a further example, the alpha-amylase variant can include, at position 386, the following amino acid residue: V (A386V). Embodiments of alpha-amylase variants having A386V include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 32, 38, 40, 42, 46, 50, 54, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162 as well as variants thereof. In some embodiments, the alpha-amylase variant comprises, at position 393 a naturally occurring amino acid residue that is different from K. For example, the alpha-amylase variant can include, at position 393, one of the following amino acid residues: A (K393A), C (K393C), D (K393D), E (K393E), F (K393F), G (K393G), H (K393H), I (K393I), L (K393L), M (K393M), N (K393N), P (K393P), Q (K393Q), R (K393R), S (K393S), T (K393T), V (K393V), W (K393W), or Y (K393Y). In another example, the alpha-amylase variant can include, at position 393, one of the following amino acid residues: S (K393S), Q (K393Q), R (K393R), or A (K393A). In a further example, the alpha-amylase variant can include, at position 393, the following amino acid residue: S (K393S). Embodiments of alpha-amylase variants having K393S include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 30, 32, 34, 36, 38, 40, 42, 48, 50, 52, 54, 56, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162 as well as variants thereof.

[0218] In some embodiments, the alpha-amylase variant comprises, at position 396 a naturally occurring amino acid residue that is different from Q. For example, the alpha-amylase variant can include, at position 396, one of the following amino acid residues: A (Q396A), C (Q396C), D (Q396D), E (Q396E), F (Q396F), G (Q396G), H (Q396H), I (Q396I), K (Q396K), L (Q396L), M (Q396M), N (Q396N), P (Q396P), R (Q396R), S (Q396S), T (Q396T), V (Q396V), W (Q396W), orY (Q396Y). In another example, the alpha-amylase variant can include, at position 396, one of the following amino acid residues: S (Q396S), T (Q396T), N (Q396N), or G (Q396G). In a further example, the alpha-amylase variant can include, at position 396, thefollowing amino acid residue: S (Q396S). Embodiments of alpha-amylase variants having Q396S include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 32, 38, 40, 42, 46, 50, 54, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162 as well as variants thereof.

[0219] In some embodiments, the alpha-amylase variant comprises, at position 397, a naturally occurring amino acid residue that is different from R. In an example, the alpha-amylase variant can comprise, at position 397, one of the following amino acid residues: A (R397A), C (R397C),

[0220]

[0221] (R397W) orY (R397Y). In a further example, the alpha-amylase variant can include, at position 397, the following amino acid residue: N (R397N). An embodiment of an alpha-amylase variant having R397N includes, but is not limited to, a polypeptide having the amino acid sequence of SEQ ID NO: 154 as well as variants thereof. In some embodiments, the alpha-amylase variant comprises, at position 402 a naturally occurring amino acid residue that is different from F. For example, the alpha-amylase variant can include, at position 402, one of the following amino acid residues: K (F402K), L (

[0222]

[0223] T (F402T), V (F402V), W (F402W), or Y (F402Y). In another example, the alpha-amylase variant can include, at position 402, one of the following amino acid residues: N (F402N), S (F402S), D (F402D), G (F402G), orT (F402T). In a further example, the alpha-amylase variant can include, at position 402, the following amino acid residue: N (F402N). Embodiments of alpha-amylase variants having F402N include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 30, 36, 40, 42, 44, 46, 48, 52, 56, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162 as well as variants thereof.

[0224] In some embodiments, the alpha-amylase variant comprises, at position 407, a naturally occurring amino acid residue that is different from N. For example, the alpha-amylase variant can comprise, at position 407, on of the following amino acid residue: A (N407A), C (N407C),

[0225]

[0226] (N407W), or Y (N407Y). In another example, the alpha-amylase variant can include, at position 407, the following amino acid residue: G (N407G) or F (N407F). In a further example, the alpha-amylase variant can include, at position 407, the following amino acid residue: G (N407G). Embodiments of alpha-amylase variants having N407G include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 134, or 136 as well as variants thereof. In a further example, the alpha-amylase variant can include, at position 407, thefollowing amino acid residue: F (N407F). An embodiment of an alpha-amylase variant having N407F includes, but is not limited to, polypeptides having the amino acid sequence of SEQ ID NO142 as well as variants thereof.

[0227] In some embodiments, the alpha-amylase variant comprises, at position 408, a naturally occurring amino acid residue that is different from K. For example, the alpha-amylase variant can comprise, at position 408, one of the following amino acid residues: A (K408A), C (K408C), D (K408D), E (K408E), F (K408F), G (K408G), H (K408H), I (K408I), L (K408L), M (K408M), N (K408N), P (K408P), Q (K408Q),R (K408R), S (K408S), T (K408T), V (K408V), W (K408W), or Y (K408Y). In a further example, the alpha-amylase variant can include, at position 408, the following amino acid residue: D (K408D). An embodiment of an alpha-amylase variants having K408D includes, but is not limited to, the polypeptide having the amino acid sequence of SEQ ID NO: 154 as well as variants thereof.

[0228] In some embodiments, the alpha-amylase variant comprises, at position 415, a naturally occurring amino acid residue that is different from R. In an example, the alpha-amylase variant can comprise, at position 415, one of the following amino acid residues: A (R415A), C (R415C),

[0229]

[0230] (R415W) orY (R415Y). In a further example, the alpha-amylase variant can include, at position 415, the following amino acid residue: M (R415M). Embodiments of alpha-amylase variants having R415M include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 154 as well as variants thereof.

[0231] In some embodiments, the alpha-amylase variant comprises, at position 416 a naturally occurring amino acid residue that is different from G. For example, the alpha-amylase variant can include, at position 416, one of the following amino acid residues: A (G416A), C (G416C),

[0232]

[0233] (G416W), or Y (G416Y). In a further example, the alpha-amylase variant can include, at position 416, the following amino acid residue: D (G416D). Embodiments of alpha-amylase variants having G416D include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 154 as well as variants thereof.

[0234] In some embodiments, the alpha-amylase variant comprises, at position 422 a naturally occurring amino acid residue that is different from G. For example, the alpha-amylase variant can include, at position 422, one of the following amino acid residues: A (G422A), C (G422C), D (G422D), E (G422E), F (G422F), H (G422H), I (G422I), K (G422K), L (G422L), M (G422M), N (G422N), P (G422P), Q (G422Q), R (G422R), S (G422S), T (G422T), V (G422V), W (G422W), or Y (G422Y). In a further example, the alpha-amylase variant can include, at position 422, the following amino acid residue: D (G422D). Embodiments of alpha-amylasevariants having G422D include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 146, or 148 as well as variants thereof.

[0235] In some embodiments, the alpha-amylase variant comprises, at position 423 a naturally occurring amino acid residue that is different from G. For example, the alpha-amylase variant can include, at position 423, one of the following amino acid residues: A (G423A), C (G423C), D (G423D), E (G423E), F (G423F), H (G423H), I (G423I), K (G423K), L (G423L), M (G423M), N (G423N), P (G423P), Q (G423Q), R (G423R), S (G423S), T (G423T), V (G423V), W (G423W), orY (G423Y). In another example, the alpha-amylase variant can include, at position 423, one of the following amino acid residues: S (G423S), N (G423N), K (G423K), A (G423A), E (G423E), or F (G423F). In a further example, the alpha-amylase variant can include, at position 423, the following amino acid residue: S (G423S). Embodiments of alpha-amylase variants having G423S include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 14, 36, 38, 40, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 144, 146, 148, 150, 152, 154, 156, 158, 160, or 162 as well as variants thereof. In a further example, the alpha-amylase variant can include, at position 423, the following amino acid residue: F (G423F). Embodiments of alpha-amylase variants having G423F include, but are not limited to, polypeptides having the amino acid sequence of SEQ ID NO: 142 as well as variants thereof. In still another embodiment, the alpha-amylase variant of the present disclosure excludes having a substitution at position 423 (e.g., it remains a G).

[0236] In some embodiments, the alpha-amylase variant comprises, at position 426, a naturally occurring amino acid residue that is different from K. For example, the alpha-amylase variant can comprise, at position 426, one of the following amino acid residues: A (K426A), C (K426C), D (K426D), E (K426E), F (K426F), G (K426G), H (K426H), I (K426I), L (K426L), M (K426M), N (K426N), P (K426P), Q (K426Q),R (K426R), S (K426S), T (K426T), V (K426V), W (K426W), or Y (K426Y). In a further example, the alpha-amylase variant can include, at position 426, the following amino acid residue: T (K426T). An embodiment of an alpha-amylase variants having K426T includes, but is not limited to, the polypeptide having the amino acid sequence of SEQ ID NO: 154 as well as variants thereof.

[0237] In some embodiments, the alpha-amylase variant comprises, at position 442 a naturally occurring amino acid residue that is different from Q. For example, the alpha-amylase variant can include, at position 442, one of the following amino acid residues: A (Q442A), C (Q442C), D (Q442D), E (Q442E), F (Q442F), G (Q442G), H (Q442H), I (Q442I), K (Q442K), L (Q442L), M (Q442M), N (Q442N), P (Q442P), R (Q442R), S (Q442S), T (Q442T), V (Q442V), W (Q442W), orY (Q442Y). In another example, the alpha-amylase variant can include, at position 442, the following amino acid residue: N (Q442N). An embodiment of an alpha-amylase varianthaving Q442N is a polypeptide having the amino acid sequence of SEQ ID NO: 6 as well as variants thereof.

[0238] In some embodiments, the alpha-amylase comprises one or more deletions. For example, the alpha-amylase variant of the present disclosure can include a deletion of position 355 (alone or in combination with other deletions).

[0239] Alpha-amylase variants of the present disclosure can include at least one of the following amino acid substitutions (and in some embodiments any combination of the following amino acid substitutions): K43Q / A / R / E, R71G / N / D / A, S79Q / K / T / R / N , N80D / S / H, T84I / V / L, S91A / E / P, E93D / S / Q, G96S / D / N / A, G103E / S / A / N / Q, I106M / F / V / L, V117M / A / T, N119P / S / A / Q, E124D / S / T, M125N / T / K / F / S, G133A / D / E / Q / S, G141D / E / T / Q / K, N143D / S / Q / K, D158G / S / E, K174Q / N / Y / R, K178S / R / Q / A, M181 V / l / L, D204G / E / P / K, W207R / S / Q / K / A / E, S209A / T / G, S211V / A / I / F, S219D / N / H / E / T, G233D / S / P / A, F242Y / W / K / D, N252G / S / D / Q, V268F / Y / M / L / I, T273A / V / L / I / S, T301A / SA / / G, A319G / S / E, Q327A / E / G / N, R336G / N / S / K, M343L / V / I, F347I / L / V / T, Q348A / S / K / T, H357A / V / N, F369Y / H / L / M, S372D / N / E / G / A, D373T / N / G / S, Q380A / E / G / D, A383D / K / R / N / S, M385IA / / L / F / A, A386V / I / T / S, K393S / Q / R / A, Q396S / T / N / G, F402N / S / D / G / T, and / or G423S / N / K / A / E / F. In some embodiments, alpha-amylase variants of the present disclosure include at least one of the following amino acid substitutions (and in some embodiments any combination of the following amino acid substitutions) : K43Q, R71G, S79Q, N80D, T84I, S91A, E93D, G96S, G103E, I106M, V117M, N119P, E124D, M125N, G133A, G141D, N143D, D158G, K174Q, K178S, M181V, D204G, W207R, S209A, S211V, S219D, G233D, F242Y, N252G, V268F, T273A, T301A, A319G, Q327A, R336G, M343L, F347I, Q348A, H357A, F369Y, S372D, D373T, Q380A, A383D, M385I, A386V, K393S, Q396S, F402N, and / or G423S.

[0240] The parental alpha-amylase (having the amino acid sequence of SEQ ID NO: 2) includes conserved amino acid residues which are involved, amongst other things, in substrate binding and catalytic activity which should preferably not be substituted. Conserved amino acid residues of the parental alpha-amylase include, but are not limited to Y12, D17, R18, F19, G39, G40, G44, L49, Y51 , G56, W61 , P64, Y73, H74, G75, Y76, D80, G89, L97, H102, D111 , N115, H116, L159, D161 , V169, D187, G188, R190, D192, G215, E216, F277, N280, H281 , D282, R285, G308, P310, Y313, G315, E317, R329 and R354. Other conserved amino acid residues that have been reported in the literature include R190 involved in catalytic nucleophile, E214 involved in catalytic acid / base, H194 involved in hydrogen bonds with substrates at subsite +1 , Y142 as a sugar tongue residue, F216 as a sugar tongue residue, Y240 involved in hydrophobic interaction with reducing end of substrates, G218 involved in hydrogen bonds with reducing end of substrates, D323 involved in hydrogen bonds with substrates at subsite -2 & -3, R327 involved in hydrogen bonds with substrates at subsite -2, W375 involved in hydrogen bonds with substrates at subsite -2, N180 involved in possible N-linked glycosylation site, N144 involved in possible N-linked glycosylation site and N412 involved in possible N-linked glycosylation site. As such, in some embodiments, the alphaamylase variants of the present disclosure can also include one or more of the conserved amino acid residues which are present in the parental alpha-amylase.

[0241] In optional embodiments, the alpha-amylase variants of the present disclosure exclude an amino acid substitution at position K43. In optional embodiments, the alpha-amylase variants of the present disclosure exclude an amino acid substitution at position L45. In optional embodiments, the alpha-amylase variants of the present disclosure exclude an amino acid substitution at position R71. In optional embodiments, the alpha-amylase variants of the present disclosure exclude an amino acid substitution at position G96. In optional embodiments, the alpha-amylase variants of the present disclosure exclude an amino acid substitution at position V117. In optional embodiments, the alpha-amylase variants of the present disclosure exclude an amino acid substitution at position N119. In optional embodiments, the alpha-amylase variants of the present disclosure exclude an amino acid substitution at position G133. In optional embodiments, the alpha-amylase variants of the present disclosure exclude an amino acid substitution at position G141. In optional embodiments, the alpha-amylase variants of the present disclosure exclude an amino acid substitution at position D158. In optional embodiments, the alpha-amylase variants of the present disclosure exclude an amino acid substitution at position W207. In optional embodiments, the alpha-amylase variants of the present disclosure exclude an amino acid substitution at position G234. In optional embodiments, the alpha-amylase variants of the present disclosure exclude an amino acid substitution at position P243. In optional embodiments, the alpha-amylase variants of the present disclosure exclude an amino acid substitution at position L244. In optional embodiments, the alpha-amylase variants of the present disclosure exclude an amino acid substitution at position Y248. In optional embodiments, the alpha-amylase variants of the present disclosure exclude an amino acid substitution at position N252. In optional embodiments, the alpha-amylase variants of the present disclosure exclude an amino acid substitution at position N298. In optional embodiments, the alpha-amylase variants of the present disclosure exclude an amino acid substitution at position A299. In a particular optional embodiment, the alpha-amylase variants of the present disclosure exclude amino acid substitutions at position G234. In optional embodiments, the alpha-amylase variants of the present disclosure exclude an amino acid substitution at position F347I. In optional embodiments, the alpha-amylase variants of the present disclosure exclude an amino acid substitution at position Q380. In optional embodiments, the alpha-amylase variants of the present disclosure exclude an amino acid substitution at position G4235.The alpha-amylase variants can be designed, for example, to be secreted, and in such embodiments, it may include a signal sequence (which is intended to be cleaved upon the secretion of the alpha-amylase variant). In some embodiments, the alpha-amylase variant can be designed to be secreted in a free form (not intended to be physically associated with the recombinant microbial host cell) or in a cell-associated form (intended to remain physically associated with the recombinant microbial host cell). In some embodiments of the secreted and cell-associated alpha-amylase variants, the alpha-amylase variants can be physically tethered to the external surface of the microbial host cell, and in some embodiment, the polypeptide can include a tethering moiety to locate it to the external surface of the microbial host cell. As it is known in the art, the design of expression of the alpha-amylase variant may require adjusting the steps and parameters of the methods to be used to make such alphaamylase variants. An embodiment of a signal sequence that can be used in the alpha-amylase variants of the present disclosure includes a linker obtained or derived from Saccharomyces sp., and in some additional embodiments, from Saccharomyces cerevisiae. In a further embodiment, the signal sequence has the amino acid sequence of SEQ ID NO: 100 or is a variant thereof. In still another embodiment, the signal sequence is encoded by the nucleic acid sequence of SEQ ID NO: 99 or a degenerate sequence encoding the amino acid sequence of SEQ ID NO: 100 ora variant thereof. In another embodiment, the signal sequence has the amino acid sequence of SEQ ID NO: 172 or is a variant thereof. In still another embodiment, the signal sequence is encoded by the nucleic acid sequence of SEQ ID NO: 171 or a degenerate sequence encoding the amino acid sequence of SEQ ID NO: 172 or a variant thereof. Additional embodiments of signal sequences that can be used in the alphaamylase variants of the present disclosure include, but are not limited to, the signal sequence described in US patent 11198881 and US patent application published under 2020 / 0087672 and are herewith incorporated in their entirety.

[0242] Fusion polypeptides

[0243] The present disclosure provides fusion polypeptides comprising the alpha-amylase variant as a moiety. The fusion polypeptides, which can be referred as “AA-CBM fusions” or as “fusions” in the present disclosure, comprises at least two moieties, each moiety having a distinct function. The fusion polypeptides of the present disclosure comprise at least one moiety exhibiting alpha-amylase (AA) activity. The moiety exhibiting alpha-amylase activity is provided by the alpha-amylase variants described herein. The fusion polypeptide of the present disclosure comprises at least one moiety exhibiting carbohydrate binding properties (e.g., a carbohydrate-binding module of CBM). Thus, the fusion polypeptides of the present disclosure exhibit both alpha-amylase (AA) activity and carbohydrate binding properties. The at least two moieties of the fusion polypeptides can be joined directly to one another. Alternatively, the atleast two moieties of the fusion polypeptides can be joined indirectly to one another by the presence of a linker. In some embodiments, the linker (L, L1 or L2) can comprise one or more amino acid residues. The fusion polypeptides can include, at their amino end, the at least one moiety having alpha-amylase amylase activity. In such embodiment, the fusion polypeptides can include, at their carboxy end, the at least one carbohydrate-binding module (CBM). Alternatively, the fusion polypeptides can include, at their amino end the at least one carbohydrate-binding module (CBM). In such embodiment, the fusion polypeptides can include, at their carboxy end, the at least one moiety having alpha-amylase activity. The fusion polypeptides can include, at both the amino and their carboxy ends carbohydrate binding modules as well as an alpha-amylase moiety between two carbohydrate binding modules. Accordingly, in some embodiments, the present disclosure provides a fusion polypeptide comprising (i) the alpha-amylase variant described herein, and (ii) a carbohydrate-binding module. In some embodiments, the fusion polypeptides exhibit improved binding to carbohydrates, and as a result, improved activity on raw starch. In some embodiments, the fusion polypeptide has one of the following formulae (la) or (lb):

[0244] NH2-AA-L-(CBM)n-COOH (la), or

[0245] NH2-AA-(L-CBM)n-COOH (lb),

[0246] wherein: NH2is the amino terminus of the fusion polypeptide;

[0247] AA is the alpha-amylase moiety;

[0248] L is an optional amino acid linker;

[0249] CBM is the carbohydrate-binding module;

[0250] n is an integral number between 1 and 5 (and in some embodiments between 1 and 3); and

[0251] COOH is the carboxy terminus of the fusion polypeptide.

[0252] In other embodiments, the fusion polypeptides exhibit improved binding to carbohydrates, and as a result, improved activity on raw starch. In some embodiments, the fusion polypeptide has one of the following formulae (Ila) or (lib):

[0253] NH2-(CBM)n-L-AA-COOH (Ila), or

[0254] NH2-(CBM-L)n-AA-COOH (lib),

[0255] wherein: NH2is the amino terminus of the fusion polypeptide;

[0256] CBM is the carbohydrate-binding module;

[0257] n is an integral number between 1 and 5 (and in some embodiments between 1 and 3);

[0258] L is an optional amino acid linker;

[0259] AA is the alpha-amylase moiety; andCOOH is the carboxy terminus of the fusion polypeptide.

[0260] In further embodiments, the fusion polypeptides exhibit improved binding to carbohydrates, and as a result, improved activity on raw starch. In some embodiments, the fusion polypeptide has one of the following formulae (Illa), (lllb), (lllc) or (Hid):

[0261] NH2-(CBM)n-L1-AA-L2-(CBM)m-COOH (Illa),

[0262] NH2-(CBM-L1)n-AA-L2-(CBM)m-COOH (lllb),

[0263] NH2-(CBM)n-L1 -AA-(L2-CBM)m-COOH (lllc),

[0264] NH2-(CBM-L1)n-AA-(L2-CBM)m-COOH (Hid), wherein: NH2is the amino terminus of the fusion polypeptide;

[0265] CBM is the carbohydrate-binding module;

[0266] n and m are integral numbers independently selected between 1 and 5 (and in some embodiments selected between 1 and 3);

[0267] L1 and L2 are optional amino acid linkers;

[0268] AA is the alpha-amylase moiety; and

[0269] COOH is the carboxy terminus of the fusion polypeptide.

[0270] In the fusion polypeptides of the present disclosure, the CBM moiety can be located at the carboxy terminus (as shown in formulae (I) and (III)), at the amino terminus (as shown in formulae (II) and (III)) or both at the carboxy and amino termini (as shown in formula (III)). In some embodiments, a single CBM moiety is included in the fusion polypeptide. In other embodiments, more than one CBM moieties are included in the fusion polypeptide. In the fusion polypeptides of formulae (I) and (II), between 1 and 5 CBM moieties can be included e.g., n can be equal to 1 , 2, 3, 4, or 5). In the fusion polypeptides of formula (III), between 1 and 5 CBM moieties can be included at each position (e.g., n can be equal to 1 , 2, 3, 4, or 5 and m can be equal to 1 , 2, 3, 4, or 5).

[0271] The CBM moiety of the fusion polypeptides can be of prokaryotic origin. For example, the CBM moiety can be obtained or derived from Salinospora sp. In some embodiments, the CBM moiety can be obtained or derived from Salinospora tropica. In a further embodiment, the CBM moiety can have the amino acid sequence of SEQ ID NO: 94 or be a variant thereof having the ability to bind to a carbohydrate such as starch (and in some further embodiments raw starch). In still another embodiment, the CBM moiety can be encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 93 or be a degenerate sequence encoding the amino acid sequence of SEQ ID NO: 94 or a variant thereof. An embodiment of a fusion polypeptide having a CBM from Salinospora tropica has the amino acid sequence of SEQ ID NO: 74 or is a variant thereof having the ability to bind to a carbohydrate such as starch (and in some further embodiments raw starch). In such embodiment, the fusionpolypeptide can be encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 73 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 74 or a variant thereof.

[0272] The CBM moiety of the fusion polypeptides can be of eukaryotic origin. For example, the CBM moiety can be obtained or derived from Humicola sp. In some embodiments, the CBM moiety can be obtained or derived from Humicola grisea. In a further embodiment, the CBM moiety can have the amino acid sequence of SEQ ID NO: 78 or be a variant thereof having the ability to bind to a carbohydrate such as starch (and in some further embodiments raw starch). In still another embodiment, the CBM moiety can be encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 77 or be a degenerate sequence encoding the amino acid sequence of SEQ ID NO: 78 or a variant thereof. An embodiment of a fusion polypeptide having a CBM from Humicola grisea has the amino acid sequence of SEQ ID NO: 58 or is a variant thereof having the ability to bind to a carbohydrate such as starch (and in some further embodiments raw starch). In such embodiment, the fusion polypeptide can be encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 57 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 58 or a variant thereof. For example, the CBM moiety can be obtained or derived from Neurospora sp. In some embodiments, the CBM moiety can be obtained or derived from Neurospora crassa. In a further embodiment, the CBM moiety can have the amino acid sequence of SEQ ID NO: 80 or be a variant thereof having the ability to bind to a carbohydrate such as starch (and in some further embodiments raw starch). In still another embodiment, the CBM moiety can be encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 79 or be a degenerate sequence encoding the amino acid sequence of SEQ ID NO: 80 or a variant thereof. An embodiment of a fusion polypeptide having a CBM from Neurospora crassa has the amino acid sequence of SEQ ID NO: 60 or is a variant thereof having the ability to bind to a carbohydrate such as starch (and in some further embodiments raw starch). In such embodiment, the fusion polypeptide can be encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 59 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 60 ora variant thereof. In a further embodiment, the CBM moiety can have the amino acid sequence of SEQ ID NO: 82 or be a variant thereof having the ability to bind to a carbohydrate such as starch (and in some further embodiments raw starch). In still another embodiment, the CBM moiety can be encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 81 or be a degenerate sequence encoding the amino acid sequence of SEQ ID NO: 82 or a variant thereof. An embodiment of a fusion polypeptide having a CBM from Neurospora crassa has the amino acid sequence of SEQ ID NO: 62 or is a variant thereof having the ability to bind to a carbohydrate such as starch (and in some further embodiments raw starch). In such embodiment, the fusionpolypeptide can be encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 61 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 62 or a variant thereof. For example, the CBM moiety can be obtained or derived from Rasamsonia sp. In some embodiments, the CBM moiety can be obtained or derived from Rasamsonia emersonii. In a further embodiment, the CBM moiety can have the amino acid sequence of SEQ ID NO: 84 or be a variant thereof having the ability to bind to a carbohydrate such as starch (and in some further embodiments raw starch). In still another embodiment, the CBM moiety can be encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 83 or be a degenerate sequence encoding the amino acid sequence of SEQ ID NO: 84 or a variant thereof. An embodiment of a fusion polypeptide having a CBM from Rasamsonia emersonii has the amino acid sequence of SEQ ID NO: 64 or is a variant thereof having the ability to bind to a carbohydrate such as starch (and in some further embodiments raw starch). In such embodiment, the fusion polypeptide can be encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 63 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 64 or a variant thereof. For example, the CBM moiety can be obtained or derived from Pyricularia sp. In some embodiments, the CBM moiety can be obtained or derived from Pyricularia oryzae. In a further embodiment, the CBM moiety can have the amino acid sequence of SEQ ID NO: 86 or be a variant thereof having the ability to bind to a carbohydrate such as starch (and in some further embodiments raw starch). In still another embodiment, the CBM moiety can be encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 85 or be a degenerate sequence encoding the amino acid sequence of SEQ ID NO: 86 or a variant thereof. An embodiment of a fusion polypeptide having a CBM from Pyricularia oryzae has the amino acid sequence of SEQ ID NO: 66 or is a variant thereof having the ability to bind to a carbohydrate such as starch (and in some further embodiments raw starch). In such embodiment, the fusion polypeptide can be encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 65 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 66 or a variant thereof. For example, the CBM moiety can be obtained or derived from Fusarium sp. In some embodiments, the CBM moiety can be obtained or derived from Fusarium graminearum. In a further embodiment, the CBM moiety can have the amino acid sequence of SEQ ID NO: 88 or be a variant thereof having the ability to bind to a carbohydrate such as starch (and in some further embodiments raw starch). In still another embodiment, the CBM moiety can be encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 87 or be a degenerate sequence encoding the amino acid sequence of SEQ ID NO: 88 or a variant thereof. An embodiment of a fusion polypeptide having a CBM from Pyricularia oryzae has the amino acid sequence of SEQ ID NO: 68 or is a variant thereof having the ability to bind to a carbohydrate such as starch (and in some further embodimentsraw starch). In such embodiment, the fusion polypeptide can be encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 67 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 68 or a variant thereof. For example, the CBM moiety can be obtained or derived from Aspergillus sp. In some embodiments, the CBM moiety can be obtained or derived from Aspergillus nidulans. In a further embodiment, the CBM moiety can have the amino acid sequence of SEQ ID NO: 90 or be a variant thereof having the ability to bind to a carbohydrate such as starch (and in some further embodiments raw starch). In still another embodiment, the CBM moiety can be encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 89 or be a degenerate sequence encoding the amino acid sequence of SEQ ID NO: 90 or a variant thereof. An embodiment of a fusion polypeptide having a CBM from Aspergillus nidulans has the amino acid sequence of SEQ ID NO: 70 or is a variant thereof having the ability to bind to a carbohydrate such as starch (and in some further embodiments raw starch). In such embodiment, the fusion polypeptide can be encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 69 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 70 or a variant thereof. For example, the CBM moiety can be obtained or derived from Streptomyces sp. In some embodiments, the CBM moiety can be obtained or derived from Streptomyces violaceus. In a further embodiment, the CBM moiety can have the amino acid sequence of SEQ ID NO: 92 or be a variant thereof having the ability to bind to a carbohydrate such as starch (and in some further embodiments raw starch). In still another embodiment, the CBM moiety can be encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 91 or be a degenerate sequence encoding the amino acid sequence of SEQ ID NO: 92 or a variant thereof. An embodiment of a fusion polypeptide having a CBM from Streptomyces violaceus has the amino acid sequence of SEQ ID NO: 72 or is a variant thereof having the ability to bind to a carbohydrate such as starch (and in some further embodiments raw starch). In such embodiment, the fusion polypeptide can be encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 71 ora degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 72 or a variant thereof. For example, the CBM moiety can be obtained or derived from Fomitopsis sp. In some embodiments, the CBM moiety can be obtained or derived from Fomitopsis palustris. In a further embodiment, the CBM moiety can have the amino acid sequence of SEQ ID NO: 96 or be a variant thereof having the ability to bind to a carbohydrate such as starch (and in some further embodiments raw starch). In still another embodiment, the CBM moiety can be encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 95 or be a degenerate sequence encoding the amino acid sequence of SEQ ID NO: 96 or a variant thereof. An embodiment of a fusion polypeptide having a CBM from Fomitopsis palustris has the amino acid sequence of SEQ ID NO: 76 or is a variant thereof having the ability to bind toa carbohydrate such as starch (and in some further embodiments raw starch). In such embodiment, the fusion polypeptide can be encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 75 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 76 or a variant thereof. For example, the CBM moiety can be obtained or derived from Aspergillus sp. In some embodiments, the CBM moiety can be obtained or derived from Aspergillus niger. In a further embodiment, the CBM moiety can have the amino acid sequence of SEQ ID NO: 164 or be a variant thereof having the ability to bind to a carbohydrate such as starch (and in some further embodiments raw starch). In still another embodiment, the CBM moiety can be encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 163 or be a degenerate sequence encoding the amino acid sequence of SEQ ID NO: 164 or a variant thereof. An embodiment of a fusion polypeptide having a CBM from Aspergillus niger has the amino acid sequence of SEQ ID NO: 166 or is a variant thereof having the ability to bind to a carbohydrate such as starch (and in some further embodiments raw starch). In such embodiment, the fusion polypeptide can be encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 165 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 166 or a variant thereof. For example, the CBM moiety can be obtained or derived from Rhizopus sp. In some embodiments, the CBM moiety can be obtained or derived from Rhizopus arrhizus. In a further embodiment, the CBM moiety can have the amino acid sequence of SEQ ID NO: 168 or be a variant thereof having the ability to bind to a carbohydrate such as starch (and in some further embodiments raw starch). In still another embodiment, the CBM moiety can be encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 167 or be a degenerate sequence encoding the amino acid sequence of SEQ ID NO: 168 or a variant thereof. An embodiment of a fusion polypeptide having a CBM from Rhizopus arrhizus has the amino acid sequence of SEQ ID NO: 170 or is a variant thereof having the ability to bind to a carbohydrate such as starch (and in some further embodiments raw starch). In such embodiment, the fusion polypeptide can be encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 169 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 170 or a variant thereof.

[0273] The amino acid linker may be absent from the fusion polypeptides of the present disclosure. However, in some embodiments, the presence of an amino acid linker (L, L1 or L2) is desirable either to provide, for example, some flexibility between the alpha-amylase moiety and the carbohydrate-binding module moiety / moieties. As used in the present disclosure, the “amino acid linker”, “L”, “L1”, or “L2” terms refer to a stretch of one or more amino acids separating the alpha-amylase moiety and the carbohydrate-binding module moiety / moieties. In some embodiments, the amino acid linker is neutral, e.g., does not interfere with the biological activity of the alpha-amylase moiety and / orthe carbohydrate-binding module moiety / moieties. In someembodiments, the amino acid linker can increase the biological activity of the alpha-amylase moiety and / or the carbohydrate-binding module moiety / moieties. In some additional embodiments, the amino acid linker can comprise between one and fifty (50) amino acid residues. When two amino acid linkers are present in the fusion polypeptides (for example in the fusion polypeptides of formula (III)), the amino acid linkers can be the same (L1 = L2) or can be different (L1 * L2). An embodiment of an amino acid linkerthat can be used in the fusion polypeptides of the present disclosure includes a linker obtained or derived from Aspergillus sp., and in some additional embodiments, from Aspergillus niger. In a further embodiment, the amino acid linker has the amino acid sequence of SEQ ID NO: 98 or is a variant thereof. In still another embodiment, the linker is encoded by the nucleic acid sequence of SEQ ID NO: 97 or a degenerate sequence encoding the amino acid sequence of SEQ ID NO: 98 or a variant thereof. In a further embodiment, the amino acid linker has the amino acid sequence of SEQ ID NO: 174 or is a variant thereof. In still another embodiment, the linker is encoded by the nucleic acid sequence of SEQ ID NO: 173 or a degenerate sequence encoding the amino acid sequence of SEQ ID NO: 174 or a variant thereof. Additional embodiments of amino acid linkers that can be used in the fusion polypeptides of the present disclosure include, but are not limited to, the linkers described in US patent application published under 2020 / 0087672 as well as PCT application published under WO2025 / 133860 herewith incorporated in its entirety. The signal sequence may be absent from the fusion polypeptides of the present disclosure. However, in some embodiments, the presence of a signal sequence is desirable to secrete the fusion polypeptide outside the recombinant yeast host cell. The signal sequence is intended to be cleaved from the fusion polypeptide once secreted by the recombinant yeast host cell. An embodiment of a signal sequence that can be used in the fusion polypeptides of the present disclosure includes a linker obtained or derived from Saccharomyces sp., and in some additional embodiments, from Saccharomyces cerevisiae. In a further embodiment, the signal sequence has the amino acid sequence of SEQ ID NO: 100 or is a variant thereof. In still another embodiment, the signal sequence is encoded by the nucleic acid sequence of SEQ ID NO: 99 or a degenerate sequence encoding the amino acid sequence of SEQ ID NO: 100 or a variant thereof. In another embodiment, the signal sequence can be derived from the alphamating factor (MATa) gene of S. cerevisiae. In some additional embodiments, the signal sequence can be derived from Rhizomucor sp., and some further embodiments, from Rhizomucor pusillus. Additional embodiments of signal sequences that can be used in the fusion polypeptides of the present disclosure include, but are not limited to, the signal sequence described in US patent 11198881 and US patent application 2020 / 0087672 and are herewith incorporated in their entirety.

[0274] As used in the context of the present disclosure the variants associated with a polypeptide such as a fusion polypeptide, a carbohydrate-biding module, a linker or a signal sequence referto polypeptide having at least one amino acid modification (addition, deletion, substitution) with respect to the parental / wildtype polypeptide. In some embodiments, the polypeptide variants exhibit at least 70% identity to the amino acid sequence of the wildtype / parental polypeptide. In some additional embodiments, the polypeptide variants exhibit at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identity to the amino acid sequence of the wildtype / parental polypeptide. The term “% identity”, as known in the art, is a relationship between two or more polypeptide sequences, as determined by comparing the sequences. The level of identity can be determined conventionally using known computer programs. Identity can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, NY (1991). Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.) or Geneious Prime. Multiple alignments of the sequences disclosed herein were performed using the Clustal Omega alignment method, which employs a Hidden Markov Model-based algorithm, using default parameters (including a cluster size = 100).

[0275] As used in the context of the present disclosure, a “fragment” is a type of variant which includes at least one deleted amino acid residues when compared to the amino acid sequence of the wildtype / parental polypeptide. In some embodiments, fragments can refer to the mature form of a secreted fusion polypeptide (from which the signal sequence has been cleaved). In additional embodiments, the fragment can be, for example, a truncation of one or more, two or more, three or more, four, five or more, six or more, seven or more, eight or more, nine or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more at the amino terminus of the non-truncated polypeptide or variant. In additional embodiments, the fragment can be, for example, a truncation of one or more, two or more, three or more, four, five or more, six or more, seven or more, eight or more, nine or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more at the carboxyl terminus of the non-truncated polypeptide or variant. Alternatively, or in combination, the fragment can be generated from removing one or more internal amino acid residues. In an embodiment, the polypeptide fragment can have at least 100, 150, 200, 250, 300, 350, 400, 450, or moreconsecutive amino acid residues of the parental / wildtype amino acid sequence or the polypeptide variant.

[0276] Variants (including fragments) exhibit a substantially similar biological activity when compared to the biological activity of the parental polypeptide. Variants of fusion polypeptides are able to exhibit both alpha-amylase activity and to bind to carbohydrates (and in some embodiments raw starch). Variants of carbohydrate-binding modules are able to bind to carbohydrates (and in some embodiments to raw starch). Variants of a linker are able to connect two moieties in a fusion polypeptide. Variants of a signal sequence are able to direct the expressed polypeptides for secretion. In some embodiments, the variants can also have at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more biological activity when compared to the biological activity of the wildtype / parental polypeptide. In some specific embodiments, the variants (including fragments) can also have more biological activity when compared to the biological activity of the wildtype / parental polypeptide.

[0277] Systems for producing the alpha-amylase variants

[0278] The present disclosure provides recombinant microbial host cells for the expression one or more of the alpha-amylase variants described herein. The recombinant microbial host cell is obtained from a microbial cell which can be a bacterium, a yeast, or a fungus. In an embodiment, the recombinant microbial host cell is obtained from a microbial cell which is a bacterium. In some embodiments, the bacterium is a Gram-positive bacterium. In other embodiments, the bacterium is a Gram-negative bacterium. In an embodiment, the recombinant microbial host cell is from Actinoplanes sp. In another embodiment, the recombinant microbial host cell is from Actinoplanes missouriensis. In an embodiment, the recombinant microbial host cell is from Aeribacillus sp. In another embodiment, the recombinant microbial host cell is from Aeribacillus pallidus. In an embodiment, the recombinant microbial host cell is from Anoxybacillus sp. In another embodiment, the recombinant microbial host cell is from Anoxybacillus caldiproteolyticus. In an embodiment, the recombinant microbial host cell is from Bacillus sp. In another embodiment, the recombinant microbial host cell is from Bacillus acidopullulyticus. In another embodiment, the recombinant microbial host cell is from Bacillus amyloliquefaciens. In another embodiment, the recombinant microbial host cell is from Bacillus licheniformis. In another embodiment, the recombinant microbial host cell is from Bacillus pumilus. In another embodiment, the recombinant microbial host cell is from Bacillus subtilis. In an embodiment, the recombinant microbial host cell is from Chryseobacterium sp. In another embodiment, the recombinant microbial host cell is from Chryseobacterium proteolyticum. In an embodiment, the recombinant microbial host cell is from Escherichia sp. In another embodiment, the recombinant microbial host cell is from Escherichia coll. In an embodiment, the recombinant microbial host cell is from Geobacillus sp. In another embodiment, the recombinant microbialhost cell is from Geobacillus stearothermophilus. In an embodiment, the recombinant microbial host cell is from Lactobacillus sp. In another embodiment, the recombinant microbial host cell is from Lactobacillus fermentum. In an embodiment, the recombinant microbial host cell is from Lactococcus sp. In another embodiment, the recombinant microbial host cell is from Lactococcus lactis. In an embodiment, the recombinant microbial host cell is from Macrococcus sp. In another embodiment, the recombinant microbial host cell is from Macrococcus caseolyticus. In an embodiment, the recombinant microbial host cell is from Microbacterium sp. In another embodiment, the recombinant microbial host cell is from Microbacterium arborescens. In an embodiment, the recombinant microbial host cell is from Micrococcus sp. In another embodiment, the recombinant microbial host cell is from Micrococcus lysodeikticus. In an embodiment, the recombinant microbial host cell is from Priestia sp. In another embodiment, the recombinant microbial host cell is from Priestia flexa. In an embodiment, the recombinant microbial host cell is from Streptomyces sp. In another embodiment, the recombinant microbial host cell is from Streptomyces mobaraensis. In another embodiment, the recombinant microbial host cell is from Streptomyces murinus. In another embodiment, the recombinant microbial host cell is from Streptomyces olivaceus. In another embodiment, the recombinant microbial host cell is from Streptomyces olivochromogenes. In another embodiment, the recombinant microbial host cell is from Streptomyces rubiginosus. In another embodiment, the recombinant microbial host cell is from Streptomyces violaceoruber. In an embodiment, the recombinant microbial host cell is from Pseudomonas sp. In another embodiment, the recombinant microbial host cell is from Pseudomonas fluorescens. In an embodiment, the recombinant microbial host cell is from Weizmannia sp. In another embodiment, the recombinant microbial host cell is from Weizmannia coagulans.

[0279] In an embodiment, the recombinant microbial host cell is obtained from a microbial cell which is a yeast. In some embodiments, the yeast is a budding yeast. In other embodiments, the yeast is methylotrophic (e.g., yeast able to utilize methanol as the sole carbon and energy source). Embodiments of methylotrophic yeasts include, but are not limited to Komagataella sp. and Ogataea sp. In some embodiments, the yeast is an oleaginous yeast (e.g., a yeast capable of accumulating more than 20% of its dry cell weight as lipids or triglycerides).

[0280] In an embodiment, the recombinant microbial host cell is from Blastobotrys sp. In another embodiment, the recombinant microbial host cell is from Blastobotrys adeninivorans (basonym Trichosporon adeninivorans). In an embodiment, the recombinant microbial host cell is from Candida sp. In another embodiment, the recombinant microbial host cell is from Candida albicans. In an embodiment, the recombinant microbial host cell is from Cyberlindnera sp. In another embodiment, the recombinant microbial host cell is from Cyberlindnera jadinii (basonym Saccharomyces jadinii). In an embodiment, the recombinant microbial host cell is from the Debaryomyces sp. In another embodiment, the recombinantmicrobial host cell is from Debaryomyces hansenii. In another embodiment, the recombinant microbial host cell is from Debaryomyces hansenii. In an embodiment, the recombinant microbial host cell is from Hanseniaspora sp. (also known as Kloeckera sp.). In another embodiment, the recombinant microbial host cell is from Hanseniaspora guilliermondii. In another embodiment, the recombinant microbial host cell is from Hanseniaspora pseudoguilliermondii. In an embodiment, the recombinant microbial host cell is from the Kazachstania sp. In another embodiment, the recombinant microbial host cell is from Kazachstania bulderi (basonym Saccharomyces bulderi). In another embodiment, the recombinant microbial host cell is from Kazachstania barnettii (basonym Saccharomyces barnettii). In another embodiment, the recombinant microbial host cell is from Kazachstania exigua (basonym Saccharomyces exiguus). In an embodiment, the recombinant microbial host cell is from Kluyveromyces sp. In another embodiment, the recombinant microbial host cell is from Kluyveromyces lactis (basonym Torulaspora lactis). In another embodiment, the recombinant microbial host cell is from Kluyveromyces marxianus also known as Kluyveromyces fragilis (basonym Saccharomyces marxianus). In an embodiment, the recombinant microbial host cell is from Komagataella sp. In another embodiment, the recombinant microbial host cell is from Komagataella phaffii. In an embodiment, the recombinant microbial host cell is from Limtongozyma sp. In another embodiment, the recombinant microbial host cell is from Limtongozyma cylindracea (basonym Candida cylindracea). In an embodiment, the recombinant microbial host cell is from Lipomyces sp. In an embodiment, the recombinant microbial host cell is from Metschnikowia sp. In another embodiment, the recombinant microbial host cell is from Metschnikowia sinensis. In another embodiment, the recombinant microbial host cell is from Metschnikowia fructicola. In another embodiment, the recombinant microbial host cell is from Metschnikowia pulcherrima. In another embodiment, the recombinant microbial host cell is from Metschnikowia zobellii. In another embodiment, the recombinant microbial host cell is from Metschnikowia shanxiensis. In an embodiment, the recombinant microbial host cell is from Ogataea sp. In another embodiment, the recombinant microbial host cell is from Ogataea polymorpha (basonym Hansenula polymorpha). In another embodiment, the recombinant microbial host cell is from Ogataea methanolica (basonym Pichia methanolica). In an embodiment, the recombinant microbial host cell is from Pichia sp. (also known as Hansenula sp.). In an embodiment, the recombinant microbial host cell is from Rasamsonia sp. In another embodiment, the recombinant microbial host cell is from Rasamsonia emersonii. In an embodiment, the recombinant microbial host cell is from Saccharomyces sp. In another embodiment, the recombinant microbial host cell is from Saccharomyces cerevisiae. In yet another embodiment, the recombinant microbial host cell is from Saccharomyces cerevisiae var. diastaticus. In another embodiment, the recombinant microbial host cell is from Saccharomyces uvarum. Inanother embodiment, the recombinant microbial host cell is from Saccharomyces boulardii. In an embodiment, the recombinant microbial host cell is from Scheffersomyces sp. In another embodiment, the recombinant microbial host cell is from Scheffersomyces stipitis (basonym Pichia stipitis). In an embodiment, the recombinant microbial host cell is from Schwanniomyces sp. In another embodiment, the recombinant microbial host cell I is from Schwanniomyces polymorphus (basonym Pichia polymorpha). In another...

Claims

WHAT IS CLAIMED IS:

1. An alpha-amylase variant (i) having at least 70% identity with the amino acid sequence of SEQ ID NO: 2, and (ii) comprising an amino acid substitution at any one of positions 3, 4, 5, 8, 18, 24, 26, 33, 43, 70, 71 , 79, 80, 84, 86, 91 , 93, 96, 103, 105, 106, 117, 119, 123, 124, 125, 132, 133, 141 , 143, 148, 151 , 152, 155, 157, 158, 168, 174, 178, 181 , 183, 185, 204, 207, 208, 209, 211 , 219, 230, 233, 242, 246, 252, 261 , 268, 273, 290, 301 , 319, 327, 334, 336, 338, 343, 345, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 397, 402, 407, 408, 415, 416, 422, 423, 426, and / or 442.

2. The alpha-amylase variant of claim 1 , comprising an amino acid substitution at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 117, 119, 124, 125, 133, 141 , 143, 158, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423.

3. The alpha-amylase variant of claim 1 , comprising an amino acid substitution at any one of positions 43, 71 , 79, 80, 84, 91 , 93, 96, 103, 106, 143, 174, 178, 181 , 204, 207, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 336, 343, 347, 348, 357, 369, 372, 373, 380, 383, 385, 386, 393, 396, 402 and / or 423.

4. The alpha-amylase variant of claim 1 , comprising an amino acid substitution at any one of positions 71 , 80, 84, 91 , 93, 96, 117, 119, 125, 133, 141 , 158, 174, 209, 211 , 252, 268, 336, 348, 357, 380, 383, 393, and / or 402.

5. The alpha-amylase variant of claim 1 , comprising an amino acid substitution at any one of positions 43, 71 , 79, 80, 93, 103, 106, 117, 124, 143, 178,181 , 204, 209, 211 , 219, 233, 242, 252, 268, 273, 301 , 319, 327, 343, 347, 369, 372, 373, 385, 386, 393, and / or 396.

6. The alpha-amylase variant of claim 1 , comprising an amino acid substitution at any one of positions 133, 207, and / or 423.

7. The alpha-amylase variant of any one of claims 1 to 6, comprising at least one of the following amino acid substitutions: K43Q / A / R / E, R71G / N / D / A, S79Q / K / T / R / N, N80D / S / H, T84I / V / L, S91A / E / P, E93D / S / Q, G96S / D / N / A, G103E / S / A / N / Q, I106M / F / V / L, V117M / A / T, N119P / S / A / Q, E124D / S / T, M125N / T / K / F / S, G133A / D / E / Q / S, G141 D / E / T / Q / K, N143D / S / Q / K, D158G / S / E, K174Q / N / Y / R, K178S / R / Q / A, M181V / I / L, D204G / E / P / K, W207R / S / Q / K / A / E, S209A / T / G, S211V / A / I / F, S219D / N / H / E / T, G233D / S / P / A, F242Y / W / K / D, N252G / S / D / Q, V268F / Y / M / L / I, T273A / V / L / I / S, T301A / S / V / G, A319G / S / E, Q327A / E / G / N, R336G / N / S / K, M343L / V / I, F347I / L / V / T, Q348A / S / K / T, H357A / V / N, F369Y / H / L / M, S372D / N / E / G / A, D373T / N / G / S, Q380A / E / G / D, A383D / K / R / N / S, M385I / V / L / F / A, A386V / I / T / S, K393S / Q / R / A, Q396S / T / N / G, F402N / S / D / G / T, and G423S / N / K / A / E / F.

8. The alpha-amylase variant of any one of claims 1 to 7, comprising at least one of the following amino acid substitutions: K43Q, R71G, S79Q, N80D, T84I, S91A, E93D, G96S,G103E, I106M, V117M, N119P, E124D, M125N, G133A, G141D, N143D, D158G, K174Q, K178S, M181V, D204G, W207R, S209A, S211V, S219D, G233D, F242Y, N252G, V268F, T273A, T301A, A319G, Q327A, R336G, M343L, F347I, Q348A, H357A, F369Y, S372D, D373T, Q380A, A383D, M385I, A386V, K393S, Q396S, F402N, and G423S.

9. The alpha-amylase variant of any one of claims 1 to 8 further comprising at least one amino acid deletion at position 355.

10. A fusion polypeptide having alpha-amylase activity, the fusion polypeptide comprising (i) the alpha-amylase variant of any one of claims 1 to 9, and (ii) a carbohydrate-binding module.

11. The fusion polypeptide of claim 10, wherein the carbohydrate-binding module has at least 70% identity with the amino acid sequence of SEQ ID NO: 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 164 or 168.

12. The fusion polypeptide of claim 10 or 11 having one of the following formula:NH2-AA-L-(CBM)n-COOH (la), orNH2-AA-(L-CBM)n-COOH (lb),wherein: NH2is the amino terminus of the fusion polypeptide;AA is the alpha-amylase moiety;L is an optional amino acid linker;CBM is the carbohydrate binding moiety;n is an integral number between 1 and 5; andCOOH is the carboxy terminus of the fusion polypeptide.

13. The fusion polypeptide of claim 10 or 11 having one of the following formula:NH2-(CBM)n-L-AA-COOH (Ila), orNH2-(CBM-L)n-AA-COOH (lib),wherein: NH2is the amino terminus of the fusion polypeptide;CBM is the carbohydrate binding moiety;n is an integral number between 1 and 5;L is an optional amino acid linker;AA is the alpha-amylase moiety; andCOOH is the carboxy terminus of the fusion polypeptide.

14. The fusion polypeptide of claim 10 or 11 having the following formula:NH2-(CBM)n-L1-AA-L2-(CBM)m-COOH (Illa),NH2-(CBM-L1)n-AA-L2-(CBM)m-COOH (lllb),NH2-(CBM)n-L1-AA-(L2-CBM)m-COOH (lllc),NH2-(CBM-L1)n-AA-(L2-CBM)m-COOH (Hid),wherein: NH2is the amino terminus of the fusion polypeptide;CBM is the carbohydrate binding moiety;n and m are integral numbers independently selected between 1 and 5; L1 and L2 are an optional amino acid linkers;AA is the alpha-amylase moiety;andCOOH is the carboxy terminus of the fusion polypeptide.

15. A heterologous nucleic acid molecule comprising an open reading frame encoding the alpha-amylase variant of any one of claims 1 to 9 orthe fusion polypeptide of any one of claims 10 to 14.

16. The heterologous nucleic acid molecule of claim 15 further comprising at least one promoter operably associated with the open reading frame.

17. The heterologous nucleic acid molecule of claim 15 or 16, wherein the open reading further encodes a signal sequence.

18. A vector comprising the heterologous nucleic acid molecule of any one of claims 15 to 17.

19. An expression cassette comprising the heterologous nucleic acid molecule of any one of claims 15 to 17.

20. A recombinant microbial host cell expressing the alpha-amylase variant of any one of claims 1 to 9 or the fusion polypeptide of any one of claims 10 to 14.

21. The recombinant microbial host cell of claim 20 comprising the heterologous nucleic acid molecule of any one of claims 15 to 17, the vector of claim 18 or the expression cassette of claim 19.

22. The recombinant microbial host cell of claim 20 or 21 being a yeast.

23. The recombinant microbial host cell of claim 22 being from Saccharomyces sp.

24. The recombinant microbial host cell of claim 23 being from Saccharomyces cerevisiae.

25. The recombinant microbial host cell of claim 22 being from Komagataella sp.

26. The recombinant microbial host cell of claim 25 being from Komagataella phaffii.

27. A method for making the alpha-amylase variant of any one of claims 1 to 9 or the fusion polypeptide of any one of claims 10 to 14, wherein the process comprises (i) expressing the heterologous nucleic acid molecule of any one of claims 15 to 17, the vector of claim 18 or theexpression cassette of claim 19 in the recombinant microbial host cell of any one of claims 20 to 26.

28. The method of claim 27, wherein the alpha-amylase variant is an intracellular polypeptide or a secreted polypeptide.

29. The method of claim 28, wherein the secreted polypeptide is in a free form or is associated to the surface of the recombinant microbial host cell.

30. The method of claim 29, wherein the polypeptide is associated to the surface of the recombinant yeast host cell is a tethered polypeptide.

31. The method of any one of claims 27 to 30, further comprising, after step (i), (ii) substantially separating the alpha-amylase variant or the fusion polypeptide from the recombinant microbial host cell.

32. The method of any one of claims 27 to 31 , further comprising, after step (i) or (ii), (iii) drying the alpha-amylase variant or the fusion polypeptide.

33. A composition comprising the alpha-amylase variant of any one of claims 1 to 9 or the fusion polypeptide of any one of claims 10 to 14, and a carrier.

34. The composition of claim 33, wherein the alpha-amylase variant is obtainable or obtained by the method of any one of claims 27 to 32.

35. The composition of claim 33 or 34 further comprising another enzyme.

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