Cleaning compositions containing polypeptides having alpha-amylase activity - Patent Application 20070123333

Abstract

Cleaning compositions containing variant α-amylases, methods of making such cleaning compositions, and methods of treating surfaces, such as fabrics, with aqueous solutions containing such compositions.

Description

[Technical field]

[0001] (Reference to array list) The present specification contains a sequence listing in computer readable form, which is incorporated herein by reference.

[0002] FIELD OF THEINVENTION The present invention relates to cleaning compositions comprising amylase enzymes, methods of making them, and methods of using them. The compositions and methods of the present invention are suitable for use in household cleaning or treatment compositions, particularly laundry compositions, such as manual and / or automatic laundry compositions and / or manual and / or automatic dishwashing compositions, and dishwashing compositions. The present invention is particularly useful for cleaning laundry. [Background technology]

[0003] Alpha-amylases (alpha-1,4-glucan-4-glucanohydrolases, EC 3.2.1.1) constitute a group of enzymes that catalyze the hydrolysis of starch and other linear and branched 1,4-glucoside oligosaccharides and polysaccharides. They have been known for many years to be used in household cleaning compositions, such as the first bacterial alpha-amylase, from B. licheniformis, also known as Termamyl. For example, those from AA560 (described in WO 2000 / 060060) and SP707 (described in Tsukamoto et al., 1988, Biochem. Biophys. Res. Comm. 151:25-31) are useful in detergents, and modifications of these and other amylases to improve their stability in detergents are known. For example, WO 96 / 23873 discloses deleting amino acids 181+182 or amino acids 183+184 of SP707 (SEQ ID NO: 7 of WO 96 / 23873) to improve the stability of this amylase. WO 96 / 23873 further discloses modifying SP707 amylase by substituting M202, for example, with leucine, to stabilize the molecule against oxidation.

[0004] It is a constant challenge for detergent formulators to improve the effectiveness of cleaning compositions, especially in an environmentally friendly way. For environmental reasons, it is also becoming increasingly important to provide detergent compositions that are effective even in the quick wash cycle, which preferably often uses less water and / or lower temperatures. It is therefore desirable to have amylolytic enzymes that exhibit improved properties, such as specific activity, when compared to the parent polypeptide. The present invention provides compositions comprising variant polypeptides, which have α-amylase activity and improved properties or enzymatic detergent effects, such as removal of starch-containing soils or specific activity, compared to their hybrid and / or parent polypeptides. In particular, preferred amylolytic enzymes can function at lower temperatures and / or in shorter wash cycles, while retaining or increasing other desirable properties, such as specific activity (amylolytic activity), stability, and / or cleaning performance to enable good cleaning. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2000 / 060060 [Patent Document 2] WO 96 / 23873 Summary of the Invention [Problem to be solved by the invention]

[0006] It is therefore an object of the present invention to provide cleaning compositions comprising α-amylase variants which exhibit improved properties such as removal or specific activity of starch-containing stains when compared to the parent polypeptide, which can be used in washing, dishwashing and / or cleaning methods. It is a further object of the present invention to provide cleaning compositions comprising α-amylase variants which have improved cleaning performance at lower wash temperatures, such as between 5°C and below 40°C, or below 30°C, or below 28°C, or below 25°C, compared to the parent α-amylase or compared to any of the α-amylases of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. [Means for solving the problem]

[0007] The present invention relates to (a) Using SEQ ID NO:1 for numbering, positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 165 , 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, 3 62, 365, 371, 383, 390, 394, 395, 405, 409, 414, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, wherein the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least and an α-amylase variant having at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to said α-amylase variant. and a cleaning adjunct.

[0008] The present invention also provides sequences for the sequences of amino acids 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 165, 167, 169, 172, 176, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 300, 309, 308, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, , 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, 362, 365, 371, 383, 390, 394, 395, 405, 409, 414, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, wherein the variant is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 140%, The invention provides a cleaning composition comprising an α-amylase variant having at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity, the variant having improved cleaning performance compared to the cleaning performance of the parent polypeptide, and a cleaning adjunct. The parent polypeptide is preferably SEQ ID NO: 1, or SEQ ID NO: 2 or SEQ ID NO: 3. The invention also provides a method of making a cleaning composition comprising obtaining an α-amylase variant as described above and mixing the α-amylase variant with a cleaning adjunct to make the cleaning composition.

[0009] The present invention also provides sequences for the nucleotides 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 16 0, 165, 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360 , 361, 362, 365, 371, 383, 390, 394, 395, 405, 409, 414, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, thereby providing an α-amylase variant, wherein the variant has at least 60%, such as at least 65%, such as at least 70%, a similarity to that of the parent polypeptide. The present invention provides a method for making a cleaning composition comprising the steps of obtaining a variant of a parent α-amylase having at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 97%, or such as at least 99%, but less than 100% sequence identity, wherein the variant has improved cleaning performance; and mixing the α-amylase variant with a cleaning adjunct to make the cleaning composition.

[0010] The present invention also provides a method for treating a surface, preferably a textile, comprising the steps of: (i) forming an aqueous cleaning solution comprising water, an α-amylase variant of a parent α-amylase described herein, and a cleaning adjunct; (ii) treating the surface with an aqueous cleaning solution at a temperature of preferably between 5° C. and 60° C., preferably between 10 and 40° C. or preferably up to 35° C., more preferably at a temperature of 30° C. or less, or at a temperature of 20° C. or less; (iii) rinsing the surface.

[0011] Preferably, the variant amylase has α-amylase activity and the variant has improved cleaning performance compared to the parent α-amylase.

[0012] The compositions and methods herein are particularly useful for treating surfaces comprising fibers or fabrics, such as cotton and / or synthetic fibers (e.g., polyester, nylon, etc.), which may be in the form of either alone or in blends (e.g., polycotton). The invention also relates to the use of the above compositions or methods for cleaning or removing stains (e.g., starch or other carbohydrate-containing stains), removing malodors, or increasing the whiteness of fabrics. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] definition In this detailed description, the following definitions apply: It should be noted that the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0014] Reference herein to "about" a value or parameter includes aspects that are directed to that value or parameter itself. For example, a statement that refers to "about X" includes the aspect "X."

[0015] Unless otherwise defined or indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0016] A domain, B domain, and C domain: The structure of α-amylase comprises three distinct domains, A, B, and C. See, for example, Machius et al., 1995, J. Mol. Biol. 246:545-559. The term "domain" refers to a region of a polypeptide that forms a separate and independent substructure of the whole molecule. α-amylase consists of a β / α-8 barrel with an active site, designated the A domain, a somewhat longer loop between β-sheet 3 and α-helix 3, designated the B domain, and a C-domain, possibly a carbohydrate-binding domain (see, for example, WO 2005 / 001064, Machius et al., supra).

[0017] The domains of an α-amylase can be determined by structural analysis, such as by using crystallographic techniques. An alternative method for determining the domains of an α-amylase is by sequence alignment of the amino acid sequence of the α-amylase with another α-amylase whose domain has been determined. For example, a sequence that aligns with the B domain sequence in an α-amylase whose B domain has been determined can be considered the B domain of the given α-amylase.

[0018] Allelic variant: The term "allelic variant" means any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variants arise naturally by mutation and can result in polymorphism within a population. A mutation in a gene can be silent (the encoded polypeptide is unchanged) or it can encode a polypeptide having an altered amino acid sequence. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

[0019] Alpha-amylase: The term "alpha-amylase" is synonymous with the term "polypeptide having alpha-amylase activity". "Alpha-amylase activity" refers to the activity of alpha-1,4-glucan-4-glucanohydrolase (EC 3.2.1.1), which constitutes a group of enzymes that catalyze the hydrolysis of starch and other linear and branched 1,4-glucoside oligosaccharides and polysaccharides. Thus, as used herein, the term "alpha-amylase" refers to an enzyme with alpha-amylase activity (enzyme class, EC 3.2.1.1) that hydrolyzes the alpha-linked bonds of large alpha-linked polysaccharides, such as starch and glycogen, to yield glucose and maltose. The terms "alpha-amylase" and "amylase" are used interchangeably herein and may constitute the same meaning and purpose. For the purposes of the present invention, alpha-amylase activity is determined according to the procedure described in the subheading "Methods" below. In one aspect, the α-amylase of the invention has at least 20%, such as at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% sequence identity to the amino acid sequence of the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0020] Amino acid: As used herein, the term "amino acid" includes the standard 20 genetically encoded amino acids and their corresponding stereoisomers in the "d" form (as compared to the natural "l" form), omega amino acids, other naturally occurring amino acids, non-conventional amino acids (e.g., α,α-disubstituted amino acids, N-alkyl amino acids, etc.), and chemically derivatized amino acids. Chemical derivatives of one or more amino acids may be obtained by reaction with a functional side group. Such derivatized molecules include, for example, molecules in which a free amino group is derivatized to form an amine hydrochloride, a p-toluenesulfonyl group, a carboxybenzoxy group, a t-butyloxycarbonyl group, a chloroacetyl group, or a formyl group. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters, or other types of esters and hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. Chemical derivatives also include peptides containing naturally occurring amino acid derivatives of the 20 standard amino acids. For example, 4-hydroxyproline may be used in place of proline, 5-hydroxylysine may be used in place of lysine, 3-methylhistidine may be used in place of histidine, homoserine may be used in place of serine, and ornithine may be used in place of lysine. Derivatives also include peptides containing one or more additions or deletions, so long as the required activity is maintained. Other included modifications are terminal modifications such as amidation, amino terminal acylation (e.g., acetylation or thioglycolic acid amidation), terminal carboxylamidation (e.g., with ammonia or methylamine).

[0021] When an amino acid is specifically recited, such as "alanine" or "Ala" or "A", the term refers to both l-alanine and d-alanine, unless otherwise specified. Other non-conventional amino acids may also be suitable components of the polypeptides of the invention, so long as the desired functional properties are retained by the polypeptide. For the peptides shown, each encoded amino acid residue is represented, where appropriate, by a single letter designation that corresponds to the trivial name of the conventional amino acid. In one embodiment, the polypeptides of the invention comprise or consist of l-amino acids.

[0022] Catalytic domain: The term "catalytic domain" refers to the region of an enzyme that contains the catalytic machinery of the enzyme.

[0023] cDNA: The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from a mature spliced ​​mRNA molecule obtained from a eukaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial primary RNA transcript is a precursor of mRNA that is processed through a series of processes, including splicing, before appearing as a spliced ​​mature mRNA.

[0024] Coding sequence: The term "coding sequence" refers to a polynucleotide that directly specifies the amino acid sequence of a variant thereof. The boundaries of the coding sequence are generally determined by an open reading frame, and usually begin with a start codon, such as ATG, GTG, and TTG, and end with a stop codon, such as TAA, TAG, and TGA. A coding sequence can be a DNA, cDNA, synthetic, or recombinant polynucleotide.

[0025] Control sequences: The term "control sequences" refers to nucleic acid sequences necessary for the expression of a polynucleotide encoding a variant of the invention. Each control sequence may be native (i.e., from the same gene) or foreign (i.e., from different genes) to the polynucleotide encoding the variant, or may be native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, control sequences include a promoter and transcriptional and translational stop signals. Control sequences may also be provided with linkers for the purpose of introducing specific restriction enzyme sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding the variant.

[0026] Corresponding: As used herein, the term "corresponding" refers to a method of determining a particular amino acid of a sequence, and a reference is made to a particular amino acid sequence. For example, for the purposes of the present invention, when a reference is made to a particular amino acid position, a person skilled in the art can align another amino acid sequence to the amino acid sequence to which the reference is made, and thereby determine which particular amino acid may be of interest in the other amino acid sequence. Alignment of another amino acid sequence with, for example, the sequence described in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, or with any other sequence listed herein, is described elsewhere herein. Alternative alignment methods can be used and are well known to those skilled in the art.

[0027] Dishwashing composition: The term "dishwashing composition" as used herein refers to all forms of compositions for cleaning dishes. Dishwashing compositions are liquid dishwashing compositions, powder dishwashing compositions, and optionally the compositions are in the form of unit doses. Unit dose dishwashing compositions may include multiple compartments, the multiple compartments including powder, liquid, or a combination thereof. In a preferred embodiment, at least one compartment includes liquid and at least one compartment includes powder.

[0028] Enzyme detergent benefits: The term "enzyme detergent benefits" as used herein refers to the beneficial effects that enzymes can add to detergents compared to the same detergents without enzymes. Important detergent benefits that enzymes can provide are stain removal with little or no visible staining after washing and / or cleaning, prevention or reduction of redeposition of soils released during the washing process (effect also called anti-redeposition), and full or partial restoration of whiteness of textiles that were originally white but have acquired a gray or yellowish appearance as a result of repeated use and cleaning (effect also called whitening). Textile care benefits that are not directly related to catalytic stain removal or prevention of redeposition of soils can also be important for enzyme detergent benefits. Examples of such textile care benefits include preventing or reducing dye transfer from one fabric to another or to another part of the same fabric (effects also called dye transfer or backsoiling), removing protruding or broken fibers from the fabric surface to reduce pilling tendency or remove existing pilling or fuzz (effects also called anti-pilling), improving fabric softness, clarifying fabric color, and removing particulate soils trapped in fabric or garment fibers. Enzyme bleaching is a further enzymatic detergent benefit, whose catalytic activity is commonly used to catalyze the formation of bleaching ingredients such as hydrogen peroxide or other peroxides.

[0029] Expression: The term "expression" includes any step involved in the production of a variant, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0030] Expression vector: The term "expression vector" refers to a linear or circular DNA molecule that contains a polynucleotide that encodes a variant, operably linked to additional nucleotides that effect its expression.

[0031] Fragment: As used herein, the term "fragment" refers to a polypeptide having one or more (e.g., several) amino acids that are not present at the amino and / or carboxyl terminus of the mature polypeptide of the parent α-amylase, e.g., any one of the parent sequences disclosed herein, such as any of SEQ ID NOs: 1-14, wherein the fragment has α-amylase activity. In one aspect, the fragment contains at least 200 contiguous amino acid residues of any of SEQ ID NOs: 1-14, e.g., at least 300 contiguous amino acid residues of any of SEQ ID NOs: 1-14, or at least 350 contiguous amino acid residues, or at least 400 contiguous amino acid residues, or at least 450 contiguous amino acid residues.

[0032] Fusion Polypeptide: As used herein, the term "fusion polypeptide" refers to a polypeptide comprising amino acid sequences derived from two or more species, such as two, three, or four species. Such fusion polypeptides can be produced by molecular techniques well known to those skilled in the art. The fusion polypeptides of the present invention have α-amylase activity. In particular, the fusion polypeptides of the present invention comprise, for example, the A and C domains of one α-amylase and the B domain from another α-amylase. Hybrid polypeptide (or "hybrid") and fusion polypeptide can be used interchangeably and constitute the same meaning and purpose.

[0033] Hard Surface Cleaning: As used herein, the term "hard surface cleaning" refers to cleaning any hard surface, such as cleaning floors, roofs, cars, kitchen or bathroom surfaces, as well as dish cleaning (dish-washing).

[0034] Heterologous Polynucleotide: The term "heterologous polynucleotide" is defined herein as a polynucleotide that is not native to a host cell, a naturally occurring polynucleotide in which structural modifications have been made to the coding region, a naturally occurring polynucleotide whose expression is quantitatively altered as a result of recombinant DNA techniques, e.g., manipulation of the DNA by a different (foreign) promoter, or a naturally occurring polynucleotide in a host cell with one or more extra copies of the polynucleotide to quantitatively alter expression. A "heterologous gene" is a gene that comprises a heterologous polynucleotide.

[0035] Heterologous: The term "heterologous" with respect to a host cell means that a polypeptide or nucleic acid does not naturally occur in the host cell. The term "heterologous" with respect to a polypeptide or nucleic acid means a regulatory sequence. For example, a promoter or domain of a polypeptide or nucleic acid is not naturally associated with the polypeptide or nucleic acid. That is, the regulatory sequence is derived from a gene other than the gene encoding the variant.

[0036] High stringency conditions: The term "high stringency conditions" refers to prehybridization and hybridization for 12-24 hours according to standard Southern blotting procedures in 5X SSPE, 0.3% SDS, 200 micrograms / mL sheared and denatured salmon sperm DNA, and 50% formamide at 42° C. for a probe at least 100 nucleotides in length. Finally, the carrier material is washed three times for 15 minutes each using 2X SSC, 0.2% SDS at 65° C.

[0037] Host cell: The term "host cell" refers to any type of cell that is susceptible to transformation, transfection, transduction, etc., with a nucleic acid construct or expression vector containing a polynucleotide described herein. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0038] Hybridization: The term "hybridization" refers to the pairing of substantially complementary strands of nucleic acid using standard Southern blotting procedures. Hybridization can be performed under medium, medium-high, high, or very high stringency conditions. Medium stringency conditions refer to prehybridization and hybridization in 5X SSPE, 0.3% SDS, 200 micrograms / mL sheared and denatured salmon sperm DNA, and 35% formamide at 42°C for 12-24 hours, followed by three washes for 15 minutes each using 0.2X SSC, 0.2% SDS at 55°C. Medium-high stringency conditions refer to prehybridization and hybridization in 5X SSPE, 0.3% SDS, 200 micrograms / mL sheared and denatured salmon sperm DNA, and 35% formamide at 42° C. for 12-24 hours, followed by three washes for 15 minutes each using 0.2X SSC, 0.2% SDS at 60° C. High stringency conditions refer to prehybridization and hybridization in 5X SSPE, 0.3% SDS, 200 micrograms / mL sheared and denatured salmon sperm DNA, and 50% formamide at 42° C. for 12-24 hours, followed by three washes for 15 minutes each using 0.2X SSC, 0.2% SDS at 65° C. Very high stringency conditions refer to 12-24 hour prehybridization and hybridization in 5X SSPE, 0.3% SDS, 200 micrograms / mL sheared and denatured salmon sperm DNA, and 50% formamide at 42° C., followed by three washes for 15 minutes each using 0.2X SSC, 0.2% SDS at 70° C. Improved properties: The term "improved properties" is defined herein as characteristics associated with an improved variant compared to the parent α-amylase.Such improved properties include, but are not limited to, increased amylolytic activity, improved catalytic efficiency, improved catalytic rate, increased chemical stability, increased oxidative stability, increased pH activity, increased pH stability, increased specific activity, increased substrate binding, increased substrate cleavage, increased substrate specificity, increased substrate stability, improved surface properties, increased thermal activity, improved thermal stability, improved cleaning performance such as soil removal performance, for example performance on starch-containing soils, stain removal, anti-graying, stability (e.g., thermal stability, pH stability, or stability in the presence of builders (including chelating agents), stability in powder, liquid, or gel detergent formulations or dishwashing compositions), modified temperature dependent performance and activity profile, pH activity, substrate specificity, product specificity, and chemical stability.

[0039] Improved cleaning performance: The term "improved cleaning performance" is defined herein as indicating an improvement in the cleaning performance of an amylase relative to the cleaning performance of a parent amylase, or relative to any of the amylases of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, for example, by increased stain removal. Improved cleaning performance may be measured by comparing so-called intensity values. Preferably, improved cleaning performance is determined according to the examples. Cleaning performance is improved if the improvement factor (IF) is greater than 1.0, preferably greater than 1.05, in one or more conditions listed in the examples. The cleaning conditions are described in the Examples section. The term "cleaning performance" generally includes cleaning of hard surface cleaning, for example, in dishwashing, but also includes cleaning performance on textiles, such as laundry, and industrial and institutional cleaning. Improved cleaning performance may be measured by comparing delta intensity as defined herein.

[0040] Isolated: The term "isolated" refers to a substance in a form or environment that does not occur in nature. Non-limiting examples of isolated substances include: (1) any substance that does not occur in nature; (2) any substance, including but not limited to, any enzyme, variant, nucleic acid, protein, peptide, or cofactor, that is at least partially removed from one or more or all of the naturally occurring components with which it is associated in nature; (3) any substance that has been artificially modified relative to the substance found in nature; or (4) any substance that has been modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., multiple copies of the gene encoding the substance; use of a stronger promoter than that naturally associated with the gene encoding the substance). An isolated substance may be present in a fermentation broth sample. In one aspect, the invention relates to an isolated α-amylase variant.

[0041] Intensity values: Wash performance is measured as brightness, which is expressed as the intensity of light reflected from a sample when illuminated with white light. When a sample is dyed, the intensity of the reflected light is lower than that of a clean sample. Therefore, the intensity of the reflected light can be used to measure wash performance, with higher intensity values ​​correlating with better wash performance. Color measurements are made with a specialized flatbed scanner (Kodak iQsmart, Kodak) that is used to capture images of the washed textiles. To extract light intensity values ​​from the scanned images, the 24-bit pixel values ​​from the images are converted to Red, Green, and Blue (RGB) values. The RGB values ​​are summed as a one-dimensional array and then the length of the resulting one-dimensional array is calculated to calculate the intensity value (Int):

[0042]

number

[0043] Low temperature: "Low temperature" is preferably a temperature of 5 to 40°C, or 5 to 35°C, or 5 to 30°C, or even 5 to 25°C, or 5 to 20°C, or 5 to 15°C, or 5 to 10°C. A preferred "low temperature" may be a temperature of 10 to 35°C, preferably 10 to 30°C, or 10 to 25°C, or 10 to 20°C, or 10 to 15°C.

[0044] Mature Polypeptide: The term "mature polypeptide" refers to a polypeptide in its final form after translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. It is known in the art that a host cell can produce a mixture of two or more different mature polypeptides (i.e., with different C-terminal and / or N-terminal amino acids) expressed by the same polynucleotide.

[0045] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" refers to a polynucleotide that encodes a mature polypeptide having alpha-amylase activity.

[0046] Moderate stringency conditions: The term "moderate stringency conditions" refers to prehybridization and hybridization for 12-24 hours according to standard Southern blotting procedures in 5X SSPE, 0.3% SDS, 200 micrograms / ml sheared and denatured salmon sperm DNA, and 35% formamide at 42° C. for a probe at least 100 nucleotides in length. Finally, the carrier material is washed three times for 15 minutes each using 2X SSC, 0.2% SDS at 55° C.

[0047] Medium-high stringency conditions: The term "medium-high stringency conditions" refers to prehybridization and hybridization for 12-24 hours according to standard Southern blotting procedures in 5X SSPE, 0.3% SDS, 200 micrograms / ml sheared and denatured salmon sperm DNA, and 35% formamide at 42° C. Finally, the carrier material is washed three times for 15 minutes each using 2X SSC, 0.2% SDS at 60° C.

[0048] Modification: In the context of the polypeptides of the invention, the term "modification" means that one or more amino acids in a reference amino acid sequence (e.g., SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14) are altered by substitution with another amino acid, by insertion of an amino acid, or by deletion, preferably by at least one deletion. The terms "modification", "alteration" and "mutation" are used interchangeably and may constitute the same meaning and purpose.

[0049] Mutant: The term "mutant" refers to a polynucleotide that encodes a variant.

[0050] Naive: The term "naive" refers to a nucleic acid or polypeptide that is naturally present in a host cell.

[0051] Nucleic acid construct: The term "nucleic acid construct" refers to a nucleic acid molecule that is either single-stranded or double-stranded, containing one or more regulatory sequences, isolated from a naturally occurring gene or modified to contain a segment of nucleic acid in a manner not otherwise found in nature, or is synthetic. The term nucleic acid construct is synonymous with the term "expression cassette" when the nucleic acid construct contains the regulatory sequences required for expression of a coding sequence of the present invention.

[0052] Operably linked: The term "operably linked" refers to a configuration in which a control sequence is positioned appropriately relative to a coding sequence of a polynucleotide such that the control sequences direct the expression of the coding sequence.

[0053] Parent or parent α-amylase: As used herein, the term "parent" or "parent α-amylase" refers to an α-amylase that is modified to produce a variant α-amylase of the invention. The term also refers to a polypeptide to which the variant of the invention is compared. A parent is preferably a naturally occurring (wild type) polypeptide, or a hybrid polypeptide thereof, prepared by any suitable means. A parent can also be a variant of a wild type polypeptide, or a variant of a hybrid polypeptide, prepared by any suitable means. For example, a parent protein can be a variant of a naturally occurring polypeptide that has been modified or altered in amino acid sequence. Thus, the parent α-amylase can have one or more (or one or more) amino acid substitutions, deletions and / or insertions. Thus, the parent α-amylase can be a variant of a naturally occurring parent α-amylase. A parent can also be an allelic variant, which is a polypeptide encoded by any of two or more alternative forms of a gene occupying the same chromosomal locus. A preferred "parent" or "parent α-amylase" as used herein is selected from the α-amylases of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14. A suitable parent may be any α-amylase having at least 60% sequence identity to any of the polypeptides of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14. A parent α-amylase may also be a polypeptide comprising a fragment of any of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14. A parent may be a bacterial or fungal α-amylase, preferably a bacterial α-amylase. An amylase having SEQ ID NO: 1, 2 or 3 may for example be an essential parent or hybrid polypeptide of the present invention.

[0054] Purified: The term "purified" refers to a nucleic acid or polypeptide that is substantially free of other components as determined by analytical techniques well known in the art (e.g., a purified polypeptide or nucleic acid may form a distinct band in an electrophoretic gel, a chromatographic eluate, and / or a medium subjected to density gradient centrifugation). A purified nucleic acid or polypeptide is at least about 50% pure, usually at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8% or more (e.g., weight percent on a molar basis). In a related sense, a composition is enriched for a molecule if the concentration of the molecule is significantly increased following application of a purification or concentration technique. The term "enriched" refers to a compound, polypeptide, cell, nucleic acid, amino acid, or other particular substance or component that is present in a composition at a higher relative or absolute concentration than in the starting composition.

[0055] Recombinant: When used in reference to a cell, nucleic acid, protein, or vector, the term "recombinant" means modified from its natural state. Thus, for example, a recombinant cell expresses genes not found in the native (non-recombinant) form of the cell, or expresses native genes at different levels or under different conditions than found in nature. A recombinant nucleic acid differs from the native sequence by one or more nucleotides and / or is operably linked to a heterologous sequence, e.g., a heterologous promoter in an expression vector. A recombinant protein may differ from the native sequence by one or more amino acids and / or may be fused to a heterologous sequence. A vector containing a nucleic acid encoding a polypeptide is a recombinant vector. The term "recombinant" is synonymous with "genetically modified" and "genetically modified."

[0056] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is expressed by the parameter "sequence identity".

[0057] For the purposes of the present invention, sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), preferably version 5.0.0 or later. The parameters used may be a gap opening penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percentage identity, which is calculated as follows: (identical residues × 100) / (length of alignment − total number of gaps in the alignment)

[0058] For the purposes of the present invention, sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, see above), as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, see above), preferably version 5.0.0 or later. The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percentage identity, which is calculated as follows: (identical deoxyribonucleotides × 100) / (length of alignment – ​​total number of gaps in alignment)

[0059] Signal peptide: The term "signal peptide" is defined herein as a peptide that is linked (fused) in-frame to the amino terminus of a biologically active polypeptide and directs the polypeptide into the secretory pathway of a cell. Signal sequences can be determined using techniques known in the art (see, e.g., Zhang and Henzel, 2004, Protein Science 13:2819-2824).

[0060] Starch binding domain: The terms "starch binding domain (SBD) or carbohydrate binding module (CBM)" are used interchangeably herein. SBDs can be divided into nine CBM families. As an energy source, starch is degraded by a large number of different amylolytic enzymes. However, only about 10 percent of them are capable of binding and degrading raw starch. These enzymes usually have a separate sequence structure module called the starch binding domain that mediates attachment to starch granules. SBDs refer to amino acid sequences that preferentially bind to starch (polysaccharide) substrates or maltosaccharides, such as α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin. They are usually motifs of about 100 amino acid residues found in about 10 percent of microbial amylolytic enzymes.

[0061] Subsequence: The term "subsequence" refers to a polynucleotide having one or more (e.g., several) nucleotides deleted from the 5'- and / or 3'-end of a mature polypeptide coding sequence, where the subsequence encodes a fragment that has α-amylase activity.

[0062] Fabric care benefits: The term "fabric care benefits" as used herein is defined as those not directly related to catalytic stain removal or prevention of redeposition of soil, but are also important for enzyme detergent benefits. Examples of such fabric care benefits are prevention or reduction of dye transfer from one fabric to another or to another part of the same fabric (anti-transfer), removal of protruding or broken fibers from the fabric surface to reduce pilling tendency or remove existing pills or fuzz (anti-pilling), improvement of fabric softness, clarification of fabric color, and removal of particulate soil trapped in fabric fibers. Enzyme bleaching is a further enzyme detergent benefit, whose catalytic activity is commonly used to catalyze the formation of bleaching components such as hydrogen peroxide or other peroxides or other bleaching substances.

[0063] Variant: As used herein, the term "variant" refers to a polypeptide having α-amylase activity that includes a modification, i.e., a substitution, an insertion, and / or a deletion, at one or more (e.g., several) positions. A substitution refers to replacing an amino acid occupying a position with a different amino acid, a deletion refers to removing an amino acid occupying a position, and an insertion refers to adding an amino acid adjacent to and immediately following the amino acid occupying a position. A variant has at least 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of one or more α-amylase activities of the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0064] Very high stringency conditions: The term "very high stringency conditions" refers to prehybridization and hybridization for 12-24 hours according to standard Southern blotting procedures in 5X SSPE, 0.3% SDS, 200 micrograms / mL sheared and denatured salmon sperm DNA, and 50% formamide at 42° C. Finally, the carrier material is washed three times for 15 minutes each using 2X SSC, 0.2% SDS at 70° C.

[0065] Very low stringency conditions: The term "very low stringency conditions" refers to prehybridization and hybridization for 12-24 hours according to standard Southern blotting procedures in 5X SSPE, 0.3% SDS, 200 micrograms / ml sheared and denatured salmon sperm DNA, and 25% formamide at 42° C. Finally, the carrier material is washed three times for 15 minutes each using 2X SSC, 0.2% SDS at 45° C.

[0066] Washing performance: In the present context, the term "washing performance" is used as the ability of an enzyme to remove starch or starch-containing stains present on an object to be washed during cleaning of hard surfaces, such as laundry or dishwashing. The term "washing performance" generally includes cleaning of hard surfaces, such as dishwashing, but also includes cleaning performance on textiles, such as laundry, as well as industrial and institutional cleaning. Washing performance can be quantified by calculating a so-called strength value.

[0067] Wild-type enzyme: The term "wild-type" α-amylase refers to an α-amylase expressed by a naturally occurring microorganism, such as a bacterium, yeast, or filamentous fungus, as found in nature. The terms "wild-type enzyme" and "parent enzyme" may be used interchangeably, where the parent enzyme is not a mutant enzyme.

[0068] nomenclature For purposes of the present invention, the nomenclature [Y / F] means that the amino acid at this position can be tyrosine (Try, Y) or phenylalanine (Phe, F). Similarly, the nomenclature [V / G / A / I] means that the amino acid at this position can be valine (Val, V), glycine (Gly, G), alanine (Ala, A), or isoleucine (Ile, I), as well as other combinations described herein. The amino acid X is defined to be any of the 20 naturally occurring amino acids, unless otherwise specified.

[0069] Mutant designation convention For the purposes of the present invention, the polypeptide disclosed in SEQ ID NO: 1 is used to determine the corresponding amino acid residue in another α-amylase, unless otherwise specified. Alternatively, the polypeptide disclosed in SEQ ID NO: 2 or SEQ ID NO: 3 is used to determine the corresponding amino acid residue in another α-amylase. The amino acid sequence of another α-amylase is aligned with the polypeptide disclosed in SEQ ID NO: 1, and based on the alignment, the amino acid position number corresponding to any amino acid residue in the polypeptide disclosed in SEQ ID NO: 2 or SEQ ID NO: 3 is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 or higher.

[0070] Identification of corresponding amino acid residues in other α-amylases can be achieved using a number of tools, including but not limited to MUSCLE (multiple sequence comparison by log prediction; version 3.5 or higher; Edgar, 2004, Nucleic Acids Research 32:1792-1797), MAFFT (version 6.857 or higher; Katoh and Kuma, 2002, Nucleic Acids Research 30:3059-3066; Katoh et al., 2005, Nucleic Acids Research 33:511-518; Katoh and Toh, 2007, Bioinformatics 23:372-374; Katoh et al., 2009, Methods in Molecular Biology 537:39-64; Katoh and Toh, 2010, Bioinformatics 26:1899-1900), and EMBOSS EMMA employing This can be determined by aligning multiple polypeptide sequences using several computer programs, including EMBOSS EMMA using ClustalW (1.83 or higher; Thompson et al., 1994, Nucleic Acids Research 22:4673-4680).

[0071] Other pairwise sequence comparison algorithms may be used when other enzymes have diverged from the polypeptide of SEQ ID NO:1, such that traditional sequence-based comparisons fail to detect these relationships (Lindahl and Elofsson, 2000, J. Mol. Biol. 295:613-615). Greater sensitivity in sequence-based searches can be obtained using search programs that utilize probabilistic representations (profiles) of polypeptide families to search databases. For example, the PSI-BLAST program can generate profiles through an iterative database search process to detect distant homologs (see Atschul et al., 1997, Nucleic Acids Res. 25:3389-3402).

[0072] Greater sensitivity can be obtained when a family or superfamily of polypeptides has more than one representation in a protein structure database. Programs such as GenTHREADER (Jones, 1999, J. Mol. Biol. 287:797-815; McGuffin and Jones, 2003, Bioinformatics 19:874-881) use information from various sources (PSI-BLAST, secondary structure prediction, structural alignment profiles, and solvation potential) as input to a neural network that predicts the structural fold of a query sequence. Similarly, sequences of unknown structure can be aligned with models of superfamilies present in the SCOP database using the method of Gough et al., 2000, J. Mol. Biol. 313:903-919. These alignments can then be used to generate homology models of the polypeptides, and such models can be assessed for accuracy using various tools developed for that purpose.

[0073] For proteins of known structure, several tools and sources are available for searching and creating structural alignments. For example, the SCOP superfamily of proteins has been structurally aligned, and these alignments are accessible and downloadable. Two or more protein structures can be aligned using various algorithms such as distance alignment matrices (Holm and Sander, 1998, Proteins 33:88-96) or combinatorial extensions (Shindyalov and Bourne, 1998, Protein Engineering 11:739-747), and implementations of these algorithms can be further used to query structural databases for structures of interest to find potential structural homologs (e.g., Holm and Park, 2000, Bioinformatics 16:566-567).

[0074] In describing the variants, the following nomenclature is adapted for ease of reference: the commonly accepted IUPAC one-letter or three-letter amino acid abbreviations are used.

[0075] Substitutions: For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Thus, a substitution of threonine at position 226 with alanine is designated as "Thr226Ala" or "T226A". In situations where the amino acid at a given position may be substituted with any other amino acid, it is designated as T226ACDEFGHIKLMNPQRSWVY. This therefore means that the threonine at position 226 may be substituted with one amino acid selected from the group A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, W, V, or Y. Similarly, in situations where the amino acid at a given position may be substituted with one amino acid selected from a particular group of amino acids, for example, the threonine at position 226 may be substituted with either tyrosine, phenylalanine, or histidine, it is designated as T226YFH. Different modifications at a given position may be separated by commas, for example, "Arg170Tyr,Glu" or "R170Y,E" represent the substitution of arginine at position 170 with tyrosine or glutamic acid. Thus, "Tyr167Gly,Ala+Arg170Gly,Ala" represents the variants "Tyr167Gly+Arg170Gly", "Tyr167Gly+Arg170Ala", "Tyr167Ala+Arg170Gly", and "Tyr167Ala+Arg170Ala".

[0076] Multiple mutations are separated by adding a mark ("+"), for example, "Gly205Arg+Ser411Phe" or "G205R+S411F" represent the substitution of glycine (G) with arginine (R) and serine (S) with phenylalanine (F) at positions 205 and 411, respectively.

[0077] Deletions. For amino acid deletions, the following nomenclature is used: original amino acid, position, and an alternating sequence (*). Thus, a deletion of glycine at position 195 is written as "Gly195*" or "G195*". Multiple deletions are separated by an additive symbol ("+"), e.g., "Gly195*+Ser411*" or "G195*+S411*".

[0078] Insertions. For amino acid insertions, the following nomenclature is used: original amino acid, position, original amino acid, inserted amino acid. Thus, an insertion of a lysine after the glycine at position 195 is designated as "Gly195GlyLys" or "G195GK." Insertions of multiple amino acids are designated as [original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2; etc.]. For example, an insertion of a lysine and an alanine after the glycine at position 195 is designated as "Gly195GlyLysAla" or "G195GKA."

[0079] In such cases, the inserted amino acid residue is numbered by adding a lower case letter to the position number of the amino acid residue preceding the inserted amino acid residue. In the above example, the sequence might be:

[0080] [Table 1]

[0081] Multiple modifications. Variants containing multiple modifications are separated by adding an addition symbol ("+"), for example, "Arg170Tyr+Gly195Glu" or "R170Y+G195E" represent the substitution of arginine and glycine with tyrosine and glutamic acid at positions 170 and 195, respectively.

[0082] Detailed Description of the Invention The present invention relates to (a) Using SEQ ID NO:1 for numbering, positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 165 , 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, 3 62, 365, 371, 383, 390, 394, 395, 405, 409, 414, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, wherein the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least and an α-amylase variant having at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to said α-amylase variant. and a cleaning adjunct.

[0083] Parent α-amylase The polypeptide having amylase activity may be a fusion (hybrid) polypeptide.

[0084] A polypeptide having α-amylase activity may comprise an A domain having at least 60% sequence identity to the A domain of any of SEQ ID NOs: 1, 2, 3, 4, or 5, a B domain having at least 60% sequence identity to the B domain of any of SEQ ID NOs: 1, 2, 10, 11, or 12, and a C domain having at least 60% sequence identity to the C domain of any of SEQ ID NOs: 1, 2, 3, 4, or 5.

[0085] The polypeptide having alpha-amylase activity may comprise a hybrid alpha-amylase of a parent polypeptide having alpha-amylase activity comprising an A domain, a B domain, and a C domain, the hybrid being a fusion polypeptide.

[0086] Suitable A domains have at least 60% sequence identity to the A domain of SEQ ID NO:1, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0087] Suitable A domains have at least 60% sequence identity to the A domain of SEQ ID NO:2, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0088] Suitable A domains have at least 60% sequence identity to the A domain of SEQ ID NO:3, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0089] Suitable A domains have at least 60% sequence identity to the A domain of SEQ ID NO:4, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0090] Suitable A domains have at least 60% sequence identity to the A domain of SEQ ID NO:5, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0091] Suitable B domains have at least 60% sequence identity to the B domain of SEQ ID NO:1, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0092] A suitable B domain has at least 60% sequence identity to the B domain of SEQ ID NO:2, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0093] A suitable B domain has at least 60% sequence identity to the B domain of SEQ ID NO:10, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0094] Suitable B domains have at least 60% sequence identity to the B domain of SEQ ID NO:11, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0095] A suitable B domain has at least 60% sequence identity to the B domain of SEQ ID NO:12, e.g. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0096] A suitable C domain has at least 60% sequence identity to the C domain of SEQ ID NO:1, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0097] A suitable C domain has at least 60% sequence identity to the C domain of SEQ ID NO:2, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0098] A suitable C domain has at least 60% sequence identity to the C domain of SEQ ID NO:3, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0099] A suitable C domain has at least 60% sequence identity to the C domain of SEQ ID NO:4, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0100] A suitable C domain has at least 60% sequence identity to the C domain of SEQ ID NO:5, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0101] Alpha-amylases useful herein can be produced by replacing the B domain, or a portion thereof, of one alpha-amylase with the B domain, or a portion thereof, of another alpha-amylase. Alpha-amylases can also be produced by replacing the A and C domains, or portions thereof, of one alpha-amylase with the A and C domains, or portions thereof, of another alpha-amylase.

[0102] In the case of Bacillus α-amylase (SEQ ID NO: 4), where the B domain is determined as amino acid residues 109-206, the sequence to be replaced by the corresponding sequence of another α-amylase starts at a position in the range of positions 93-113 and ends at a position in the range of positions 195-215. For example, it could start at a position in the range of positions 97-109 and end at a position in the range of positions 199-215, or it could start at a position in the range of positions 100-106 and end at a position in the range of positions 202-208, in particular at position 109-206 of SEQ ID NO: 12. The A and C domains of Bacillus α-amylase were determined to be amino acid residues 1-108 (A1)+207-396 (A2) and 397-485, respectively. The α-amylase of the invention may comprise an A1 domain starting at a position in the range of positions 1-5 and ending at a position in the range of positions 94-114, for example starting at a position in the range of positions 1-3 and ending at a position in the range of positions 99-110, or starting at a position in the range of positions 1-3 and ending at a position in the range of positions 104-109, particularly in the range of positions 1-108 of SEQ ID NO: 4. The α-amylase of the invention may comprise an A2 domain and a C domain starting at a position in the range of positions 201-221 and ending at a position in the range of positions 478-514, for example starting at a position in the range of positions 203-211 and ending at a position in the range of positions 480-510, or starting at a position in the range of positions 205-216 and ending at a position 482-484, particularly in the range of positions 207-485 of SEQ ID NO: 4.

[0103] The hybrid polypeptide may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 1 or 2, or SEQ ID NO: 3. In another embodiment, the hybrid polypeptide is SEQ ID NO: 1, 2, or 3, or an allelic variant of the polypeptide of SEQ ID NO: 1 or 2.

[0104] In a preferred embodiment, the parent polypeptide may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 1. As noted elsewhere herein, the parent polypeptide may be any polypeptide that has α-amylase activity and has at least 60% sequence identity to any one of the amino acid sequences set forth in any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0105] In a preferred embodiment, the parent polypeptide has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, and the parent polypeptide has α-amylase activity. In one embodiment, the amino acid sequence of the parent polypeptide differs from the amino acid sequence set forth in SEQ ID NO: 1-14 by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

[0106] According to the present invention, a value of 1.0 corresponds to the performance observed for the parent polypeptide. A value greater than 1.0 indicates an improved performance of the tested hybrid compared to the parent polypeptide. Thus, a value >1.0 indicates an improved property of the hybrid polypeptide, e.g., an increased specific activity, compared to the parent polypeptide.

[0107] In one embodiment, the hybrid polypeptide is the parent polypeptide.

[0108] The parent α-amylase can be a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to any one of the polypeptides of SEQ ID NOs: 1-14.

[0109] In one aspect, the parent has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 1 and has α-amylase activity. In one embodiment, the amino acid sequence of the parent differs from the polypeptide of SEQ ID NO: 1 by 10 or less amino acids, e.g., 5 amino acids, 4 amino acids, 3 amino acids, 2 amino acids, and 1 amino acid.

[0110] The parent preferably comprises or consists of the amino acid sequence of SEQ ID NO: 1. In one embodiment, the parent comprises or consists of the polypeptide of SEQ ID NO: 1. In another embodiment, the parent is an allelic variant of the polypeptide of SEQ ID NO: 1.

[0111] In one aspect, the parent has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 2 and has α-amylase activity. In one embodiment, the amino acid sequence of the parent differs from the polypeptide of SEQ ID NO: 2 by 10 or less amino acids, e.g., 5 amino acids, 4 amino acids, 3 amino acids, 2 amino acids, and 1 amino acid.

[0112] The parent preferably comprises or consists of the amino acid sequence of SEQ ID NO: 2. In one embodiment, the parent comprises or consists of the polypeptide of SEQ ID NO: 2. In another embodiment, the parent is an allelic variant of the polypeptide of SEQ ID NO: 2.

[0113] In one aspect, the parent has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 3 and has α-amylase activity. In one embodiment, the amino acid sequence of the parent differs from the polypeptide of SEQ ID NO: 3 by 10 or less amino acids, e.g., 5 amino acids, 4 amino acids, 3 amino acids, 2 amino acids, and 1 amino acid.

[0114] The parent preferably comprises or consists of the amino acid sequence of SEQ ID NO: 3. In one embodiment, the parent comprises or consists of the polypeptide of SEQ ID NO: 3. In another embodiment, the parent is an allelic variant of the polypeptide of SEQ ID NO: 3.

[0115] In one aspect, the parent has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 4 and has α-amylase activity. In one embodiment, the amino acid sequence of the parent differs from the polypeptide of SEQ ID NO: 4 by 10 or less amino acids, e.g., 5 amino acids, 4 amino acids, 3 amino acids, 2 amino acids, and 1 amino acid.

[0116] The parent preferably comprises or consists of the amino acid sequence of SEQ ID NO: 4. In one embodiment, the parent comprises or consists of the polypeptide of SEQ ID NO: 4. In another embodiment, the parent is an allelic variant of the polypeptide of SEQ ID NO: 4.

[0117] In one aspect, the parent has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 5 and has α-amylase activity. In one embodiment, the amino acid sequence of the parent differs from the polypeptide of SEQ ID NO: 5 by 10 or less amino acids, e.g., 5 amino acids, 4 amino acids, 3 amino acids, 2 amino acids, and 1 amino acid.

[0118] The parent preferably comprises or consists of the amino acid sequence of SEQ ID NO: 5. In one embodiment, the parent comprises or consists of the polypeptide of SEQ ID NO: 5. In another embodiment, the parent is an allelic variant of the polypeptide of SEQ ID NO: 5.

[0119] In one aspect, the parent has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 6 and has α-amylase activity. In one embodiment, the amino acid sequence of the parent differs from the polypeptide of SEQ ID NO: 6 by 10 or less amino acids, e.g., 5 amino acids, 4 amino acids, 3 amino acids, 2 amino acids, and 1 amino acid.

[0120] The parent preferably comprises or consists of the amino acid sequence of SEQ ID NO: 6. In one embodiment, the parent comprises or consists of the polypeptide of SEQ ID NO: 6. In another embodiment, the parent is an allelic variant of the polypeptide of SEQ ID NO: 6.

[0121] In one aspect, the parent has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 7 and has α-amylase activity. In one embodiment, the amino acid sequence of the parent differs from the polypeptide of SEQ ID NO: 7 by 10 or less amino acids, e.g., 5 amino acids, 4 amino acids, 3 amino acids, 2 amino acids, and 1 amino acid.

[0122] The parent preferably comprises or consists of the amino acid sequence of SEQ ID NO: 7. In one embodiment, the parent comprises or consists of the polypeptide of SEQ ID NO: 7. In another embodiment, the parent is an allelic variant of the polypeptide of SEQ ID NO: 7.

[0123] In one aspect, the parent has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 8 and has α-amylase activity. In one embodiment, the amino acid sequence of the parent differs from the polypeptide of SEQ ID NO: 8 by 10 or less amino acids, e.g., 5 amino acids, 4 amino acids, 3 amino acids, 2 amino acids, and 1 amino acid.

[0124] The parent preferably comprises or consists of the amino acid sequence of SEQ ID NO: 8. In one embodiment, the parent comprises or consists of the polypeptide of SEQ ID NO: 8. In another embodiment, the parent is an allelic variant of the polypeptide of SEQ ID NO: 8.

[0125] In one aspect, the parent has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 9 and has α-amylase activity. In one embodiment, the amino acid sequence of the parent differs from the polypeptide of SEQ ID NO: 9 by 10 or less amino acids, e.g., 5 amino acids, 4 amino acids, 3 amino acids, 2 amino acids, and 1 amino acid.

[0126] The parent preferably comprises or consists of the amino acid sequence of SEQ ID NO: 9. In one embodiment, the parent comprises or consists of the polypeptide of SEQ ID NO: 9. In another embodiment, the parent is an allelic variant of the polypeptide of SEQ ID NO: 9.

[0127] In one aspect, the parent has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 10 and has α-amylase activity. In one embodiment, the amino acid sequence of the parent differs from the polypeptide of SEQ ID NO: 10 by 10 or less amino acids, e.g., 5 amino acids, 4 amino acids, 3 amino acids, 2 amino acids, and 1 amino acid.

[0128] The parent preferably comprises or consists of the amino acid sequence of SEQ ID NO: 10. In one embodiment, the parent comprises or consists of the polypeptide of SEQ ID NO: 10. In another embodiment, the parent is an allelic variant of the polypeptide of SEQ ID NO: 10.

[0129] In one aspect, the parent has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 11 and has α-amylase activity. In one embodiment, the amino acid sequence of the parent differs from the polypeptide of SEQ ID NO: 11 by 10 or less amino acids, e.g., 5 amino acids, 4 amino acids, 3 amino acids, 2 amino acids, and 1 amino acid.

[0130] The parent preferably comprises or consists of the amino acid sequence of SEQ ID NO: 11. In one embodiment, the parent comprises or consists of the polypeptide of SEQ ID NO: 11. In another embodiment, the parent is an allelic variant of the polypeptide of SEQ ID NO: 11.

[0131] In one aspect, the parent has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 12 and has α-amylase activity. In one embodiment, the amino acid sequence of the parent differs from the polypeptide of SEQ ID NO: 12 by 10 or less amino acids, e.g., 5 amino acids, 4 amino acids, 3 amino acids, 2 amino acids, and 1 amino acid.

[0132] The parent preferably comprises or consists of the amino acid sequence of SEQ ID NO: 12. In one embodiment, the parent comprises or consists of the polypeptide of SEQ ID NO: 12. In another embodiment, the parent is an allelic variant of the polypeptide of SEQ ID NO: 12.

[0133] In one aspect, the parent has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 13 and has α-amylase activity. In one embodiment, the amino acid sequence of the parent differs from the polypeptide of SEQ ID NO: 13 by 10 or less amino acids, e.g., 5 amino acids, 4 amino acids, 3 amino acids, 2 amino acids, and 1 amino acid.

[0134] The parent preferably comprises or consists of the amino acid sequence of SEQ ID NO: 13. In one embodiment, the parent comprises or consists of the polypeptide of SEQ ID NO: 13. In another embodiment, the parent is an allelic variant of the polypeptide of SEQ ID NO: 13.

[0135] In one aspect, the parent has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 14 and has α-amylase activity. In one embodiment, the amino acid sequence of the parent differs from the polypeptide of SEQ ID NO: 14 by 10 or less amino acids, e.g., 5 amino acids, 4 amino acids, 3 amino acids, 2 amino acids, and 1 amino acid.

[0136] The parent preferably comprises or consists of the amino acid sequence of SEQ ID NO: 14. In one embodiment, the parent comprises or consists of the polypeptide of SEQ ID NO: 14. In another embodiment, the parent is an allelic variant of the polypeptide of SEQ ID NO: 14.

[0137] The amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or fragments thereof, can be used to design nucleic acid probes to identify and clone DNA encoding the parent from strains of different genera or species, according to methods well known in the art. In particular, such probes can be used to hybridize the genome or cDNA of the desired genus or species, followed by a conventional Southern blotting procedure to identify and isolate the corresponding genes therein. Such probes can be significantly shorter than the entire sequence, but must be at least 14, e.g., at least 25, at least 35, or at least 70 nucleotides in length. Preferably, the nucleic acid probe is at least 100 nucleotides in length, e.g., at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, or at least 900 nucleotides in length. Both DNA and RNA can be used. The probes are usually labeled to detect the corresponding gene (e.g., with 32P, 3H, 35S, biotin, or avidin). Such probes are encompassed by the present invention.

[0138] Genomic DNA or cDNA libraries prepared from such other strains can be screened for DNA that hybridizes with the above-mentioned probes and encodes the parent. Genomic or other DNA from such other strains can be separated by agarose or polyacrylamide electrophoresis, or other separation techniques. DNA from the library or separated DNA can be transferred and immobilized on nitrocellulose or other suitable carrier material. The carrier material is used in a Southern blot procedure to identify clones or DNA that hybridize to the parent or its subsequence.

[0139] For purposes of the present invention, hybridization refers to the hybridization of a polynucleotide under low to very high stringency conditions to a labeled nucleotide probe corresponding to a polynucleotide encoding SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or a subsequence thereof. Molecules to which the probe hybridizes can be detected, for example, using x-ray film or other detection means known in the art.

[0140] In one aspect, the nucleic acid probe is a polynucleotide encoding a polypeptide of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or a fragment thereof.

[0141] For long probes at least 100 nucleotides in length, very low to very high stringency conditions are optimally defined as prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms / mL sheared denatured salmon sperm DNA, and either 25% formamide for very low and low stringency, 35% formamide for medium and above-medium stringency, or 50% formamide for high and very high stringency, followed by standard Southern blotting procedures for 12 to 24 hours. Finally, the carrier material is washed three times for 15 minutes each with 2xSSC, 0.2% SDS at 45°C (very low stringency), 50°C (low stringency), 55°C (medium stringency), 60°C (medium-upper stringency), 65°C (high stringency), or 70°C (very high stringency).

[0142] For short probes, ranging from about 15 nucleotides to about 70 nucleotides in length, stringency conditions are optimally defined as prehybridization and hybridization for 12 to 24 hours following standard Southern blotting procedures at about 5° C. to about 10° C. below the Tm calculated using calculations according to Bolton and McCarthy (1962, Proc. Natl. Acad. Sci. USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40, 1× Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg yeast RNA per ml. Finally, the support material is washed once for 15 minutes in 6X SSC and 0.1% SDS, and twice for 15 minutes each with 6X SSC at a temperature about 5° C. to about 10° C. below the calculated Tm.

[0143] A parent can be obtained from any genus of microorganism. For purposes of the present invention, the term "obtained from" as used herein in reference to a given source shall mean that the parent encoded by the polynucleotide is produced by the source or by the strain into which the polynucleotide from the source has been inserted. In one embodiment, the parent is secreted extracellularly.

[0144] The parent may be a bacterial α-amylase, for example, the parent may be a gram-positive bacterial polypeptide, such as a Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces. or a Gram-negative bacterial polypeptide, such as an alpha-amylase from Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neissera, Pseudomonas, Salmonella, or Ureaplasma.

[0145] In one embodiment, the parent is Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis α-amylase.

[0146] In another embodiment, the parent is an α-amylase from Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus.

[0147] In another embodiment, the parent is an α-amylase from Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans.

[0148] In another aspect, the parent is a Bacillus sp. α-amylase, for example, the α-amylase of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14.

[0149] For the aforementioned species, it will be understood that the invention encompasses perfect and imperfect states, as well as other taxonomic equivalents, such as anamorphs, regardless of the species name by which they are known. Those of skill in the art will readily recognize the identity of appropriate equivalents.

[0150] Strains of these species are readily available to the public in numerous culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and the Agricultural Research Service, Culture Collection, Northern Regional Research Center (NRRL).

[0151] Parents can be identified and obtained from other sources, including microorganisms isolated from natural materials (e.g., soil, compost, water, etc.), or DNA samples obtained directly from natural materials (e.g., soil, compost, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. Polynucleotides encoding the parents can then be obtained by similarly screening genomic or cDNA libraries of other microorganisms, or mixed DNA samples. Once the polynucleotides encoding the parents are detected with the probes, the polynucleotides can be isolated or cloned by utilizing techniques known to those skilled in the art (see, for example, Sambrook et al., 1989, supra).

[0152] The parent may also be a hybrid polypeptide in which a portion of one polypeptide is fused at the N-terminus or C-terminus of a portion of another polypeptide.

[0153] The parent may also be a fusion polypeptide or a cleavable fusion polypeptide, in which one polypeptide is fused at the N-terminus or C-terminus of another polypeptide. A fusion polypeptide is produced by fusing a polynucleotide encoding one polypeptide to a polynucleotide encoding another polypeptide. Techniques for producing fusion polypeptides are known in the art and include ligating coding sequences encoding the polypeptides such that they are in frame, and the expression of the fused polypeptides is under the control of the same promoter and terminator. Fusion proteins may also be constructed using intein technology, in which the fusion is generated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266:776-779).

[0154] The fusion polypeptide may further comprise a cleavage site between the two polypeptides, which is cleaved upon secretion of the fusion protein, releasing the two polypeptides. Examples of cleavage sites include Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3:568-576, Svetina et al., 2000, J. Biotechnol. 76: 245-251, Rasmussen-Wilson et al. al., 1997, Appl.Environ.Microbiol.63:3488-3493, Ward et al., 1995, Biotechnology 13:498-503 and Contreras et al., 1991, Biotechnology 9:378-381, Eaton et al., 1986, Biochemistry 25:505-512, Collins-Racie et al., 1995, Biotechnology 13:982-987, Carter et al. al., 1989, Proteins: Structure, Function, and Genetics 6:240-248 and Stevens, 2003, Drug Discovery World 4:35-48.

[0155] Preparation of mutants The present invention relates to a method for obtaining a cleaning composition, the method comprising obtaining a variant having α-amylase activity, the variant comprising: (a) a nucleotide sequence corresponding to positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 118, 119, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, , 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 165, 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, 362, 365, 371, 383, 390, 394, 395, 405, 409, 414, 420, 422, 4 and (b) introducing modifications into a parent α-amylase at one or more positions corresponding to 27, 455, 457, 458, 470, 472, 473, 474, 475, and 476, wherein each modification is independently a substitution or deletion, and wherein the variant has α-amylase activity; and (b) recovering the variant; and combining the variant with a cleaning adjunct.

[0156] A method for obtaining a variant having α-amylase activity includes: (a) cloning the amino acid sequence at positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 165, 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202, 203, 205 , 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, 362, 365, 371, 383, 390, 394, 395, 405, 409, 41 introducing modifications into a parent α-amylase at one or more positions corresponding to 4, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, where each modification is independently a substitution or deletion, and the variant has α-amylase activity; and (b) recovering the variant.

[0157] In one aspect, a method for obtaining a variant having α-amylase activity comprises: (a) isolating a variant of the amino acid sequence set forth in any of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 at positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 108, 109, 210, 211, 212, 213, or 214, using SEQ ID NO: 1 for numbering; , 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 165, 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 1 88, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, 362, 365, 371, 383, 390, 39 introducing modifications into a parent α-amylase at one or more positions corresponding to 4, 395, 405, 409, 414, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, where each modification is independently a substitution or deletion, and the variant has α-amylase activity; and (b) recovering the variant.

[0158] In one embodiment, the modification is a substitution. In one embodiment, the modification is a deletion.

[0159] In another embodiment, a method for obtaining a variant with α-amylase activity comprises (a) introducing modifications into a parent α-amylase at one or more positions, the modifications being H1*, H2*, N3D, N3G, N3S, G7A, G7H, G7S, R26Y, A31S, R37A, R37N, R37V, T40G, T40N, A41D, N54S, V56T, A60V, A60P, L63F, K72R, G73L, K93R, N94D, Q98L, V103A, V103I, V103L, V104M, M105I, M105L, or the like of the amino acid sequence set forth in SEQ ID NO:1. N106D, A109G, G110K, A111G, A111L, G113Q, G113S, G113A, T114K, F116L, F1 16N, F116Q, F116W, V117G, D118Q, D118T, D123N, N126D, N128Y, T131I, T131V , S132D, S132L, T134D, Y135E, Q136T, Q136K, I137F, Q138V, W140S, W140M, W 140Y, K142E, K142G, K142P, D144N, P146S, P146H, T151A, Y152L, F155W, W159 H, W159L, Y160P, T165V, W167A, W167D, W167E, W167F, W167P, W167S, E169I, E169Q, E169V, K172G, K172Q, L173F, N174K, N174S, I176V, Y177D, K178A, R18 0*, R180Q, R180S, S181*, S181L, S181T, T182*, T182D, T182E, T182G, T182I , T182L, T182M, T182S, G183*, G183P, A185D, A185E, A185K, A185N, A185Q, A1 85R, W188C, E189P, E193I, N194F, F202Y, F202Y, A203F, L205F, L205N, L205 Q, L205R, L205V, L205Y, T224F, T224Y, N225S, Y242F, Y242L, S243Q, F244I, F 244L, V252I, T256A, T256D, K258V, E259G, E259V, M260D, M260W, F261K, F26 1P, F261S, F266Y, N276F, N276W, N276Y, N279S, N280F, N280W, N280Y, W283N,S286K, S286L, S302T, S302Y, G303R, T315A, H320T, P321H, P321Y, M322G, M322Y, H323A, H323N, H323W, W346N, L354V, E359W, Q360M, Q360W, Q360Y, G361M, Y362V, V365I, Y371S, Y371N, A383D, E390A, E390D, N394W, N394D, F 395Y, D405G, I409L, R414M, H420P, H420A, H420Q, H420Y, N422V, T427L, G455R, K457N, K457R, R458S, N470A, N470M, S472R, V473C, N474F, N474L, G475E, G475K, G476H, G476E, and G476A; and (b) recovering the mutant.

[0160] In another embodiment, a method for obtaining a variant with α-amylase activity comprises (a) introducing substitutions into a parent α-amylase at one or more positions, the substitutions being N3D, N3G, N3S, G7A, G7H, G7S, R26Y, A31S, R37A, R37N, R37V, T40G, T40N, A41D, N54S, V56T, A60V, A60P, L63F, K72R, G73L, K93R, N94D, or N94H of the amino acid sequence set forth in SEQ ID NO:2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, using SEQ ID NO:1 for numbering. , Q98L, V103A, V103I, V103L, V104M, M105I, M105L, N106D, A109G, G110K, A1 11G, A111L, G113Q, G113S, G113A, T114K, F116L, F116N, F116Q, F116W, V117G , D118Q, D118T, D123N, N126D, N128Y, T131I, T131V, S132D, S132L, T134D, Y 135E, Q136T, Q136K, I137F, Q138V, W140S, W140M, W140Y, K142E, K142G, K142 P, D144N, P146S, P146H, T151A, Y152L, F155W, W159H, W159L, Y160P, T165V, W167A, W167D, W167E, W167F, W167P, W167S, E169I, E169Q, E169V, K172G, K17 2Q, L173F, N174K, N174S, I176V, Y177D, K178A, R180Q, R180S, S181L, S181T , T182D, T182E, T182G, T182I, T182L, T182M, T182S, G183P, A185D, A185E, A1 85K, A185N, A185Q, A185R, W188C, E189P, E193I, N194F, F202Y, F202Y, A203 F, L205F, L205N, L205Q, L205R, L205V, L205Y, T224F, T224Y, N225S, Y242F, Y 242L, S243Q, F244I, F244L, V252I, T256A, T256D, K258V, E259G, E259V, M26 0D, M260W, F261K, F261P, F261S, F266Y, N276F, N276W, N276Y, N279S, N280F,N280W, N280Y, W283N, S286K, S286L, S302T, S302Y, G303R, T315A, H320T, P321H, P321Y, M322G, M322Y, H323A, H323N , H323W, W346N, L354V, E359W, Q360M, Q360W, Q360Y, G361M, Y362V, V365I, Y371S, Y371N, A383D, E390A, E390D, N394 W, N394D, F395Y, D405G, I409L, R414M, H420P, H420A, H420Q, H420Y, N422V, T427L, G455R, K457N, K457R, R458S, N470A, N470M, S472R, V473C, N474F, N474L, G475E, G475K, G476H, G476E, and G476A; and (b) recovering the mutant.

[0161] In one embodiment, the method further comprises introducing deletions into the parent α-amylase at one or more positions, the deletions being selected from H1*, H2*, R180*, S181*, T182*, and G183* of the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, using SEQ ID NO: 1 for numbering, and recovering the mutants.

[0162] Variants can be prepared using any mutagenesis means known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, and the like.

[0163] Site-directed mutagenesis is a technique in which one or more (several) mutations are made at one or more defined sites in a parent coding polynucleotide.

[0164] Site-directed mutagenesis can be performed in vitro by PCR with the use of oligonucleotide primers containing the desired mutation. Site-directed mutagenesis can also be performed in vitro by cassette mutagenesis, which involves cutting a site in a plasmid containing a parent encoding polynucleotide with a restriction enzyme, followed by ligation of an oligonucleotide containing the mutation in the polynucleotide. Usually, the restriction enzyme digested in the plasmid and the oligonucleotide is the same, allowing the sticky ends of the plasmid and the insert to ligate together. See, e.g., Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA 76:4949-4955 and Barton et al., 1990, Nucleic Acids Res. 18:7349-4966.

[0165] Site-directed mutagenesis can also be performed in vivo by methods known in the art, see, e.g., U.S. Patent Application Publication No. 2004 / 0171154; Storici et al., 2001, Nature Biotechnol. 19:773-776; Kren et al., 1998, Nat. Med. 4:285-290; and Calissano and Macino, 1996, Fungal Genet. Newslett. 43:15-16.

[0166] Any site-directed mutagenesis procedure can be used in the present invention. Many commercial kits are available that can be used to prepare mutants.

[0167] Synthetic gene construction requires the in vitro synthesis of polynucleotide molecules designed to encode a polypeptide of interest. Gene synthesis can be performed using a number of techniques, such as the multiplexed microchip-based technique described by Tian et al. (2004, Nature 432:1050-1054) and similar techniques, in which oligonucleotides are synthesized and assembled on a photoprogrammable microfluidic chip.

[0168] Single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested using known mutagenesis, recombination, and / or shuffling methods followed by associated screening procedures such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86:2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:10832-10837; U.S. Pat. No. 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0169] Mutagenesis / shuffling methods may be combined with high-throughput automated screening methods to detect activity of cloned mutant polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17:893-896). Mutant DNA molecules encoding active polypeptides can be recovered from the host cells and rapidly sequenced using methods standard in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.

[0170] Semi-synthetic gene construction is achieved by combining aspects of synthetic gene construction and / or site-directed mutagenesis and / or random mutagenesis and / or shuffling. Semi-synthetic construction is typified by a process that utilizes synthetic polynucleotide fragments combined with PCR technology. Thus, defined regions of a gene can be synthesized de novo, while other regions may be amplified using site-directed mutagenesis primers, and still other regions may be subjected to error-prone or non-error-prone PCR amplification. Subsequences of the polynucleotides may then be shuffled.

[0171] α-Amylase variants Suitable α-amylase variants of a parent α-amylase for use herein are those that are represented by the sequences at positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 165, 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 1 85, 188, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 35 9, 360, 361, 362, 365, 371, 383, 390, 394, 395, 405, 409, 414, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, and the variants have α-amylase activity.

[0172] The α-amylase variants are numbered as follows, using SEQ ID NO:1 for numbering: positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 165, 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, The variants may include modifications at one or more positions corresponding to 361, 362, 365, 371, 383, 390, 394, 395, 405, 409, 414, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, and such variants have α-amylase activity.

[0173] The α-amylase variants are identified by the following numbers using SEQ ID NO:1: positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 300, 309, 309, 309 5, 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, 362, 365 , 371, 383, 390, 394, 395, 405, 409, 414, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, wherein the variants have α-amylase activity, and the variants have a sequence similar to that of any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, at least 60%, at least 65%, at least 70%, at least at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity.

[0174] Suitable α-amylase variants include those at positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 165, 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, 362, 365, 371, 383, 390, 39 and wherein the variants have α-amylase activity, and the variants have a similar affinity to any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, and the variants have a similar affinity to any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, and the variants have a similar affinity to any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, The α-amylase variants may have at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100%, and the α-amylase variants have improved properties compared to the parent peptide.

[0175] Suitable α-amylase variants include those having the amino acid sequence at positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 127, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 8, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 165, 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202 , 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, 362, 365, 371, 383, 390 , 394, 395, 405, 409, 414, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, wherein the variants have α-amylase activity, and the α-amylase variants have improved properties compared to the parent peptide.

[0176] In one embodiment, the α-amylase variants are selected from the group consisting of α-amylases at positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 13 1, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 165, 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202, 203, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 27 24, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, 362, 365, 371, 383, 390, 394, 395, 405, 409, The variants comprise modifications at one or more positions corresponding to 414, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, and the variants have α-amylase activity, and the α-amylase variants have improved properties compared to the parent peptide, wherein the parent peptide is SEQ ID NO:1, 2, or 3.

[0177] Preferred variants comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the parent polypeptide. In another embodiment, the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the parent polypeptide having α-amylase activity.

[0178] Preferred variants have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the hybrid polypeptide.

[0179] Preferred variants have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to a hybrid polypeptide having α-amylase activity.

[0180] Preferred variants have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the polypeptide of SEQ ID NO:1.

[0181] Preferred variants have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the polypeptide of SEQ ID NO:2.

[0182] Preferred variants have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the polypeptide of SEQ ID NO:3.

[0183] Preferred variants have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the polypeptide of SEQ ID NO:4.

[0184] Preferred variants have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the polypeptide of SEQ ID NO:5.

[0185] Preferred variants have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the polypeptide of SEQ ID NO:6.

[0186] Preferred variants have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the polypeptide of SEQ ID NO:7.

[0187] Preferred variants have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the polypeptide of SEQ ID NO:8.

[0188] Preferred variants have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the polypeptide of SEQ ID NO:9.

[0189] Preferred variants have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the polypeptide of SEQ ID NO:10.

[0190] In another embodiment, the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the polypeptide of SEQ ID NO:11.

[0191] In another embodiment, the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the polypeptide of SEQ ID NO:12.

[0192] Preferred variants have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the polypeptide of SEQ ID NO:13.

[0193] Preferred variants have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the polypeptide of SEQ ID NO:14.

[0194] In one aspect, the substituted amino acid residue is different from the naturally occurring amino acid residue at that position, hi one embodiment, the substitution is selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, with the proviso that the substituted amino acid residue is different from the naturally occurring amino acid residue at that position.

[0195] In one aspect the number of modifications is 1 to 50, for example 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, or 1 to 5, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 modifications.

[0196] In one aspect, the number of substitutions is 1 to 50, for example, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, or 1 to 5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 substitutions.

[0197] In one aspect, the number of deletions is 1 to 50, e.g., 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, or 1 to 5, being 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 deletions.

[0198] The above-mentioned variants having substitutions should be understood to encompass all possible combinations of one or more substitutions at the specified positions.

[0199] In one embodiment, the α-amylase variants are located at positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 165, 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, 3 and comprising a modification at one or more positions corresponding to SEQ ID NOs: 62, 365, 371, 383, 390, 394, 395, 405, 409, 414, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, wherein the variants have α-amylase activity, and the variants have a sequence identity that is at least 60%, at least 65%, at least 70%, at least at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity.

[0200] In one aspect, the α-amylase variants are selected from the group consisting of α-amylase variants at positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 475 and 476, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204 5, 159, 160, 165, 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, 3 and the variants comprise modifications at one or more (e.g., several) positions corresponding to 62, 365, 371, 383, 390, 394, 395, 405, 409, 414, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, and the variants have α-amylase activity, and the variants have a homology of at least 60%, at least 65%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 10 ... 0%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity.

[0201] In another embodiment, the variants are located at positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 165, 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, 362 , 365, 371, 383, 390, 394, 395, 405, 409, 414, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, wherein the variants have α-amylase activity, and the variants have a sequence similar to that of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, and 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity.

[0202] In another embodiment, the variants are located at positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 165, 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, 362 , 365, 371, 383, 390, 394, 395, 405, 409, 414, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, wherein the variant has α-amylase activity, and the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity.

[0203] In another embodiment, the variants are located at positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 165, 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, 362 , 365, 371, 383, 390, 394, 395, 405, 409, 414, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, wherein the variants have α-amylase activity, and the variants have a sequence similar to that of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, respectively. 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity.

[0204] In another embodiment, the variants are located at positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 165, 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, 362 , 365, 371, 383, 390, 394, 395, 405, 409, 414, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, wherein the variants have α-amylase activity, and the variants have a sequence similar to that of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, respectively, and 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity.

[0205] In another embodiment, the variants are located at positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 165, 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, 362 , 365, 371, 383, 390, 394, 395, 405, 409, 414, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, wherein the variants have α-amylase activity, and the variants have a sequence similar to that of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, respectively, and 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity.

[0206] In another embodiment, the variants are located at positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 165, 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, 362 , 365, 371, 383, 390, 394, 395, 405, 409, 414, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, wherein the variants have α-amylase activity, and the variants have a sequence similar to that of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, and 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity.

[0207] In another embodiment, the variants are located at positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 165, 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, 362 , 365, 371, 383, 390, 394, 395, 405, 409, 414, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, wherein the variants have α-amylase activity, and the variants have a sequence similar to that of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, and 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity.

[0208] In another embodiment, the variants are located at positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 165, 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, 362 , 365, 371, 383, 390, 394, 395, 405, 409, 414, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, wherein the variants have α-amylase activity, and the variants have a sequence similar to that of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, and 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity.

[0209] In another embodiment, the variants are located at positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 165, 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, 362 , 365, 371, 383, 390, 394, 395, 405, 409, 414, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, wherein the variants have α-amylase activity, and the variants have a sequence similar to that of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, and 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity.

[0210] In another embodiment, the variants are located at positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 165, 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, 362 , 365, 371, 383, 390, 394, 395, 405, 409, 414, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, wherein the variants have α-amylase activity, and the variants have a sequence identity that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity.

[0211] In another embodiment, the variants are located at positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 165, 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, 362 , 365, 371, 383, 390, 394, 395, 405, 409, 414, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, wherein the variants have α-amylase activity, and the variants have a sequence identity that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity.

[0212] In another embodiment, the variants are located at positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 165, 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, 362 , 365, 371, 383, 390, 394, 395, 405, 409, 414, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, wherein the variants have α-amylase activity, and the variants have a sequence identity that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity.

[0213] In another embodiment, the variants are located at positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 165, 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, 362 , 365, 371, 383, 390, 394, 395, 405, 409, 414, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, wherein the variants have α-amylase activity, and the variants have a sequence identity that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity.

[0214] In another embodiment, the variants are located at positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 165, 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, 362 , 365, 371, 383, 390, 394, 395, 405, 409, 414, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, wherein the variants have α-amylase activity, and the variants have a sequence similar to that of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, respectively, and 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity.

[0215] In another embodiment, the variants are located at positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 165, 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, and comprising modifications at 16 positions corresponding to 362, 365, 371, 383, 390, 394, 395, 405, 409, 414, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, wherein the variants have α-amylase activity, and the variants have a sequence identity that is at least 60%, at least 65%, at least 70%, at least at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity.

[0216] In another embodiment, the variants are located at positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 165, 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, 362 , 365, 371, 383, 390, 394, 395, 405, 409, 414, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, wherein the variants have α-amylase activity, and the variants have a sequence identity that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity.

[0217] In another embodiment, the variants are located at positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 165, 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, 362 , 365, 371, 383, 390, 394, 395, 405, 409, 414, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, wherein the variants have α-amylase activity, and the variants have a sequence identity that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity.

[0218] In another embodiment, the variants are located at positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 300, 309, 308, 309, 309, 309, 3 5, 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, 362, 365 , 371, 383, 390, 394, 395, 405, 409, 414, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, wherein the variants have α-amylase activity, and the variants have a sequence similar to that of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, at least 60%, at least 65%, at least 70%, at least at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity.

[0219] In another embodiment, the variants are located at positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 165, 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, 362 , 365, 371, 383, 390, 394, 395, 405, 409, 414, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, wherein the variants have α-amylase activity, and the variants have a sequence similar to that of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, respectively, and 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity.

[0220] In another embodiment, the variants are located at positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 165, 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, 362 , 365, 371, 383, 390, 394, 395, 405, 409, 414, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, wherein the variants have α-amylase activity, and the variants have a sequence identity that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity.

[0221] In another embodiment, the variants are located at positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 165, 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, 362 , 365, 371, 383, 390, 394, 395, 405, 409, 414, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, wherein the variants have α-amylase activity, and the variants have a sequence identity that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity.

[0222] In another embodiment, the variants are located at positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 165, 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, 362 , 365, 371, 383, 390, 394, 395, 405, 409, 414, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, wherein the variants have α-amylase activity, and the variants have a sequence identity that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity.

[0223] In another embodiment, the variants are located at positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 165, 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, 362 , 365, 371, 383, 390, 394, 395, 405, 409, 414, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, wherein the variants have α-amylase activity, and the variants have a sequence identity that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity.

[0224] In another embodiment, the variants are located at positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 165, 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, 362 , 365, 371, 383, 390, 394, 395, 405, 409, 414, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, wherein the variants have α-amylase activity, and the variants have a sequence identity that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity.

[0225] In another embodiment, the variants are located at positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 165, 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, 362 , 365, 371, 383, 390, 394, 395, 405, 409, 414, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, wherein the variants have α-amylase activity, and the variants have a sequence identity that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity.

[0226] In another embodiment, the variants are located at positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 165, 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, 362 , 365, 371, 383, 390, 394, 395, 405, 409, 414, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, wherein the variants have α-amylase activity, and the variants have a sequence identity that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity.

[0227] In another embodiment, the variants are located at positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 2000, 201, 201, 202, 203, 204, 205, 206, 207, 208, 209, 300, 309, 308, 309, , 165, 167, 169, 172, 173, 174, 176, 177, 178, 180, 181, 182, 183, 185, 188, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, 36 and comprising a modification at each of positions corresponding to SEQ ID NOs: 2, 365, 371, 383, 390, 394, 395, 405, 409, 414, 420, 422, 427, 455, 457, 458, 470, 472, 473, 474, 475, and 476, wherein the variants have α-amylase activity, and the variants have a sequence similar to that of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, wherein the variants have a sequence similar to that of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity.

[0228] In another aspect, the variant comprises or consists of a deletion at a position corresponding to position 1. In another aspect, an amino acid at a position corresponding to position 1 is deleted in the polypeptide of SEQ ID NO: 1. In another aspect, the variant comprises or consists of the deletion H1* in the polypeptide of SEQ ID NO: 1.

[0229] In another aspect, the variant comprises or consists of a deletion at a position corresponding to position 2. In another aspect, an amino acid at a position corresponding to position 2 is deleted in the polypeptide of SEQ ID NO: 1. In another aspect, the variant comprises or consists of the deletion H2* in the polypeptide of SEQ ID NO: 1.

[0230] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 3 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 3 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution N3S or N3D or N3G of the polypeptide of SEQ ID NO: 1.

[0231] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 7 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 7 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution G7A or G7H or G7S of the polypeptide of SEQ ID NO: 1.

[0232] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 26 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 26 is substituted with Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution R26Y in the polypeptide of SEQ ID NO: 1.

[0233] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 31 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 31 is substituted with Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution A31S in the polypeptide of SEQ ID NO: 1.

[0234] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 37 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 37 is substituted with Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution R37A or R37N or R37V of the polypeptide of SEQ ID NO: 1.

[0235] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 40 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 40 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution T40N or T40G in the polypeptide of SEQ ID NO: 1.

[0236] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 41 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 41 is substituted with Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Thr, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution A41D of the polypeptide of SEQ ID NO: 1.

[0237] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 54 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 54 is substituted with Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution N54S of the polypeptide of SEQ ID NO: 1.

[0238] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 56 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 56 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr. In another aspect, the variant comprises or consists of the substitution V56T of the polypeptide of SEQ ID NO: 1.

[0239] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 60 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 60 is substituted with Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution A60P or A60V in the polypeptide of SEQ ID NO: 1.

[0240] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 63 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 63 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution L63F in the polypeptide of SEQ ID NO: 1.

[0241] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 72 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 72 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution K72R in the polypeptide of SEQ ID NO: 1.

[0242] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 73 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 73 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution G73L in the polypeptide of SEQ ID NO: 1.

[0243] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 93 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 93 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution K93D in the polypeptide of SEQ ID NO: 1.

[0244] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 94 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 94 is substituted with Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution N94D of the polypeptide of SEQ ID NO: 1.

[0245] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 98 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 98 is substituted with Ala, Arg, Asn, Asp, Cys, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution Q98L in the polypeptide of SEQ ID NO: 1.

[0246] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 103 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 103 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr. In another aspect, the variant comprises or consists of the substitution V103 or V103I of the polypeptide of SEQ ID NO: 1.

[0247] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 104 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 104 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr. In another aspect, the variant comprises or consists of the substitution V104M of the polypeptide of SEQ ID NO: 1.

[0248] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 105 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 105 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution M105I or M105L in the polypeptide of SEQ ID NO: 1.

[0249] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 106 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 106 is substituted with Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution N106D of the polypeptide of SEQ ID NO: 1.

[0250] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 109 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 109 is substituted with Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution A109G in the polypeptide of SEQ ID NO: 1.

[0251] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 110 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 110 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution G110K of the polypeptide of SEQ ID NO: 1.

[0252] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 111 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 111 is substituted with Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution A111G or A111L of the polypeptide of SEQ ID NO: 1.

[0253] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 113 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 113 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution G113A or G113Q or G113S of the polypeptide of SEQ ID NO: 1.

[0254] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 114 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 114 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution T114K of the polypeptide of SEQ ID NO: 1.

[0255] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 116 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 116 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution F116L or F116N or F116Q or F116W of the polypeptide of SEQ ID NO: 1.

[0256] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 117 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 117 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr. In another aspect, the variant comprises or consists of the substitution V117G of the polypeptide of SEQ ID NO: 1.

[0257] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 118 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 118 is substituted with Ala, Arg, Asn, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution D118T or D118Q of the polypeptide of SEQ ID NO: 1.

[0258] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 123 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 123 is substituted with Ala, Arg, Asn, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution D123N in the polypeptide of SEQ ID NO: 1.

[0259] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 126 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 126 is substituted with Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution N126D of the polypeptide of SEQ ID NO: 1.

[0260] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 128 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 128 is substituted with Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution N128Y in the polypeptide of SEQ ID NO: 1.

[0261] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 131 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 131 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution T131I or T131V in the polypeptide of SEQ ID NO: 1.

[0262] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 132 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 132 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution S132D or S132L of the polypeptide of SEQ ID NO: 1.

[0263] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 134 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 134 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution T134D in the polypeptide of SEQ ID NO: 1.

[0264] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 135 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 135 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Val. In another aspect, the variant comprises or consists of the substitution Y135E in the polypeptide of SEQ ID NO: 1.

[0265] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 136 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 136 is substituted with Ala, Arg, Asn, Asp, Cys, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution Q136T or Q136K of the polypeptide of SEQ ID NO: 1.

[0266] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 137 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 137 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution I137F in the polypeptide of SEQ ID NO: 1.

[0267] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 138 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 138 is substituted with Ala, Arg, Asn, Asp, Cys, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution Q138V in the polypeptide of SEQ ID NO: 1.

[0268] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 140 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 140 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution W140S or W140Y or W140M of the polypeptide of SEQ ID NO: 1.

[0269] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 142 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 142 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution K142E or K142G or K142P of the polypeptide of SEQ ID NO: 1.

[0270] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 144 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 144 is substituted with Ala, Arg, Asn, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution D144N in the polypeptide of SEQ ID NO: 1.

[0271] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 146 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 146 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution P146H or P146S of the polypeptide of SEQ ID NO: 1.

[0272] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 151 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 151 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution T151A in the polypeptide of SEQ ID NO: 1.

[0273] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 152 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 152 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Val. In another aspect, the variant comprises or consists of the substitution Y152L of the polypeptide of SEQ ID NO: 1.

[0274] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 155 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 155 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution F155W in the polypeptide of SEQ ID NO: 1.

[0275] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 159 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 159 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution W159H or W159L in the polypeptide of SEQ ID NO: 1.

[0276] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 160 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 160 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Val. In another aspect, the variant comprises or consists of the substitution Y160P in the polypeptide of SEQ ID NO: 1.

[0277] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 165 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 165 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution T165V in the polypeptide of SEQ ID NO: 1.

[0278] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 167 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 167 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution W167A or W167D or W167E or W167F or W167P or W167S of the polypeptide of SEQ ID NO: 1.

[0279] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 169 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 169 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Gly, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution E169V or E169I or E169Q of the polypeptide of SEQ ID NO: 1.

[0280] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 172 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 172 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution K172G or K172Q of the polypeptide of SEQ ID NO: 1.

[0281] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 173 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 173 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution L173F in the polypeptide of SEQ ID NO: 1.

[0282] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 174 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 174 is substituted with Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution N174K or N174S of the polypeptide of SEQ ID NO: 1.

[0283] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 176 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 176 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution I176V in the polypeptide of SEQ ID NO: 1.

[0284] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 177 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 177 is substituted with Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Val. In another aspect, the variant comprises or consists of the substitution Y177D in the polypeptide of SEQ ID NO: 1.

[0285] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 178 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 178 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution K178A in the polypeptide of SEQ ID NO: 1.

[0286] In another aspect, the variant comprises or consists of a deletion or substitution at a position corresponding to position 180. In another aspect, the amino acid at the position corresponding to position 180 is deleted in the polypeptide of SEQ ID NO: 1. In another aspect, the variant comprises or consists of the deletion R180* in the polypeptide of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 180 is substituted with Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution R180Q or R180S in the polypeptide of SEQ ID NO: 1.

[0287] In another aspect, the variant comprises or consists of a deletion or substitution at a position corresponding to position 181. In another aspect, the amino acid at a position corresponding to position 181 is deleted in the polypeptide of SEQ ID NO: 1. In another aspect, the variant comprises or consists of the deletion S181* in the polypeptide of SEQ ID NO: 1. In another aspect, the amino acid at a position corresponding to position 181 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution S181L or S181T in the polypeptide of SEQ ID NO: 1.

[0288] In another aspect, the variant comprises or consists of a deletion or substitution at a position corresponding to position 182 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 182 is deleted in the polypeptide of SEQ ID NO: 1. In another aspect, the variant comprises or consists of the deletion T182* in the polypeptide of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 182 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution T182D or T182E or T182G or T182I or T182L or T182M or T182S in the polypeptide of SEQ ID NO: 1.

[0289] In another aspect, the variant comprises or consists of a deletion or substitution at a position corresponding to position 183 of SEQ ID NO: 1. In another aspect, the amino acid at a position corresponding to position 183 is deleted in the polypeptide of SEQ ID NO: 1. In another aspect, the variant comprises or consists of the deletion G183* in the polypeptide of SEQ ID NO: 1. In another aspect, the amino acid at a position corresponding to position 183 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution G183P in the polypeptide of SEQ ID NO: 1.

[0290] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 185 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 185 is substituted with Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution A186D or A186E or A186K or A186N or A186Q or A186R of the polypeptide of SEQ ID NO: 1.

[0291] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 188 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 188 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution W188C in the polypeptide of SEQ ID NO: 1.

[0292] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 189 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 189 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution E189P in the polypeptide of SEQ ID NO: 1.

[0293] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 193 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 193 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution E193I in the polypeptide of SEQ ID NO: 1.

[0294] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 194 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 194 is substituted with Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution N194F in the polypeptide of SEQ ID NO: 1.

[0295] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 196 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 196 is substituted with Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution N196F or N196I of the polypeptide of SEQ ID NO: 1.

[0296] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 202 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 202 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution F202Y of the polypeptide of SEQ ID NO: 1.

[0297] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 203 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 203 is substituted with Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution A203F in the polypeptide of SEQ ID NO: 1.

[0298] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 205 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 205 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution L205F or L205N or L205Q or L205R or L205V or L205Y of the polypeptide of SEQ ID NO: 1.

[0299] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 224 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 224 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution T224F or T244Y of the polypeptide of SEQ ID NO: 1.

[0300] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 225 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 225 is substituted with Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution N225S of the polypeptide of SEQ ID NO: 1.

[0301] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 242 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 242 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Val. In another aspect, the variant comprises or consists of the substitution Y242F or Y242L of the polypeptide of SEQ ID NO: 1.

[0302] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 243 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 243 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution S243Q of the polypeptide of SEQ ID NO: 1.

[0303] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 244 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 244 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Ser, Pro, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution F244I or F244L in the polypeptide of SEQ ID NO: 1.

[0304] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 252 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 252 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr. In another aspect, the variant comprises or consists of the substitution V252I in the polypeptide of SEQ ID NO: 1.

[0305] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 256 of SEQ ID NO: 1. In another aspect, the amino acid at the position corresponding to position 256 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution T256A or T256D of the polypeptide of SEQ ID NO: 1.

[0306] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 258. In another aspect, the amino acid at the position corresponding to position 258 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution K258V in the polypeptide of SEQ ID NO:1.

[0307] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 259. In another aspect, the amino acid at the position corresponding to position 259 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution E259G or E259V in the polypeptide of SEQ ID NO:1.

[0308] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 260. In another aspect, the amino acid at the position corresponding to position 260 is substituted with Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution M260D or M260W in the polypeptide of SEQ ID NO:1.

[0309] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 261. In another aspect, the amino acid at the position corresponding to position 261 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution F261K or F261P or F261S of the polypeptide of SEQ ID NO:1.

[0310] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 266. In another aspect, the amino acid at the position corresponding to position 266 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution F266Y in the polypeptide of SEQ ID NO:1.

[0311] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 276. In another aspect, the amino acid at the position corresponding to position 276 is substituted with Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Phe, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution N276F or N276W or N276Y in the polypeptide of SEQ ID NO:1.

[0312] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 279. In another aspect, the amino acid at the position corresponding to position 279 is substituted with Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Phe, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution N279S in the polypeptide of SEQ ID NO:1.

[0313] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 280. In another aspect, the amino acid at the position corresponding to position 280 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Phe, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution K280F or K280W or K280Y of the polypeptide of SEQ ID NO:1.

[0314] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 283. In another aspect, the amino acid at the position corresponding to position 283 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Phe, Ser, Thr, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution W283N in the polypeptide of SEQ ID NO:1.

[0315] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 286. In another aspect, the amino acid at the position corresponding to position 286 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Phe, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution S286K or S286L in the polypeptide of SEQ ID NO:1.

[0316] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 302. In another aspect, the amino acid at the position corresponding to position 302 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Phe, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution S302T or S302Y in the polypeptide of SEQ ID NO:1.

[0317] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 303. In another aspect, the amino acid at the position corresponding to position 303 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Pro, Phe, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution G303R in the polypeptide of SEQ ID NO:1.

[0318] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 315. In another aspect, the amino acid at the position corresponding to position 315 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Phe, Ser, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution T315A in the polypeptide of SEQ ID NO:1.

[0319] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 320. In another aspect, the amino acid at the position corresponding to position 320 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Pro, Phe, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution H320T in the polypeptide of SEQ ID NO:1.

[0320] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 321. In another aspect, the amino acid at the position corresponding to position 321 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution P321H or P321Y in the polypeptide of SEQ ID NO:1.

[0321] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 322. In another aspect, the amino acid at the position corresponding to position 322 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Pro, Phe, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution M322G or M322Y in the polypeptide of SEQ ID NO:1.

[0322] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 323. In another aspect, the amino acid at the position corresponding to position 323 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Pro, Phe, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution H323A or H323N or H323W in the polypeptide of SEQ ID NO:1.

[0323] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 346. In another aspect, the amino acid at the position corresponding to position 346 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Phe, Ser, Thr, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution W346N in the polypeptide of SEQ ID NO:1.

[0324] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 354. In another aspect, the amino acid at the position corresponding to position 354 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Lys, Met, Pro, Phe, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution L354V in the polypeptide of SEQ ID NO:1.

[0325] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 359. In another aspect, the amino acid at the position corresponding to position 359 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Pro, Phe, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution E359W in the polypeptide of SEQ ID NO:1.

[0326] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 361. In another aspect, the amino acid at the position corresponding to position 361 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr. In another aspect, the variant comprises or consists of a substitution G361M in the polypeptide of SEQ ID NO: 1. In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 362. In another aspect, the amino acid at the position corresponding to position 362 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Phe, Ser, Thr, Trp, or Val. In another aspect, the variant comprises or consists of a substitution Y362V in the polypeptide of SEQ ID NO: 1.

[0327] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 365. In another aspect, the amino acid at the position corresponding to position 365 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Phe, Ser, Thr, Tyr, or Trp. In another aspect, the variant comprises or consists of the substitution V365I in the polypeptide of SEQ ID NO:1.

[0328] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 371. In another aspect, the amino acid at the position corresponding to position 371 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Phe, Ser, Thr, Tyr, or Trp. In another aspect, the variant comprises or consists of the substitution Y371S or Y371N in the polypeptide of SEQ ID NO:1.

[0329] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 383. In another aspect, the amino acid at the position corresponding to position 383 is substituted with Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Phe, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution A383D in the polypeptide of SEQ ID NO:1.

[0330] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 390. In another aspect, the amino acid at the position corresponding to position 390 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Pro, Phe, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution E390A or E390D of the polypeptide of SEQ ID NO:1.

[0331] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 394. In another aspect, the amino acid at the position corresponding to position 394 is substituted with Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Phe, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution N394W or N394D in the polypeptide of SEQ ID NO:1.

[0332] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 395. In another aspect, the amino acid at the position corresponding to position 395 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution F395Y in the polypeptide of SEQ ID NO:1.

[0333] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 405. In another aspect, the amino acid at the position corresponding to position 405 is substituted with Ala, Arg, Asn, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Phe, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution D405G in the polypeptide of SEQ ID NO:1.

[0334] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 409. In another aspect, the amino acid at the position corresponding to position 409 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Lys, Met, Pro, Phe, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution I409L in the polypeptide of SEQ ID NO:1.

[0335] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 414. In another aspect, the amino acid at the position corresponding to position 414 is substituted with Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution R414M in the polypeptide of SEQ ID NO:1.

[0336] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 420. In another aspect, the amino acid at the position corresponding to position 420 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Pro, Phe, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution H420A or H420P or H420Q or H420Y of the polypeptide of SEQ ID NO:1.

[0337] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 422. In another aspect, the amino acid at the position corresponding to position 422 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr. In another aspect, the variant comprises or consists of the substitution N422V in the polypeptide of SEQ ID NO:1.

[0338] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 427. In another aspect, the amino acid at the position corresponding to position 427 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Phe, Ser, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution T427L in the polypeptide of SEQ ID NO:1.

[0339] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 455. In another aspect, the amino acid at the position corresponding to position 455 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Pro, Phe, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution G455R in the polypeptide of SEQ ID NO:1.

[0340] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 457. In another aspect, the amino acid at the position corresponding to position 457 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Phe, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution K457N or K457R in the polypeptide of SEQ ID NO:1.

[0341] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 458. In another aspect, the amino acid at the position corresponding to position 458 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution P458S in the polypeptide of SEQ ID NO:1.

[0342] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 470. In another aspect, the amino acid at the position corresponding to position 470 is substituted with Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Phe, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution N470A or N470M of the polypeptide of SEQ ID NO:1.

[0343] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 472. In another aspect, the amino acid at the position corresponding to position 472 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Phe, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution S472R in the polypeptide of SEQ ID NO:1.

[0344] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 473. In another aspect, the amino acid at the position corresponding to position 473 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Phe, Ser, Thr, Tyr, or Tyr. In another aspect, the variant comprises or consists of the substitution V473C in the polypeptide of SEQ ID NO:1.

[0345] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 474. In another aspect, the amino acid at the position corresponding to position 474 is substituted with Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Phe, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution N474F or N474L of the polypeptide of SEQ ID NO:1.

[0346] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 475. In another aspect, the amino acid at the position corresponding to position 475 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Pro, Phe, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution G475E or G475K in the polypeptide of SEQ ID NO:1.

[0347] In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 476. In another aspect, the amino acid at the position corresponding to position 476 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the variant comprises or consists of the substitution G476A or G476E or G476H of the polypeptide of SEQ ID NO:1.

[0348] In another aspect, the variants are represented by the following sequences using SEQ ID NO: 1 for numbering: positions H1, H2, N3, G7, R26, A31, R37, T40, A41, N54, V56, A60, L63, K72, G73, K93, N94, Q98, V103, V104, M105, N106, A109, G110, A111, G113, T114, F116, V117, D118, D123, N126, N128, T131, S132, T134, Y135, Q136, I137, Q138, W140, K142, D144, P146, T151, Y152, Y154, Y156, Y158, Y159, Y200, Y201, Y202, Y203, Y204, Y205, Y206, Y207, Y208, Y209, Y300, Y310, Y320, Y331, Y332, Y333, Y334, Y335, Y336, Y337, Y338, Y340, Y342, Y344, Y346, Y348, Y349, Y350, Y351, Y352, Y353, Y354, Y355, Y356, Y357, Y358, Y359, Y360, Y361, Y362, Y363, Y364, Y365, Y366, Y367, Y368, Y369, Y370, Y371, Y372, Y373, Y3 152, F155, W159, Y160, T165, W167, E169, K172, L173, N174, I176, Y177, K178, R180, S181, T182, G183, A185, W188, E189, E193, N194, N196, F202, A203, L205, T224, N225, Y242, S243, F244, V252, T256, K258, E259, M260 , F261, F266, N276, N279, K280, W283, S286, S302, G303, T315, H320, P321 , M322, H323, W346, L354, E359, Q360, G361, Y362, V365, Y371, A383, E390, N394, F395, D405, I409, R414, H420, N422, T427, G455, K457, P458, N470, S472, V473, N474, G475, and G476, wherein the variant has α-amylase activity, and the variant has a sequence similar to that of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity.

[0349] In another aspect, the variants are selected from the group consisting of positions H1*, H2*, N3D, N3G, N3S, G7A, G7H, G7S, R26Y, A31S, R37A, R37N, R37V, T40G, T40N, A41D, N54S, V56T, A60V, A60P, L63F, K72R, G73L, K93R, N94D, Q98L, V103A, V103I, V103L, V104M, M105I, M105L, N106D, A109G, G110K, A111G, A111L, G113Q, G113S, G113A, T114K, F11 6L, F116N, F116Q, F116W, V117G, D118Q, D118T, D123N, N126D, N128Y, T131I , T131V, S132D, S132L, T134D, Y135E, Q136T, Q136K, I137F, Q138V, W140S, W 140M, W140Y, K142E, K142G, K142P, D144N, P146S, P146H, T151A, Y152L, F15 5W, W159H, W159L, Y160P, T165V, W167A, W167D, W167E, W167F, W167P, W167S, E169I, E169Q, E169V, K172G, K172Q, L173F, N174K, N174S, I176V, Y177D, K1 78A, R180*, R180Q, R180S, S181*, S181L, S181T, T182*, T182D, T182E, T182 G, T182I, T182L, T182M, T182S, G183*, G183P, A185D, A185E, A185K, A185N, A185Q, A185R, W188C, E189P, E193I, N194F, F202Y, F202Y, A203F, L205F, L20 5N, L205Q, L205R, L205V, L205Y, T224F, T224Y, N225S, Y242F, Y242L, S243Q , F244I, F244L, V252I, T256A, T256D, K258V, E259G, E259V, M260D, M260W, F 261K, F261P, F261S, F266Y, N276F, N276W, N276Y, N279S, N280F, N280W, N28 0Y, W283N, S286K, S286L, S302T, S302Y, G303R, T315A, H320T, P321H, P321Y,M322G, M322Y, H323A, H323N, H323W, W346N, L354V, E359W, Q360M, Q360W, Q360Y, G361M, Y36 2V, V365I, Y371S, Y371N, A383D, E390A, E390D, N394W, N394D, F395Y, D405G, I409L, R414M, H and comprising a modification at one or more positions corresponding to 420P, H420A, H420Q, H420Y, N422V, T427L, G455R, K457N, K457R, R458S, N470A, N470M, S472R, V473C, N474F, N474L, G475E, G475K, G476H, G476E, and G476A, The organism has α-amylase activity, and the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0350] In another aspect, the variants are selected from the group consisting of positions H1*, H2*, N3D, N3G, N3S, G7A, G7H, G7S, R26Y, A31S, R37A, R37N, R37V, T40G, T40N, A41D, N54S, V56T, A60V, A60P, L63F, K72R, G73L, K93R, N94D, Q98L, V103A, V103I, V103L, V104M, M105I, M105L, N106D, A109G, G110K, A111G, A111L, G113Q, G113S, G113A, T114K, F11 6L, F116N, F116Q, F116W, V117G, D118Q, D118T, D123N, N126D, N128Y, T131I , T131V, S132D, S132L, T134D, Y135E, Q136T, Q136K, I137F, Q138V, W140S, W 140M, W140Y, K142E, K142G, K142P, D144N, P146S, P146H, T151A, Y152L, F15 5W, W159H, W159L, Y160P, T165V, W167A, W167D, W167E, W167F, W167P, W167S, E169I, E169Q, E169V, K172G, K172Q, L173F, N174K, N174S, I176V, Y177D, K1 78A, R180*, R180Q, R180S, S181*, S181L, S181T, T182*, T182D, T182E, T182 G, T182I, T182L, T182M, T182S, G183*, G183P, A185D, A185E, A185K, A185N, A185Q, A185R, W188C, E189P, E193I, N194F, F202Y, F202Y, A203F, L205F, L20 5N, L205Q, L205R, L205V, L205Y, T224F, T224Y, N225S, Y242F, Y242L, S243Q , F244I, F244L, V252I, T256A, T256D, K258V, E259G, E259V, M260D, M260W, F 261K, F261P, F261S, F266Y, N276F, N276W, N276Y, N279S, N280F, N280W, N28 0Y, W283N, S286K, S286L, S302T, S302Y, G303R, T315A, H320T, P321H, P321Y,M322G, M322Y, H323A, H323N, H323W, W346N, L354V, E359W, Q360M, Q360W, Q360Y, G361M , Y362V, V365I, Y371S, Y371N, A383D, E390A, E390D, N394W, N394D, F395Y, D405G, I409L , R414M, H420P, H420A, H420Q, H420Y, N422V, T427L, G455R, K457N, K457R, R458S, N470A, N470M, S472R, V473C, N474F, N474L, G475E, G475K, G476H, G476E, and G476A. and a modification at position 144 of the ribozyme, wherein the variant has α-amylase activity, and the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to SEQ ID NO:1.

[0351] In another aspect, the variants are selected from the group consisting of positions H1*, H2*, N3D, N3G, N3S, G7A, G7H, G7S, R26Y, A31S, R37A, R37N, R37V, T40G, T40N, A41D, N54S, V56T, A60V, A60P, L63F, K72R, G73L, K93R, N94D, Q98L, V103A, V103I, V103L, V104M, M105I, M105L, N106D, A109G, G110K, A111G, A111L, G113Q, G113S, G113A, T114K, F11 6L, F116N, F116Q, F116W, V117G, D118Q, D118T, D123N, N126D, N128Y, T131I , T131V, S132D, S132L, T134D, Y135E, Q136T, Q136K, I137F, Q138V, W140S, W 140M, W140Y, K142E, K142G, K142P, D144N, P146S, P146H, T151A, Y152L, F15 5W, W159H, W159L, Y160P, T165V, W167A, W167D, W167E, W167F, W167P, W167S, E169I, E169Q, E169V, K172G, K172Q, L173F, N174K, N174S, I176V, Y177D, K1 78A, R180*, R180Q, R180S, S181*, S181L, S181T, T182*, T182D, T182E, T182 G, T182I, T182L, T182M, T182S, G183*, G183P, A185D, A185E, A185K, A185N, A185Q, A185R, W188C, E189P, E193I, N194F, F202Y, F202Y, A203F, L205F, L20 5N, L205Q, L205R, L205V, L205Y, T224F, T224Y, N225S, Y242F, Y242L, S243Q , F244I, F244L, V252I, T256A, T256D, K258V, E259G, E259V, M260D, M260W, F 261K, F261P, F261S, F266Y, N276F, N276W, N276Y, N279S, N280F, N280W, N28 0Y, W283N, S286K, S286L, S302T, S302Y, G303R, T315A, H320T, P321H, P321Y,M322G, M322Y, H323A, H323N, H323W, W346N, L354V, E359W, Q360M, Q360W, Q360Y, G361M , Y362V, V365I, Y371S, Y371N, A383D, E390A, E390D, N394W, N394D, F395Y, D405G, I409L , R414M, H420P, H420A, H420Q, H420Y, N422V, T427L, G455R, K457N, K457R, R458S, N470A, N470M, S472R, V473C, N474F, N474L, G475E, G475K, G476H, G476E, and G476A. and a modification at position 20 of the ribozyme A polypeptide, the variant having α-amylase activity, the variant having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to SEQ ID NO:2.

[0352] In another aspect, the variants are selected from the group consisting of positions H1*, H2*, N3D, N3G, N3S, G7A, G7H, G7S, R26Y, A31S, R37A, R37N, R37V, T40G, T40N, A41D, N54S, V56T, A60V, A60P, L63F, K72R, G73L, K93R, N94D, Q98L, V103A, V103I, V103L, V104M, M105I, M105L, N106D, A109G, G110K, A111G, A111L, G113Q, G113S, G113A, T114K, F11 6L, F116N, F116Q, F116W, V117G, D118Q, D118T, D123N, N126D, N128Y, T131I , T131V, S132D, S132L, T134D, Y135E, Q136T, Q136K, I137F, Q138V, W140S, W 140M, W140Y, K142E, K142G, K142P, D144N, P146S, P146H, T151A, Y152L, F15 5W, W159H, W159L, Y160P, T165V, W167A, W167D, W167E, W167F, W167P, W167S, E169I, E169Q, E169V, K172G, K172Q, L173F, N174K, N174S, I176V, Y177D, K1 78A, R180*, R180Q, R180S, S181*, S181L, S181T, T182*, T182D, T182E, T182 G, T182I, T182L, T182M, T182S, G183*, G183P, A185D, A185E, A185K, A185N, A185Q, A185R, W188C, E189P, E193I, N194F, F202Y, F202Y, A203F, L205F, L20 5N, L205Q, L205R, L205V, L205Y, T224F, T224Y, N225S, Y242F, Y242L, S243Q , F244I, F244L, V252I, T256A, T256D, K258V, E259G, E259V, M260D, M260W, F 261K, F261P, F261S, F266Y, N276F, N276W, N276Y, N279S, N280F, N280W, N28 0Y, W283N, S286K, S286L, S302T, S302Y, G303R, T315A, H320T, P321H, P321Y,M322G, M322Y, H323A, H323N, H323W, W346N, L354V, E359W, Q360M, Q360W, Q360Y, G361M , Y362V, V365I, Y371S, Y371N, A383D, E390A, E390D, N394W, N394D, F395Y, D405G, I409L , R414M, H420P, H420A, H420Q, H420Y, N422V, T427L, G455R, K457N, K457R, R458S, N470A, N470M, S472R, V473C, N474F, N474L, G475E, G475K, G476H, G476E, and G476A. and a modification at position 144 of the ribozyme, wherein the variant has α-amylase activity, and the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO:3.

[0353] In another aspect, the variants are selected from the group consisting of positions H1*, H2*, N3D, N3G, N3S, G7A, G7H, G7S, R26Y, A31S, R37A, R37N, R37V, T40G, T40N, A41D, N54S, V56T, A60V, A60P, L63F, K72R, G73L, K93R, N94D, Q98L, V103A, V103I, V103L, V104M, M105I, M105L, N106D, A109G, G110K, A111G, A111L, G113Q, G113S, G113A, T114K, F11 6L, F116N, F116Q, F116W, V117G, D118Q, D118T, D123N, N126D, N128Y, T131I , T131V, S132D, S132L, T134D, Y135E, Q136T, Q136K, I137F, Q138V, W140S, W 140M, W140Y, K142E, K142G, K142P, D144N, P146S, P146H, T151A, Y152L, F15 5W, W159H, W159L, Y160P, T165V, W167A, W167D, W167E, W167F, W167P, W167S, E169I, E169Q, E169V, K172G, K172Q, L173F, N174K, N174S, I176V, Y177D, K1 78A, R180*, R180Q, R180S, S181*, S181L, S181T, T182*, T182D, T182E, T182 G, T182I, T182L, T182M, T182S, G183*, G183P, A185D, A185E, A185K, A185N, A185Q, A185R, W188C, E189P, E193I, N194F, F202Y, F202Y, A203F, L205F, L20 5N, L205Q, L205R, L205V, L205Y, T224F, T224Y, N225S, Y242F, Y242L, S243Q , F244I, F244L, V252I, T256A, T256D, K258V, E259G, E259V, M260D, M260W, F 261K, F261P, F261S, F266Y, N276F, N276W, N276Y, N279S, N280F, N280W, N28 0Y, W283N, S286K, S286L, S302T, S302Y, G303R, T315A, H320T, P321H, P321Y,M322G, M322Y, H323A, H323N, H323W, W346N, L354V, E359W, Q360M, Q360W, Q360Y, G361M , Y362V, V365I, Y371S, Y371N, A383D, E390A, E390D, N394W, N394D, F395Y, D405G, I409L , R414M, H420P, H420A, H420Q, H420Y, N422V, T427L, G455R, K457N, K457R, R458S, N470A, N470M, S472R, V473C, N474F, N474L, G475E, G475K, G476H, G476E, and G476A. and a modification at position 144 of SEQ ID NO:4, wherein the variant has α-amylase activity, and the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO:4.

[0354] In another aspect, the variants are selected from the group consisting of positions H1*, H2*, N3D, N3G, N3S, G7A, G7H, G7S, R26Y, A31S, R37A, R37N, R37V, T40G, T40N, A41D, N54S, V56T, A60V, A60P, L63F, K72R, G73L, K93R, N94D, Q98L, V103A, V103I, V103L, V104M, M105I, M105L, N106D, A109G, G110K, A111G, A111L, G113Q, G113S, G113A, T114K, F11 6L, F116N, F116Q, F116W, V117G, D118Q, D118T, D123N, N126D, N128Y, T131I , T131V, S132D, S132L, T134D, Y135E, Q136T, Q136K, I137F, Q138V, W140S, W 140M, W140Y, K142E, K142G, K142P, D144N, P146S, P146H, T151A, Y152L, F15 5W, W159H, W159L, Y160P, T165V, W167A, W167D, W167E, W167F, W167P, W167S, E169I, E169Q, E169V, K172G, K172Q, L173F, N174K, N174S, I176V, Y177D, K1 78A, R180*, R180Q, R180S, S181*, S181L, S181T, T182*, T182D, T182E, T182 G, T182I, T182L, T182M, T182S, G183*, G183P, A185D, A185E, A185K, A185N, A185Q, A185R, W188C, E189P, E193I, N194F, F202Y, F202Y, A203F, L205F, L20 5N, L205Q, L205R, L205V, L205Y, T224F, T224Y, N225S, Y242F, Y242L, S243Q , F244I, F244L, V252I, T256A, T256D, K258V, E259G, E259V, M260D, M260W, F 261K, F261P, F261S, F266Y, N276F, N276W, N276Y, N279S, N280F, N280W, N28 0Y, W283N, S286K, S286L, S302T, S302Y, G303R, T315A, H320T, P321H, P321Y,M322G, M322Y, H323A, H323N, H323W, W346N, L354V, E359W, Q360M, Q360W, Q360Y, G361M , Y362V, V365I, Y371S, Y371N, A383D, E390A, E390D, N394W, N394D, F395Y, D405G, I409L , R414M, H420P, H420A, H420Q, H420Y, N422V, T427L, G455R, K457N, K457R, R458S, N470A, N470M, S472R, V473C, N474F, N474L, G475E, G475K, G476H, G476E, and G476A. and a modification at position 104 of the ribozyme, wherein the variant has α-amylase activity, and the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO:5.

[0355] In another aspect, the variants are selected from the group consisting of positions H1*, H2*, N3D, N3G, N3S, G7A, G7H, G7S, R26Y, A31S, R37A, R37N, R37V, T40G, T40N, A41D, N54S, V56T, A60V, A60P, L63F, K72R, G73L, K93R, N94D, Q98L, V103A, V103I, V103L, V104M, M105I, M105L, N106D, A109G, G110K, A111G, A111L, G113Q, G113S, G113A, T114K, F11 6L, F116N, F116Q, F116W, V117G, D118Q, D118T, D123N, N126D, N128Y, T131I , T131V, S132D, S132L, T134D, Y135E, Q136T, Q136K, I137F, Q138V, W140S, W 140M, W140Y, K142E, K142G, K142P, D144N, P146S, P146H, T151A, Y152L, F15 5W, W159H, W159L, Y160P, T165V, W167A, W167D, W167E, W167F, W167P, W167S, E169I, E169Q, E169V, K172G, K172Q, L173F, N174K, N174S, I176V, Y177D, K1 78A, R180*, R180Q, R180S, S181*, S181L, S181T, T182*, T182D, T182E, T182 G, T182I, T182L, T182M, T182S, G183*, G183P, A185D, A185E, A185K, A185N, A185Q, A185R, W188C, E189P, E193I, N194F, F202Y, F202Y, A203F, L205F, L20 5N, L205Q, L205R, L205V, L205Y, T224F, T224Y, N225S, Y242F, Y242L, S243Q , F244I, F244L, V252I, T256A, T256D, K258V, E259G, E259V, M260D, M260W, F 261K, F261P, F261S, F266Y, N276F, N276W, N276Y, N279S, N280F, N280W, N28 0Y, W283N, S286K, S286L, S302T, S302Y, G303R, T315A, H320T, P321H, P321Y,M322G, M322Y, H323A, H323N, H323W, W346N, L354V, E359W, Q360M, Q360W, Q360Y, G361M , Y362V, V365I, Y371S, Y371N, A383D, E390A, E390D, N394W, N394D, F395Y, D405G, I409L , R414M, H420P, H420A, H420Q, H420Y, N422V, T427L, G455R, K457N, K457R, R458S, N470A, N470M, S472R, V473C, N474F, N474L, G475E, G475K, G476H, G476E, and G476A. and a modification at position 60, wherein the variant has α-amylase activity, and the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO:6.

[0356] In another aspect, the variants are selected from the group consisting of positions H1*, H2*, N3D, N3G, N3S, G7A, G7H, G7S, R26Y, A31S, R37A, R37N, R37V, T40G, T40N, A41D, N54S, V56T, A60V, A60P, L63F, K72R, G73L, K93R, N94D, Q98L, V103A, V103I, V103L, V104M, M105I, M105L, N106D, A109G, G110K, A111G, A111L, G113Q, G113S, G113A, T114K, F11 6L, F116N, F116Q, F116W, V117G, D118Q, D118T, D123N, N126D, N128Y, T131I , T131V, S132D, S132L, T134D, Y135E, Q136T, Q136K, I137F, Q138V, W140S, W 140M, W140Y, K142E, K142G, K142P, D144N, P146S, P146H, T151A, Y152L, F15 5W, W159H, W159L, Y160P, T165V, W167A, W167D, W167E, W167F, W167P, W167S, E169I, E169Q, E169V, K172G, K172Q, L173F, N174K, N174S, I176V, Y177D, K1 78A, R180*, R180Q, R180S, S181*, S181L, S181T, T182*, T182D, T182E, T182 G, T182I, T182L, T182M, T182S, G183*, G183P, A185D, A185E, A185K, A185N, A185Q, A185R, W188C, E189P, E193I, N194F, F202Y, F202Y, A203F, L205F, L20 5N, L205Q, L205R, L205V, L205Y, T224F, T224Y, N225S, Y242F, Y242L, S243Q , F244I, F244L, V252I, T256A, T256D, K258V, E259G, E259V, M260D, M260W, F 261K, F261P, F261S, F266Y, N276F, N276W, N276Y, N279S, N280F, N280W, N28 0Y, W283N, S286K, S286L, S302T, S302Y, G303R, T315A, H320T, P321H, P321Y,M322G, M322Y, H323A, H323N, H323W, W346N, L354V, E359W, Q360M, Q360W, Q360Y, G361M , Y362V, V365I, Y371S, Y371N, A383D, E390A, E390D, N394W, N394D, F395Y, D405G, I409L , R414M, H420P, H420A, H420Q, H420Y, N422V, T427L, G455R, K457N, K457R, R458S, N470A, N470M, S472R, V473C, N474F, N474L, G475E, G475K, G476H, G476E, and G476A. and a modification at position 144 of the ribozyme having α-amylase activity, the variant having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO:7.

[0357] In another aspect, the variants are selected from the group consisting of positions H1*, H2*, N3D, N3G, N3S, G7A, G7H, G7S, R26Y, A31S, R37A, R37N, R37V, T40G, T40N, A41D, N54S, V56T, A60V, A60P, L63F, K72R, G73L, K93R, N94D, Q98L, V103A, V103I, V103L, V104M, M105I, M105L, N106D, A109G, G110K, A111G, A111L, G113Q, G113S, G113A, T114K, F11 6L, F116N, F116Q, F116W, V117G, D118Q, D118T, D123N, N126D, N128Y, T131I , T131V, S132D, S132L, T134D, Y135E, Q136T, Q136K, I137F, Q138V, W140S, W 140M, W140Y, K142E, K142G, K142P, D144N, P146S, P146H, T151A, Y152L, F15 5W, W159H, W159L, Y160P, T165V, W167A, W167D, W167E, W167F, W167P, W167S, E169I, E169Q, E169V, K172G, K172Q, L173F, N174K, N174S, I176V, Y177D, K1 78A, R180*, R180Q, R180S, S181*, S181L, S181T, T182*, T182D, T182E, T182 G, T182I, T182L, T182M, T182S, G183*, G183P, A185D, A185E, A185K, A185N, A185Q, A185R, W188C, E189P, E193I, N194F, F202Y, F202Y, A203F, L205F, L20 5N, L205Q, L205R, L205V, L205Y, T224F, T224Y, N225S, Y242F, Y242L, S243Q , F244I, F244L, V252I, T256A, T256D, K258V, E259G, E259V, M260D, M260W, F 261K, F261P, F261S, F266Y, N276F, N276W, N276Y, N279S, N280F, N280W, N28 0Y, W283N, S286K, S286L, S302T, S302Y, G303R, T315A, H320T, P321H, P321Y,M322G, M322Y, H323A, H323N, H323W, W346N, L354V, E359W, Q360M, Q360W, Q360Y, G361M , Y362V, V365I, Y371S, Y371N, A383D, E390A, E390D, N394W, N394D, F395Y, D405G, I409L , R414M, H420P, H420A, H420Q, H420Y, N422V, T427L, G455R, K457N, K457R, R458S, N470A, N470M, S472R, V473C, N474F, N474L, G475E, G475K, G476H, G476E, and G476A. and a modification at position 144 of the ribozyme, wherein the variant has α-amylase activity, and the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to SEQ ID NO:8.

[0358] In another aspect, the variants are selected from the group consisting of positions H1*, H2*, N3D, N3G, N3S, G7A, G7H, G7S, R26Y, A31S, R37A, R37N, R37V, T40G, T40N, A41D, N54S, V56T, A60V, A60P, L63F, K72R, G73L, K93R, N94D, Q98L, V103A, V103I, V103L, V104M, M105I, M105L, N106D, A109G, G110K, A111G, A111L, G113Q, G113S, G113A, T114K, F11 6L, F116N, F116Q, F116W, V117G, D118Q, D118T, D123N, N126D, N128Y, T131I , T131V, S132D, S132L, T134D, Y135E, Q136T, Q136K, I137F, Q138V, W140S, W 140M, W140Y, K142E, K142G, K142P, D144N, P146S, P146H, T151A, Y152L, F15 5W, W159H, W159L, Y160P, T165V, W167A, W167D, W167E, W167F, W167P, W167S, E169I, E169Q, E169V, K172G, K172Q, L173F, N174K, N174S, I176V, Y177D, K1 78A, R180*, R180Q, R180S, S181*, S181L, S181T, T182*, T182D, T182E, T182 G, T182I, T182L, T182M, T182S, G183*, G183P, A185D, A185E, A185K, A185N, A185Q, A185R, W188C, E189P, E193I, N194F, F202Y, F202Y, A203F, L205F, L20 5N, L205Q, L205R, L205V, L205Y, T224F, T224Y, N225S, Y242F, Y242L, S243Q , F244I, F244L, V252I, T256A, T256D, K258V, E259G, E259V, M260D, M260W, F 261K, F261P, F261S, F266Y, N276F, N276W, N276Y, N279S, N280F, N280W, N28 0Y, W283N, S286K, S286L, S302T, S302Y, G303R, T315A, H320T, P321H, P321Y,M322G, M322Y, H323A, H323N, H323W, W346N, L354V, E359W, Q360M, Q360W, Q360Y, G361M , Y362V, V365I, Y371S, Y371N, A383D, E390A, E390D, N394W, N394D, F395Y, D405G, I409L , R414M, H420P, H420A, H420Q, H420Y, N422V, T427L, G455R, K457N, K457R, R458S, N470A, N470M, S472R, V473C, N474F, N474L, G475E, G475K, G476H, G476E, and G476A. and a modification at position 144 of the ribozyme, wherein the variant has α-amylase activity, and the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to SEQ ID NO:9.

[0359] In another aspect, the variants are selected from the group consisting of positions H1*, H2*, N3D, N3G, N3S, G7A, G7H, G7S, R26Y, A31S, R37A, R37N, R37V, T40G, T40N, A41D, N54S, V56T, A60V, A60P, L63F, K72R, G73L, K93R, N94D, Q98L, V103A, V103I, V103L, V104M, M105I, M105L, N106D, A109G, G110K, A111G, A111L, G113Q, G113S, G113A, T114K, F11 6L, F116N, F116Q, F116W, V117G, D118Q, D118T, D123N, N126D, N128Y, T131I , T131V, S132D, S132L, T134D, Y135E, Q136T, Q136K, I137F, Q138V, W140S, W 140M, W140Y, K142E, K142G, K142P, D144N, P146S, P146H, T151A, Y152L, F15 5W, W159H, W159L, Y160P, T165V, W167A, W167D, W167E, W167F, W167P, W167S, E169I, E169Q, E169V, K172G, K172Q, L173F, N174K, N174S, I176V, Y177D, K1 78A, R180*, R180Q, R180S, S181*, S181L, S181T, T182*, T182D, T182E, T182 G, T182I, T182L, T182M, T182S, G183*, G183P, A185D, A185E, A185K, A185N, A185Q, A185R, W188C, E189P, E193I, N194F, F202Y, F202Y, A203F, L205F, L20 5N, L205Q, L205R, L205V, L205Y, T224F, T224Y, N225S, Y242F, Y242L, S243Q , F244I, F244L, V252I, T256A, T256D, K258V, E259G, E259V, M260D, M260W, F 261K, F261P, F261S, F266Y, N276F, N276W, N276Y, N279S, N280F, N280W, N28 0Y, W283N, S286K, S286L, S302T, S302Y, G303R, T315A, H320T, P321H, P321Y,M322G, M322Y, H323A, H323N, H323W, W346N, L354V, E359W, Q360M, Q360W, Q360Y, G361M , Y362V, V365I, Y371S, Y371N, A383D, E390A, E390D, N394W, N394D, F395Y, D405G, I409L , R414M, H420P, H420A, H420Q, H420Y, N422V, T427L, G455R, K457N, K457R, R458S, N470A, N470M, S472R, V473C, N474F, N474L, G475E, G475K, G476H, G476E, and G476A. and a modification at position 104 of the ribozyme, wherein the variant has α-amylase activity, and the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 10.

[0360] In another aspect, the variants are selected from the group consisting of positions H1*, H2*, N3D, N3G, N3S, G7A, G7H, G7S, R26Y, A31S, R37A, R37N, R37V, T40G, T40N, A41D, N54S, V56T, A60V, A60P, L63F, K72R, G73L, K93R, N94D, Q98L, V103A, V103I, V103L, V104M, M105I, M105L, N106D, A109G, G110K, A111G, A111L, G113Q, G113S, G113A, T114K, F11 6L, F116N, F116Q, F116W, V117G, D118Q, D118T, D123N, N126D, N128Y, T131I , T131V, S132D, S132L, T134D, Y135E, Q136T, Q136K, I137F, Q138V, W140S, W 140M, W140Y, K142E, K142G, K142P, D144N, P146S, P146H, T151A, Y152L, F15 5W, W159H, W159L, Y160P, T165V, W167A, W167D, W167E, W167F, W167P, W167S, E169I, E169Q, E169V, K172G, K172Q, L173F, N174K, N174S, I176V, Y177D, K1 78A, R180*, R180Q, R180S, S181*, S181L, S181T, T182*, T182D, T182E, T182 G, T182I, T182L, T182M, T182S, G183*, G183P, A185D, A185E, A185K, A185N, A185Q, A185R, W188C, E189P, E193I, N194F, F202Y, F202Y, A203F, L205F, L20 5N, L205Q, L205R, L205V, L205Y, T224F, T224Y, N225S, Y242F, Y242L, S243Q , F244I, F244L, V252I, T256A, T256D, K258V, E259G, E259V, M260D, M260W, F 261K, F261P, F261S, F266Y, N276F, N276W, N276Y, N279S, N280F, N280W, N28 0Y, W283N, S286K, S286L, S302T, S302Y, G303R, T315A, H320T, P321H, P321Y,M322G, M322Y, H323A, H323N, H323W, W346N, L354V, E359W, Q360M, Q360W, Q360Y, G361M , Y362V, V365I, Y371S, Y371N, A383D, E390A, E390D, N394W, N394D, F395Y, D405G, I409L , R414M, H420P, H420A, H420Q, H420Y, N422V, T427L, G455R, K457N, K457R, R458S, N470A, N470M, S472R, V473C, N474F, N474L, G475E, G475K, G476H, G476E, and G476A. and a modification at position 16 of the ribozyme A polypeptide, wherein the variant has α-amylase activity and the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to SEQ ID NO:11.

[0361] In another aspect, the variants are selected from the group consisting of positions H1*, H2*, N3D, N3G, N3S, G7A, G7H, G7S, R26Y, A31S, R37A, R37N, R37V, T40G, T40N, A41D, N54S, V56T, A60V, A60P, L63F, K72R, G73L, K93R, N94D, Q98L, V103A, V103I, V103L, V104M, M105I, M105L, N106D, A109G, G110K, A111G, A111L, G113Q, G113S, G113A, T114K, F11 6L, F116N, F116Q, F116W, V117G, D118Q, D118T, D123N, N126D, N128Y, T131I , T131V, S132D, S132L, T134D, Y135E, Q136T, Q136K, I137F, Q138V, W140S, W 140M, W140Y, K142E, K142G, K142P, D144N, P146S, P146H, T151A, Y152L, F15 5W, W159H, W159L, Y160P, T165V, W167A, W167D, W167E, W167F, W167P, W167S, E169I, E169Q, E169V, K172G, K172Q, L173F, N174K, N174S, I176V, Y177D, K1 78A, R180*, R180Q, R180S, S181*, S181L, S181T, T182*, T182D, T182E, T182 G, T182I, T182L, T182M, T182S, G183*, G183P, A185D, A185E, A185K, A185N, A185Q, A185R, W188C, E189P, E193I, N194F, F202Y, F202Y, A203F, L205F, L20 5N, L205Q, L205R, L205V, L205Y, T224F, T224Y, N225S, Y242F, Y242L, S243Q , F244I, F244L, V252I, T256A, T256D, K258V, E259G, E259V, M260D, M260W, F 261K, F261P, F261S, F266Y, N276F, N276W, N276Y, N279S, N280F, N280W, N28 0Y, W283N, S286K, S286L, S302T, S302Y, G303R, T315A, H320T, P321H, P321Y,M322G, M322Y, H323A, H323N, H323W, W346N, L354V, E359W, Q360M, Q360W, Q360Y, G361M , Y362V, V365I, Y371S, Y371N, A383D, E390A, E390D, N394W, N394D, F395Y, D405G, I409L , R414M, H420P, H420A, H420Q, H420Y, N422V, T427L, G455R, K457N, K457R, R458S, N470A, N470M, S472R, V473C, N474F, N474L, G475E, G475K, G476H, G476E, and G476A. and a modification at position 144 of the present invention, wherein the variant has α-amylase activity, and the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 12.

[0362] In another aspect, the variants are selected from the group consisting of positions H1*, H2*, N3D, N3G, N3S, G7A, G7H, G7S, R26Y, A31S, R37A, R37N, R37V, T40G, T40N, A41D, N54S, V56T, A60V, A60P, L63F, K72R, G73L, K93R, N94D, Q98L, V103A, V103I, V103L, V104M, M105I, M105L, N106D, A109G, G110K, A111G, A111L, G113Q, G113S, G113A, T114K, F11 6L, F116N, F116Q, F116W, V117G, D118Q, D118T, D123N, N126D, N128Y, T131I , T131V, S132D, S132L, T134D, Y135E, Q136T, Q136K, I137F, Q138V, W140S, W 140M, W140Y, K142E, K142G, K142P, D144N, P146S, P146H, T151A, Y152L, F15 5W, W159H, W159L, Y160P, T165V, W167A, W167D, W167E, W167F, W167P, W167S, E169I, E169Q, E169V, K172G, K172Q, L173F, N174K, N174S, I176V, Y177D, K1 78A, R180*, R180Q, R180S, S181*, S181L, S181T, T182*, T182D, T182E, T182 G, T182I, T182L, T182M, T182S, G183*, G183P, A185D, A185E, A185K, A185N, A185Q, A185R, W188C, E189P, E193I, N194F, F202Y, F202Y, A203F, L205F, L20 5N, L205Q, L205R, L205V, L205Y, T224F, T224Y, N225S, Y242F, Y242L, S243Q , F244I, F244L, V252I, T256A, T256D, K258V, E259G, E259V, M260D, M260W, F 261K, F261P, F261S, F266Y, N276F, N276W, N276Y, N279S, N280F, N280W, N28 0Y, W283N, S286K, S286L, S302T, S302Y, G303R, T315A, H320T, P321H, P321Y,M322G, M322Y, H323A, H323N, H323W, W346N, L354V, E359W, Q360M, Q360W, Q360Y, G361M , Y362V, V365I, Y371S, Y371N, A383D, E390A, E390D, N394W, N394D, F395Y, D405G, I409L , R414M, H420P, H420A, H420Q, H420Y, N422V, T427L, G455R, K457N, K457R, R458S, N470A, N470M, S472R, V473C, N474F, N474L, G475E, G475K, G476H, G476E, and G476A. and a modification at position 13, wherein the variant has α-amylase activity, and the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 13.

[0363] In another aspect, the variants are selected from the group consisting of positions H1*, H2*, N3D, N3G, N3S, G7A, G7H, G7S, R26Y, A31S, R37A, R37N, R37V, T40G, T40N, A41D, N54S, V56T, A60V, A60P, L63F, K72R, G73L, K93R, N94D, Q98L, V103A, V103I, V103L, V104M, M105I, M105L, N106D, A109G, G110K, A111G, A111L, G113Q, G113S, G113A, T114K, F11 6L, F116N, F116Q, F116W, V117G, D118Q, D118T, D123N, N126D, N128Y, T131I , T131V, S132D, S132L, T134D, Y135E, Q136T, Q136K, I137F, Q138V, W140S, W 140M, W140Y, K142E, K142G, K142P, D144N, P146S, P146H, T151A, Y152L, F15 5W, W159H, W159L, Y160P, T165V, W167A, W167D, W167E, W167F, W167P, W167S, E169I, E169Q, E169V, K172G, K172Q, L173F, N174K, N174S, I176V, Y177D, K1 78A, R180*, R180Q, R180S, S181*, S181L, S181T, T182*, T182D, T182E, T182 G, T182I, T182L, T182M, T182S, G183*, G183P, A185D, A185E, A185K, A185N, A185Q, A185R, W188C, E189P, E193I, N194F, F202Y, F202Y, A203F, L205F, L20 5N, L205Q, L205R, L205V, L205Y, T224F, T224Y, N225S, Y242F, Y242L, S243Q , F244I, F244L, V252I, T256A, T256D, K258V, E259G, E259V, M260D, M260W, F 261K, F261P, F261S, F266Y, N276F, N276W, N276Y, N279S, N280F, N280W, N28 0Y, W283N, S286K, S286L, S302T, S302Y, G303R, T315A, H320T, P321H, P321Y,M322G, M322Y, H323A, H323N, H323W, W346N, L354V, E359W, Q360M, Q360W, Q360Y, G361M , Y362V, V365I, Y371S, Y371N, A383D, E390A, E390D, N394W, N394D, F395Y, D405G, I409L , R414M, H420P, H420A, H420Q, H420Y, N422V, T427L, G455R, K457N, K457R, R458S, N470A, N470M, S472R, V473C, N474F, N474L, G475E, G475K, G476H, G476E, and G476A. and a modification at position 14, wherein the variant has α-amylase activity, and the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 14.

[0364] As used herein, the term "pairwise deletion" refers to the deletion of amino acids at two positions that can be adjacent to each other, but can also be separated by one, two, three, four, or five amino acids.

[0365] In one embodiment, the variant comprises a pair-wise deletion within the B domain of the parent polypeptide.

[0366] In one embodiment, the variant comprises a pair-wise deletion within the B domain of a parent polypeptide that has α-amylase activity.

[0367] As used herein, the term "pairwise deletion" refers to the deletion of amino acids at two positions that can be adjacent to each other, but can also be separated by one, two, three, four, or five amino acids.

[0368] In one aspect, the α-amylase variant of the invention comprises a deletion at one or more positions selected from the group consisting of R180*+S181*, R180*+T182*, R180*+G183*, S181*+T182*, S181*+G183*, and T182*+G183*, preferably at R180*S181*, using SEQ ID NO:1 for numbering.

[0369] In one aspect, the invention relates to a variant of SEQ ID NO: 1 comprising a deletion at the position corresponding to R180*+S181*, using SEQ ID NO: 1 for numbering, which variant has α-amylase activity, and which variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0370] Thus, in one embodiment, the variant is a) deletions and / or substitutions at two or more positions corresponding to positions 180, 181, 182, and 183, using SEQ ID NO:1 for numbering; b) using SEQ ID NO:1 for numbering, positions 1, 2, 3, 7, 26, 31, 37, 40, 41, 54, 56, 60, 63, 72, 73, 93, 94, 98, 103, 104, 105, 106, 109, 110, 111, 113, 114, 116, 117, 118, 123, 126, 128, 131, 132, 134, 135, 136, 137, 138, 140, 142, 144, 146, 151, 152, 155, 159, 160, 165, 167, 169, 172, 173, 174, 176, 177, 178, 185, 188, 189, 193, 194, 196, 202, 203, 205, 224, 225, 242, 243, 244, 252, 256, 258, 259, 260, 261, 266, 276, 279, 280, 283, 286, 302, 303, 315, 320, 321, 322, 323, 346, 354, 359, 360, 361, 362, 365, 371, 383, 39 and a modification at one or more positions corresponding to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, wherein the variants have α-amylase activity, and the variants have at least 60%, at least 65%, at least 70%, at least 75%, at least at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity.

[0371] In one embodiment, the variant is a) deletions and / or substitutions at two or more positions corresponding to positions R180, S181, T182, and G183, using SEQ ID NO:1 for numbering; b) Using SEQ ID NO: 1 for numbering, positions H1, H2*, N3, G7, R26, A31, R37, T40, A41, N54, V56, A60, L63, K72, G73, K93, N94, Q98, V103, V104, M105, N106, A109, G110, A111, G113, T114, F116, V117, D118, D123, N126, N128, T131, S132, T134, Y135, Q136, I137, Q138, W140, K142, D144, P146, T151, Y152 , F155, W159, Y160, T165, W167, E169, K172, L173, N174, I176, Y177, K178, A185, W188, E189, E193, N194, N196, F202, A203, L205, T224, N225 , Y242, S243, F244, V252, T256, K258, E259, M260, F261, F266, N276, N2 79, K280, W283, S286, S302, G303, T315, H320, P321, M322, H323, W346, and a modification at one or more positions corresponding to L354, E359, Q360, G361, Y362, V365, Y371, A383, E390, N394, F395, D405, I409, R414, H420, N422, T427, G455, K457, P458, N470, S472, V473, N474, G475, and G476, wherein the variant has α-amylase activity, and the variant has at least 60% repeat sequence relative to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. %, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity.

[0372] In one embodiment, the variant is a) deletions and / or substitutions at two or more positions corresponding to positions R180, S181, T182, and G183, using SEQ ID NO:1 for numbering; b) Using SEQ ID NO:1 for numbering, positions H1*, H2*, N3D, N3G, N3S, G7A, G7H, G7S, R26Y, A31S, R37A, R37N, R37V, T40G, T40N, A41D, N54S, V56T, A60V, A60P, L63F, K72R, G73L, K93R, N94D, Q98L, V103A, V103I, V103L, V104M, M105I, M105L, N106D, A109G, G110K, A111G, A111L, G113Q, G113S, G113A, T114K, F116L, F116N, F116R, F116S ... 16Q, F116W, V117G, D118Q, D118T, D123N, N126D, N128Y, T131I, T131V, S132 D, S132L, T134D, Y135E, Q136T, Q136K, I137F, Q138V, W140S, W140M, W140Y, K142E, K142G, K142P, D144N, P146S, P146H, T151A, Y152L, F155W, W159H, W1 59L, Y160P, T165V, W167A, W167D, W167E, W167F, W167P, W167S, E169I, E169Q , E169V, K172G, K172Q, L173F, N174K, N174S, I176V, Y177D, K178A, A185D, A 185E, A185K, A185N, A185Q, A185R, W188C, E189P, E193I, N194F, F202Y, F20 2Y, A203F, L205F, L205N, L205Q, L205R, L205V, L205Y, T224F, T224Y, N225S , Y242F, Y242L, S243Q, F244I, F244L, V252I, T256A, T256D, K258V, E259G, E2 59V, M260D, M260W, F261K, F261P, F261S, F266Y, N276F, N276W, N276Y, N279 S, N280F, N280W, N280Y, W283N, S286K, S286L, S302T, S302Y, G303R, T315A, H 320T, P321H, P321Y, M322G, M322Y, H323A, H323N, H323W, W346N, L354V, E35 9W, Q360M, Q360W, Q360Y, G361M, Y362V, V365I, Y371S, Y371N, A383D, E390A,and modifications at one or more positions corresponding to E390D, N394W, N394D, F395Y, D405G, I409L, R414M, H420P, H420A, H420Q, H420Y, N422V, T427L, G455R, K457N, K457R, R458S, N470A, N470M, S472R, V473C, N474F, N474L, G475E, G475K, G476H, G476E, and G476A, wherein the variant has alpha-amylase activity, and the variant has the sequence set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity.

[0373] In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: a) a deletion at positions corresponding to positions R180* and S181*, using SEQ ID NO:1 for numbering, b) Using SEQ ID NO:1 for numbering, positions H1*+D118T, H1*+W140Y, H1*+T182S, H1*+A185D, H1*+E189P, H1*+Y242F, H1*+E259G, H1*+F266Y, H1*+N279S, H1*+K280Y, H1*+G303R, H1*+E390A, H1*+G475K, H1*+A185Q, H1*+N194F, D118T+W140Y, D118T+T182S, D118T+A185D, D118T+E189P, D118T+Y242F, D118T+E259G, D1 18T+F266Y, D118T+N279S, D118T+K280Y, D118T+G303R, D118T+E390A, D118 T+G475K, D118T+A185Q, D118T+N194F, W140Y+T182S, W140Y+A185D, W140Y+ E189P, W140Y+Y242F, W140Y+E259G, W140Y+F266Y, W140Y+N279S, W140Y+K2 80Y, W140Y+G303R, W140Y+E390A, W140Y+G475K, W140Y+A185Q, W140Y+N194 F, T182S+A185D, T182S+E189P, T182S+Y242F, T182S+E259G, T182S+F266Y, T182S+N279S, T182S+K280Y, T182S+G303R, T182S+E390A, T182S+G475K, T1 82S+A185Q, T182S+N194F, A185D+E189P, A185D+Y242F, A185D+E259G, A185 D+F266Y, A185D+N279S, A185D+K280Y, A185D+G303R, A185D+E390A, A185D+ G475K, A185D, +A185Q, A185D, +N194F, E189P+Y242F, E189P+E259G, E189P+ F266Y, E189P+N279S, E189P+K280Y, E189P+G303R, E189P+E390A, E189P+G4 75K, E189P+A185Q, E189P+N194F, Y242F+E259G, Y242F+F266Y, Y242F+N279 S, Y242F+K280Y, Y242F+G303R, Y242F+E390A, Y242F+G475K, Y242F+A185Q,Y242F+N194F, E259G+F266Y, E259G+N279S, E259G+K280Y, E259G+G303R, E259G+E390A, E259G+G475K, E259G+A185Q, E 259G+N194F, F266Y+N279S, F266Y+K280Y, F266Y+G303R, F266Y+E390A, F266Y+G475K, F266Y+A185Q, F266Y+N194F, N27 9+K280Y, N279S+G303R, N279S+E390A, N279S+G475K, N279S+A185Q, N279S+N194F, K280Y+G303R, K280Y+E390A, K280Y+ G475K, K280Y+A185Q, K280Y+N194F, G303R+E390A, G303R+G475K, G303R+A185Q, G303R+N194F, E390A+G475K, E390A+A1 and a modification at a position corresponding to 85Q, E390A+N194F, G475K+A185Q, G475K+N194F, A185Q+N194F, the variant having a similar structure to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 ... , at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, and the variant has α-amylase activity.

[0374] In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: a) a deletion at positions corresponding to positions R180* and S181*, using SEQ ID NO:1 for numbering, b) Using SEQ ID NO:1 for numbering, positions H1*+D118T+W140Y, H1*+D118T+T182S, H1*+D118T+A185D, H1*+D118T+E189P, H1*+D118T+Y242F, H1*+D118T+E259G, H1*+D118T+F266Y, H1*+D118T+N279S, H1*+D118T+K280Y, H1*+D118T+G303R, H1*+D118T+E390A, H1*+D118T+G475K, H1*+D118T+A185Q, H1*+D118T+N194F, H1 *+W140Y+T182S, H1*+W140Y+A185D, H1*+W140Y+E189P, H1*+W140Y+Y242F, H1*+W140Y+E259G, H1*+W140Y+F266Y, H1*+W140Y+N279S, H1*+W140Y+K280Y , H1*+W140Y+G303R, H1*+W140Y+E390A, H1*+W140Y+G475K, H1*+W140Y+A18 5Q, H1*+W140Y+N194F, H1*+T182S+A185D, H1*+T182S+E189P, H1*+T182S+Y2 42F, H1*+T182S+E259G, H1*+T182S+F266Y, H1*+T182S+N279S, H1*+T182S+ K280Y, H1*+T182S+G303R, H1*+T182S+E390A, H1*+T182S+G475K, H1*+T182S +A185Q, H1*+T182S+N194F, H1*+A185D+E189P, H1*+A185D+Y242F, H1*+A18 5D+E259G, H1*+A185D+F266Y, H1*+A185D+N279S, H1*+A185D+K280Y, H1*+A1 85D+G303R, H1*+A185D+E390A, H1*+A185D+G475K, H1*+A185D+A185Q, H1*+ A185D+N194F, H1*+E189P+Y242F, H1*+E189P+E259G, H1*+E189P+F266Y, H1* +E189P+N279S, H1*+E189P+K280Y, H1*+E189P+G303R, H1*+E189P+E390A, H 1*+E189P+G475K, H1*+E189P+A185Q, H1*+E189P+N194F, H1*+Y242F+E259G,H1*+Y242F+F266Y、H1*+Y242F+N279S、H1*+Y242F+K280Y、H1*+Y242F+G303R、H1*+Y242F+E390A、H1*+Y242F+G475K、H1*+Y242F+A185Q、H1*+Y242F+N194F、H1*+E259G+F266Y、H1*+E259G+N279S、H1*+E259G+K280Y、H1*+E259G+G303R、H1*+E259G+E390A、H1*+E259G+G475K、H1*+E259G+A185Q、H1*+E259G+N194F、H1*+F266Y+N279S、H1*+F266Y+K280Y、H1*+F266Y+G303R、H1*+F266Y+E390A、H1*+F266Y+G475K、H1*+F266Y+A185Q、H1*+F266Y+N194F、H1*+N279S+K280Y、H1*+N279S+G303R、H1*+N279S+E390A、H1*+N279S+G475K、H1*+N279S+A185Q、H1*+N279S+N194F、H1*+K280Y+G303R、H1*+K280Y+E390A、H1*+K280Y+G475K、H1*+K280Y+A185Q、H1*+K280Y+N194F、H1*+G303R+E390A、H1*+G303R+G475K、H1*+G303R+A185Q、H1*+G303R+N194F、H1*+E390A+G475K、H1*+E390A+A185Q、H1*+E390A+N194F、H1*+G475K+A185Q、H1*+G475K+N194F、H1*+A185Q+N194F、D118T+W140Y+T182S、D118T+W140Y+A185D、D118T+W140Y+E189P、D118T+W140Y+Y242F、D118T+W140Y+E259G、D118T+W140Y+F266Y、D118T+W140Y+N279S、D118T+W140Y+K280Y、D118T+W140Y+G303R、D118T+W140Y+E390A、D118T+W140Y+G475K、D118T+W140Y+A185Q、D118T+W140Y+N194F、D118T+T182S+A185D、D118T+T182S+E189P、D118T+T182S+Y242F、D118T+T182S+E259G、D118T+T182S+F266Y、D118T+T182S+N279S、D118T+T182S+K280Y、D118T+T182S+G303R、D118T+T182S+E390A、D118T+T182S+G475K、D118T+T182S+A185Q、D118T+T182S+N194F、D118T+A185D+E189P、D118T+A185D+Y242F、D118T+A185D+E259G、D118T+A185D+F266Y、D118T+A185D+N279S、D118T+A185D+K280Y、D118T+A185D+G303R、D118T+A185D+E390A、D118T+A185D+G475K、D118T+A185D+A185Q、D118T+A185D+N194F、D118T+E189P+Y242F、D118T+E189P+E259G、D118T+E189P+F266Y、D118T+E189P+N279S、D118T+E189P+K280Y、D118T+E189P+G303R、D118T+E189P+E390A、D118T+E189P+G475K、D118T+E189P+A185Q、D118T+E189P+N194F、D118T+Y242F+E259G、D118T+Y242F+F266Y、D118T+Y242F+N279S、D118T+Y242F+K280Y、D118T+Y242F+G303R、D118T+Y242F+E390A、D118T+Y242F+G475K、D118T+Y242F+A185Q、D118T+Y242F+N194F、D118T+E259G+F266Y、D118T+E259G+N279S、D118T+E259G+K280Y、D118T+E259G+G303R、D118T+E259G+E390A、D118T+E259G+G475K、D118T+E259G+A185Q、D118T+E259G+N194F、D118T+F266Y+N279S、D118T+F266Y+K280Y、D118T+F266Y+G303R、D118T+F266Y+E390A、D118T+F266Y+G475K、D118T+F266Y+A185Q、D118T+F266Y+N194F、D118T+N279S+K280Y、D118T+N279S+G303R、D118T+N279S+E390A、D118T+N279S+G475K、D118T+N279S+A185Q、D118T+N279S+N194F、D118T+K280Y+G303R、D118T+K280Y+E390A、D118T+K280Y+G475K、D118T+K280Y+A185Q、D118T+K280Y+N194F、D118T+G303R+E390A、D118T+G303R+G475K、D118T+G303R+A185Q、D118T+G303R+N194F、D118T+E390A+G475K、D118T+E390A+A185Q、D118T+E390A+N194F、D118T+G475K+A185Q、D118T+G475K+N194F、D118T+A185Q+N194F、W140Y+T182S+A185D、W140Y+T182S+E189P、W140Y+T182S+Y242F、W140Y+T182S+E259G、W140Y+T182S+F266Y、W140Y+T182S+N279S、W140Y+T182S+K280Y、W140Y+T182S+G303R、W140Y+T182S+E390A、W140Y+T182S+G475K、W140Y+T182S+A185Q、W140Y+T182S+N194F、W140Y+A185D+E189P、W140Y+A185D+Y242F、W140Y+A185D+E259G、W140Y+A185D+F266Y、W140Y+A185D+N279S、W140Y+A185D+K280Y、W140Y+A185D+G303R、W140Y+A185D+E390A、W140Y+A185D+G475K、W140Y+A185D+A185Q、W140Y+A185D+N194F、W140Y+E189P+Y242F、W140Y+E189P+E259G、W140Y+E189P+F266Y、W140Y+E189P+N279S、W140Y+E189P+K280Y、W140Y+E189P+G303R、W140Y+E189P+E390A、W140Y+E189P+G475K、W140Y+E189P+A185Q、W140Y+E189P+N194F、W140Y+Y242F+E259G、W140Y+Y242F+F266Y、W140Y+Y242F+N279S、W140Y+Y242F+K280Y、W140Y+Y242F+G303R、W140Y+Y242F+E390A、W140Y+Y242F+G475K、W140Y+Y242F+A185Q、W140Y+Y242F+N194F、W140Y+E259G+F266Y、W140Y+E259G+N279S、W140Y+E259G+K280Y、W140Y+E259G+G303R、W140Y+E259G+E390A、W140Y+E259G+G475K、W140Y+E259G+A185Q、W140Y+E259G+N194F、W140Y+F266Y+N279S、W140Y+F266Y+K280Y、W140Y+F266Y+G303R、W140Y+F266Y+E390A、W140Y+F266Y+G475K、W140Y+F266Y+A185Q、W140Y+F266Y+N194F、W140Y+N279S+K280Y、W140Y+N279S+G303R、W140Y+N279S+E390A、W140Y+N279S+G475K、W140Y+N279S+A185Q、W140Y+N279S+N194F、W140Y+K280Y+G303R、W140Y+K280Y+E390A、W140Y+K280Y+G475K、W140Y+K280Y+A185Q、W140Y+K280Y+N194F、W140Y+G303R+E390A、W140Y+G303R+G475K、W140Y+G303R+A185Q、W140Y+G303R+N194F、W140Y+E390A+G475K、W140Y+E390A+A185Q、W140Y+E390A+N194F、W140Y+G475K+A185Q、W140Y+G475K+N194F、W140Y+A185Q+N194F、T182S+A185D+E189P、T182S+A185D+Y242F、T182S+A185D+E259G、T182S+A185D+F266Y、T182S+A185D+N279S、T182S+A185D+K280Y、T182S+A185D+G303R、T182S+A185D+E390A、T182S+A185D+G45K、T182S+A185D+A185Q、T182S+A185D+N194F、T182S+E189P+Y242F、T182S+E189P+E259G、T182S+E189P+F266Y、T182S+E189P+N279S、T182S+E189P+K280Y、T182S+E189P+G303R、T182S+E189P+E390A、T182S+E189P+G475K、T182S+E189P+A185Q、T182S+E189P+N194、 F、T182S+Y242F+E259G、T182S+Y242F+F266Y、T182S+Y242F+N279S、T182S+Y242F+K280Y、T182S+Y242F+G303R、T182S+Y242F+E390A、T182S+Y242F+G475K、T182S+Y242F+A185Q、T182S+Y242F+N194F、T182S+E259G+F266Y、T182S+E259G+N279S、T182S+E259G+K280Y、T182S+E259G+G303R、T182S+E259G+E390A、T182S+E259G+G475K、T182S+E259G+A185Q、T182S+E259G+N194F、T182S+F266Y+N279S、T182S+F266Y+K280Y、T182S+F266Y+G303R、T182S+F266Y+E390A、T182S+F266Y+G475K、T182S+F266Y+A185Q、T182S+F266Y+N194F、T182S+N279S+K280Y、T182S+N279S+G303R、T182S+N279S+E390A、T182S+N279S+G475K、T182S+N279S+A185Q、T182S+N279S+N194F、T182S+K280Y+G303R、T182S+K280Y+E390A、T182S+K280Y+G475K、T182S+K280Y+A185Q、T182S+K280Y+N194F、T182S+G303R+E390A、T182S+G303R+G475K、T182S+G303R+A185Q、T182S+G303R+N194F、T182S+E390A+G475K、T182S+E390A+A185Q、T182S+E390A+N194F、T182S+G475K+A185Q、T182S+G475K+N194F、T182S+A185Q+N194F、A185D+E189P+Y242F、A185D+E189P+E259G、A185D+E189P+F266Y、A185D+E189P+N279S、A185D+E189P+K280Y、A185D+E189P+G303R、A185D+E189P+E390A、A185D+E189P+G475K、A185D+E189P+A185Q、A185D+E189P+N194F、A185D+Y242F+E259G、A185D+Y242F+F266Y、A185D+Y242F+N279S、A185D+Y242F+K280Y、A185D+Y242F+G303R、A185D+Y242F+E390A、A185D+Y242F+G475K、A185D+Y242F+A185Q、A185D+Y242F+N194F、A185D+E259G+F266Y、A185D+E259G+N279S、A185D+E259G+K280Y、A185D+E259G+G303R、A185D+E259G+E390A、A185D+E259G+G475K、A185D+E259G+A185Q、A185D+E259G+N194F、A185D+F266Y+N279S、A185D+F266Y+K280Y、A185D+F266Y+G303R、A185D+F266Y+E390A、A185D+F266Y+G475K、A185D+F266Y+A185Q、A185D+F266Y+N194F、A185D+N279S+K280Y、A185D+N279S+G303R、A185D+N279S+E390A、A185D+N279S+G475K、A185D+N279S+A185Q、A185D+N279S+N194F、A185D+K280Y+G303R、A185D+K280Y+E390A、A185D+K280Y+G475K、A185D+K280Y+A185Q、A185D+K280Y+N194F、A185D+G303R+E390A、A185D+G303R+G475K、A185D+G303R+A185Q、A185D+G303R+N194F、A185D+E390A+G475K、A185D+E390A+A185Q、A185D+E390A+N194F、A185D+G475K+A185Q、A185D+G475K+N194F、A185D+A185Q+N194F、E189P+Y242F+E259G、E189P+Y242F+F266Y、E189P+Y242F+N279S、E189P+Y242F+K280Y、E189P+Y242F+G303R、E189P+Y242F+E390A、E189P+Y242F+G475K、E189P+Y242F+A185Q、E189P+Y242F+N194F、E189P+E259G+F266Y、E189P+E259G+N279S、E189P+E259G+K280Y、E189P+E259G+G303R、E189P+E259G+E390A、E189P+E259G+G475K、E189P+E259G+A185Q、E189P+E259G+N194F、E189P+F266Y+N279S、E189P+F266Y+K280Y、E189P+F266Y+G303R、E189P+F266Y+E390A、E189P+F266Y+G475K、E189P+F266Y+A185Q、E189P+F266Y+N194F、E189P+N279S+K280Y、E189P+N279S+G303R、E189P+N279S+E390A、E189P+N279S+G475K、E189P+N279S+A185Q、E189P+N279S+N194F、E189P+K280Y+G303R、E189P+K280Y+E390A、E189P+K280Y+G475K、E189P+K280Y+A185Q、E189P+K280Y+N194F、E189P+G303R+E390A、E189P+G303R+G475K、E189P+G303R+A185Q、E189P+G303R+N194F、E189P+E390A+G475K、E189P+E390A+A185Q、E189P+E390A+N194F、E189P+G475K+A185Q、E189P+G475K+N194F、E189P+A185Q+N194F、Y242F+E259G+F266Y、Y242F+E259G+N279S、Y242F+E259G+K280Y、Y242F+E259G+G303R、Y242F+E259G+E390A、Y242F+E259G+G475K、Y242F+E259G+A185Q、Y242F+E259G+N194F、Y242F+F266Y+N279S、Y242F+F266Y+K280Y、Y242F+F266Y+G303R、Y242F+F266Y+E390A、Y242F+F266Y+G475K、Y242F+F266Y+A185Q、Y242F+F266Y+N194F、Y242F+N279S+K280Y、Y242F+N279S+G303R、Y242F+N279S+E390A、Y242F+N279S+G475K、Y242F+N279S+A185Q、<h2 style=";text-align:left;direction:ltr">Y242F+N279S+N194F、Y242F+K280Y+G303R、Y242F+K280Y+E390A、Y242F+K 280Y+G475K、Y242F+K280Y+A185Q、Y242F+K280Y+N194F、Y242F+G303R+E39 0A、Y242F+G303R+G475K、Y242F+G303R+A185Q、Y242F+G303R+N194F、Y242F +E390A+G475K、Y242F+E390A+A185Q、Y242F+E390A+N194F、Y242F+G475K+A 185Q、Y242F+G475K+N194F、Y242F+A185Q+N194F、E259G+F266Y+N279S、E25 9G+F266Y+K280Y、E259G+F266Y+G303R、E259G+F266Y+E390A、E259G+F266Y +G475K, E259G+F266Y+A185Q, E259G+F266Y+N194F, E259G+N279S+K280Y, E259G+N279S+G303R, E259G+N279S+E390A, E259G+N279S+G475K, E259G+N27 9S+A185Q、E259G+N279S+N194F、E259G+K280Y+G303R、E259G+K280Y+E390A、E259G+K280Y+G475K、E259G+K280Y+A185Q、E259G+K280Y+N194F、E259G+ G303R+E390A, E259G+G303R+G475K, E259G+G303R+A185Q, E259G+G303R+N194F, E259G+E390A+G475K, E259G+E390A+A185Q, E259G+E390A+N194F, E259 G+G475K+A185Q, E259G+G475K+N194F, E259G+A185Q+N194F, F266Y+N279S+K280Y, F266Y+N279S+G303R, F266Y+N279S+E390A, F266Y+N279S+G475K, F2 66Y+N279S+A185Q、F266Y+N279S+N194F、F266Y+K280Y+G303R、F266Y+K280 Y+E390A、F266Y+K280Y+G475K、F266Y+K280Y+A185Q、F266Y+K280Y+N194F、F266Y+G303R+E390A、F266Y+G303R+G475K、F266Y+G303R+A185Q、F266Y+G303R+N194F、F266Y+E390A+G475K、F266Y+E390A+A185Q、F266Y+E390A+N194F、F266Y+G475K+A185Q、F266Y+G475K+N194F、F266Y+A185Q+N194F、N279S+K280Y+G303R、N279S+K280Y+E390A、N279S+K280Y+G475K、N279S+K280Y+A185Q、N279S+K280Y+N194F、N279S+G303R+E390A、N279S+G303R+G475K、N279S+G303R+A185Q、N279S+G303R+N194F、N279S+E390A+G475K、N279S+E390A+A185Q、N279S+E390A+N194F、N279S+G475K+A185Q、 N279S+G475K+N194F, N279S+A185Q+N194F, K280Y+G303R+E390A, K280Y+G303R+G475K, K280Y+G303R+A185Q, K 280Y+G303R+N194F, K280Y+E390A+G475K, K280Y+E390A+A185Q, K280Y+E390A+N194F, K280Y+G475K+A185Q, K28 0Y+G475K+N194F, K280Y+A185Q+N194F, G303R+E390A+G475K, G303R+E390A+A185Q, G303R+E390A+N194F, G303R +G475K+A185Q, G303R+G475K+N194F, G303R+A185Q+N194F, E390A+G475K+A185Q, E390A+G475K+N194F, E390A+A and modifications at positions corresponding to G475K+A185Q+N194F, G475K+A185Q+N194F, wherein the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, and wherein the variant has α-amylase activity.

[0375] In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: a) a deletion at positions corresponding to positions R180* and S181*, using SEQ ID NO:1 for numbering, b) using SEQ ID NO: 1 for numbering, positions H1*+G7A+W140Y+E189P, H1*+G7A+W140Y+Y242F, H1*+G7A+W140Y+E259G, H1*+G7A+W140Y+N279S, H1*+G7A+W140Y+K280Y, H1*+G7A+W140Y+G303R, H1*+G7A+W140Y+E390A, H1*+G7A+E189P+Y242F, H1*+G7A+E189P+E259G, H1*+G7A+E189P+N279S, H1*+G7A+E189P+K280Y, H1*+G7A +E189P+G303R, H1*+G7A+E189P+E390A, H1*+G7A+Y242F+E259G, H1*+G7A+Y 242F+N279S, H1*+G7A+Y242F+K280Y, H1*+G7A+Y242F+G303R, H1*+G7A+Y242 F+E390A, H1*+G7A+E259G+N279S, H1*+G7A+E259G+K280Y, H1*+G7A+E259G+ G303R, H1*+G7A+E259G+E390A, H1*+G7A+N279S+K280Y, H1*+G7A+N279S+G30 3R, H1*+G7A+N279S+E390A, H1*+G7A+K280Y+G303R, H1*+G7A+K280Y+E390A , H1*+G7A+G303R+E390A, H1*+W140Y+E189P+Y242F, H1*+W140Y+E189P+E259 G, H1*+W140Y+E189P+N279S, H1*+W140Y+E189P+K280Y, H1*+W140Y+E189P+ G303R, H1*+W140Y+E189P+E390A, H1*+W140Y+Y242F+E259G, H1*+W140Y+Y24 2F+N279S, H1*+W140Y+Y242F+K280Y, H1*+W140Y+Y242F+G303R, H1*+W140Y +Y242F+E390A, H1*+W140Y+E259G+N279S, H1*+W140Y+E259G+K280Y, H1*+W1 40Y+E259G+G303R, H1*+W140Y+E259G+E390A, H1*+W140Y+N279S+K280Y, H1* +W140Y+N279S+G303R, H1*+W140Y+N279S+E390A, H1*+W140Y+K280Y+G303R,H1*+W140Y+K280Y+E390A、H1*+W140Y+G303R+E390A、H1*+E189P+Y242F+E259G、H1*+E189P+Y242F+N279S、H1*+E189P+Y242F+K280Y、H1*+E189P+Y242F+G303R、H1*+E189P+Y242F+E390A、H1*+E189P+E259G+N279S、H1*+E189P+E259G+K280Y、H1*+E189P+E259G+G303R、H1*+E189P+E259G+E390A、H1*+E189P+N279S+K280Y、H1*+E189P+N279S+G303R、H1*+E189P+N279S+E390A、H1*+E189P+K280Y+G303R、H1*+E189P+K280Y+E390A、H1*+E189P+G303R+E390A、H1*+Y242F+E259G+N279S、H1*+Y242F+E259G+K280Y、H1*+Y242F+E259G+G303R、H1*+Y242F+E259G+E390A、H1*+Y242F+N279S+K280Y、H1*+Y242F+N279S+G303R、H1*+Y242F+N279S+E390A、H1*+Y242F+K280Y+G303R、H1*+Y242F+K280Y+E390A、H1*+Y242F+G303R+E390A、H1*+E259G+N279S+K280Y、H1*+E259G+N279S+G303R、H1*+E259G+N279S+E390A、H1*+E259G+K280Y+G303R、H1*+E259G+K280Y+E390A、H1*+E259G+G303R+E390A、H1*+N279S+K280Y+G303R、H1*+N279S+K280Y+E390A、H1*+N279S+G303R+E390A、H1*+K280Y+G303R+E390A、G7A+W140Y+E189P+Y242F、G7A+W140Y+E189P+E259G、G7A+W140Y+E189P+N279S、G7A+W140Y+E189P+K280Y、G7A+W140Y+E189P+G303R、G7A+W140Y+E189P+E390A、G7A+W140Y+Y242F+E259G、G7A+W140Y+Y242F+N279S、<h2 style=";text-align:left;direction:ltr">G7A+W140Y+Y242F+K280Y、G7A+W140Y+Y242F+G303R、G7A+W140Y+Y242F+E390A、G7A+W140Y+E259G+N279S、G7A+W140Y+E259G+K280Y、G7A+W140Y+E25 9G+G303R、G7A+W140Y+E259G+E390A、G7A+W140Y+N279S+K280Y、G7A+W140Y +N279S+G303R、G7A+W140Y+N279S+E390A、G7A+W140Y+K280Y+G303R、G7A+W 140Y+K280Y+E390A、G7A+W140Y+G303R+E390A、G7A+E189P+Y242F+E259G、G7A+E189P+Y242F+N279S、G7A+E189P+Y242F+K280Y、G7A+E189P+Y242F+G30 3R、G7A+E189P+Y242F+E390A、G7A+E189P+E259G+N279S、G7A+E189P+E259G+K280Y、G7A+E189P+E259G+G303R、G7A+E189P+E259G+E390A、G7A+E189P+N 279S+K280Y、G7A+E189P+N279S+G303R、G7A+E189P+N279S+E390A、G7A+E1 89P+K280Y+G303R、G7A+E189P+K280Y+E390A、G7A+E189P+G303R+E390A、G7 A+Y242F+E259G+N279S、G7A+Y242F+E259G+K280Y、G7A+Y242F+E259G+G303R、G7A+Y242F+E259G+E390A、G7A+Y242F+N279S+K280Y、G7A+Y242F+N279S+ G303R、G7A+Y242F+N279S+E390A、G7A+Y242F+K280Y+G303R、G7A+Y242F+K280Y+E390A、G7A+Y242F+G303R+E390A、G7A+E259G+N279S+K280Y、G7A+E259 G+N279S+G303R、G7A+E259G+N279S+E390A、G7A+E259G+K280Y+G303R、G7A+E259G+K280Y+E390A、G7A+E259G+G303R+E390A、G7A+N279S+K280Y+G303R、G7A+N279S+K280Y+E390A、G7A+N279S+G303R+E390A、G7A+K280Y+G303R+E390A、W140Y+E189P+Y242F+E259G、W140Y+E189P+Y242F+N279S、W140Y+E189P+Y242F+K280Y、W140Y+E189P+Y242F+G303R、W140Y+E189P+Y242F+E390A、W140Y+E189P+E259G+N279S、W140Y+E189P+E259G+K280Y、W140Y+E189P+E259G+G303R、W140Y+E189P+E259G+E390A、W140Y+E189P+N279S+K280Y、W140Y+E189P+N279S+G303R、W140Y+E189P+N279S+E390A、W140Y+E189P+K280Y+G303R、W140Y+E189P+K280Y+E390A、W140Y+E189P+G303R+E390A、W140Y+Y242F+E259G+N279S、W140Y+Y242F+E259G+K280Y、W140Y+Y242F+E259G+G303R、W140Y+Y242F+E259G+E390A、W140Y+Y242F+N279S+K280Y、W140Y+Y242F+N279S+G303R、W140Y+Y242F+N279S+E390A、W140Y+Y242F+K280Y+G303R、W140Y+Y242F+K280Y+E390A、W140Y+Y242F+G303R+E390A、W140Y+E259G+N279S+K280Y、W140Y+E259G+N279S+G303R、W140Y+E259G+N279S+E390A、W140Y+E259G+K280Y+G303R、W140Y+E259G+K280Y+E390A、W140Y+E259G+G303R+E390A、W140Y+N279S+K280Y+G303R、W140Y+N279S+K280Y+E390AW140Y+N279S+G303R+E390A、W140Y+K280Y+G303R+E390A、E189P+Y242F+E259G+N279S、E189P+Y242F+E259G+K280Y、E189P+Y242F+E259G+G303R、E189P+Y242F+E259G+E390A、E189P+Y242F+N279S+K280Y, E189P+Y242F+N279S+G303R, E189P+Y242F+N2 79S+E390A, E189P+Y242F+K280Y+G303R, E189P+Y242F+K280Y+E390A, E189 P+Y242F+G303R+E390A, E189P+E259G+N279S+K280Y, E189P+E259G+N279S+ G303R, E189P+E259G+N279S+E390A, E189P+E259G+K280Y+G303R, E189P+E2 59G+K280Y+E390A, E189P+E259G+G303R+E390A, E189P+N279S+K280Y+G303 R, E189P+N279S+K280Y+E390A, E189P+N279S+G303R+E390A, E189P+K280Y+ G303R+E390A, Y242F+E259G+N279S+K280Y, Y242F+E259G+N279S+G303R, Y2 42F+E259G+N279S+E390A, Y242F+E259G+K280Y+G303R, Y242F+E259G+K280Y +E390A, Y242F+E259G+G303R+E390A, Y242F+N279S+K280Y+G303R, Y242F+N 279S+K280Y+E390A, Y242F+N279S+G303R+E390A, Y242F+K280Y+G303R+E39 Compatible with 0A, E259G+N279S+K280Y+G303R, E259G+N279S+K280Y+E390A, E259G+N279S+G303R+E390A, E259G+K280Y+G303R+E390A, N279S+K280Y+G303R+E390A and a modification at a position corresponding to at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%,At least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity, and the variant has α-amylase activity.

[0376] In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: a) a deletion at positions corresponding to positions R180* and S181*, using SEQ ID NO:1 for numbering, b) using SEQ ID NO:1 for numbering, positions H1*+G7A+W140Y+E189P+Y242F, H1*+G7A+W140Y+E189P+E259G, H1*+G7A+W140Y+E189P+N279S, H1*+G7A+W140Y+E189P+K280Y, H1*+G7A+W140Y+E189P+G303R, H1*+G7A+W140Y+E189P+E390A, H1*+G7A+W140Y+Y242F+E259G, H1*+G7A+W140Y+Y242F+N279S, H1*+G7A+W140Y+Y 242F+K280Y, H1*+G7A+W140Y+Y242F+G303R, H1*+G7A+W140Y+Y242F+E390 A, H1*+G7A+W140Y+E259G+N279S, H1*+G7A+W140Y+E259G+K280YH1*+G7A+W 140Y+E259G+G303R, H1*+G7A+W140Y+E259G+E390A, H1*+G7A+W140Y+N279 S+K280Y, H1*+G7A+W140Y+N279S+G303R, H1*+G7A+W140Y+N279S+E390A, H1 *+G7A+W140Y+K280Y+G303R, H1*+G7A+W140Y+K280Y+E390A, H1*+G7A+W14 0Y+G303R+E390A, H1*+G7A+E189P+Y242F+E259G, H1*+G7A+E189P+Y242F+N 279S, H1*+G7A+E189P+Y242F+K280Y, H1*+G7A+E189P+Y242F+G303R, H1*+ G7A+E189P+Y242F+E390A, H1*+G7A+E189P+E259G+N279S, H1*+G7A+E189P+ E259G+K280Y, H1*+G7A+E189P+E259G+G303R, H1*+G7A+E189P+E259G+E39 0A, H1*+G7A+E189P+N279S+K280Y, H1*+G7A+E189P+N279S+G303R, H1*+G7A +E189P+N279S+E390A, H1*+G7A+E189P+K280Y+G303R, H1*+G7A+E189P+K2 80Y+E390A, H1*+G7A+E189P+G303R+E390A, H1*+G7A+Y242F+E259G+N279S,H1*+G7A+Y242F+E259G+K280Y、H1*+G7A+Y242F+E259G+G303R、H1*+G7A+Y242F+E259G+E390A、H1*+G7A+Y242F+N279S+K280Y、H1*+G7A+Y242F+N279S+G303R、H1*+G7A+Y242F+N279S+E390A、H1*+G7A+Y242F+K280Y+G303R、H1*+G7A+Y242F+K280Y+E390A、H1*+G7A+Y242F+G303R+E390A、H1*+G7A+E259G+N279S+K280Y、H1*+G7A+E259G+N279S+G303R、H1*+G7A+E259G+N279S+E390A、H1*+G7A+E259G+K280Y+G303R、H1*+G7A+E259G+K280Y+E390A、H1*+G7A+E259G+G303R+E390A、H1*+G7A+N279S+K280Y+G303R、H1*+G7A+N279S+K280Y+E390A、H1*+G7A+N279S+G303R+E390A、H1*+G7A+K280Y+G303R+E390A、H1*+W140Y+E189P+Y242F+E259G、H1*+W140Y+E189P+Y242F+N279S、H1*+W140Y+E189P+Y242F+K280Y、H1*+W140Y+E189P+Y242F+G303R、H1*+W140Y+E189P+Y242F+E390A、H1*+W140Y+E189P+E259G+N279S、H1*+W140Y+E189P+E259G+K280Y、H1*+W140Y+E189P+E259G+G303R、H1*+W140Y+E189P+E259G+E390A、H1*+W140Y+E189P+N279S+K280Y、H1*+W140Y+E189P+N279S+G303R、H1*+W140Y+E189P+N279S+E390A、H1*+W140Y+E189P+K280Y+G303R、H1*+W140Y+E189P+K280Y+E390A、H1*+W140Y+E189P+G303R+E390A、H1*+W140Y+Y242F+E259G+N279S、H1*+W140Y+Y242F+E259G+K280Y、H1*+W140Y+Y242F+E259G+G303R、H1*+W140Y+Y242F+E259G+E390A、H1*+W140Y+Y242F+N279S+K280Y、H1*+W140Y+Y242F+N279S+G303R、H1*+W140Y+Y242F+N279S+E390A、H1*+W140Y+Y242F+K280Y+G303R、H1*+W140Y+Y242F+K280Y+E390A、H1*+W140Y+Y242F+G303R+E390A、H1*+W140Y+E259G+N279S+K280Y、H1*+W140Y+E259G+N279S+G303R、H1*+W140Y+E259G+N279S+E390A、H1*+W140Y+E259G+K280Y+G303R、H1*+W140Y+E259G+K280Y+E390A、H1*+W140Y+E259G+G303R+E390A、H1*+W140Y+N279S+K280Y+G303R、H1*+W140Y+N279S+K280Y+E390A、H1*+W140Y+N279S+G303R+E390A、H1*+W140Y+K280Y+G303R+E390A、H1*+E189P+Y242F+E259G+N279S、H1*+E189P+Y242F+E259G+K280Y、H1*+E189P+Y242F+E259G+G303R、H1*+E189P+Y242F+E259G+E390A、H1*+E189P+Y242F+N279S+K280Y、H1*+E189P+Y242F+N279S+G303R、H1*+E189P+Y242F+N279S+E390A、H1*+E189P+Y242F+K280Y+G303R、H1*+E189P+Y242F+K280Y+E390A、H1*+E189P+Y242F+G303R+E390A、H1*+E189P+E259G+N279S+K280Y、H1*+E189P+E259G+N279S+G303R、H1*+E189P+E259G+N279S+E390A、H1*+E189P+E259G+K280Y+G303R、H1*+E189P+E259G+K280Y+E390A、H1*+E189P+E259G+G303R+E390A、H1*+E189P+N279S+K280Y+G303R、H1*+E189P+N279S+K280Y+E390A、<h2 style=";text-align:left;direction:ltr">H1*+E189P+N279S+G303R+E390A、H1*+E189P+K280Y+G303R+E390A、H1*+Y242F+E259G+N279S+K280Y、H1*+Y242F+E259G+N279S+G303R、H1*+Y242F+ E259G+N279S+E390A、H1*+Y242F+E259G+K280Y+G303R、H1*+Y242F+E259G+K280Y+E390A、H1*+Y242F+E259G+G303R+E390A、H1*+Y242F+N279S+K280Y +G303R、H1*+Y242F+N279S+K280Y+E390A、H1*+Y242F+N279S+G303R+E390A、H1*+Y242F+K280Y+G303R+E390A、H1*+E259G+N279S+K280Y+G303R、H1* +E259G+N279S+K280Y+E390A、H1*+E259G+N279S+G303R+E390A、H1*+E259G+K280Y+G303R+E390A、H1*+N279S+K280Y+G303R+E390A、G7A+W140Y+E189 P+Y242F+E259G、G7A+W140Y+E189P+Y242F+N279S、G7A+W140Y+E189P+Y242F+K280Y、G7A+W140Y+E189P+Y242F+G303R、G7A+W140Y+E189P+Y242F+E3 90A、G7A+W140Y+E189P+E259G+N279S、G7A+W140Y+E189P+E259G+K280Y、G 7A+W140Y+E189P+E259G+G303R、G7A+W140Y+E189P+E259G+E390A、G7A+W14 0Y+E189P+N279S+K280Y、G7A+W140Y+E189P+N279S+G303R、G7A+W140Y+E1 89P+N279S+E390A、G7A+W140Y+E189P+K280Y+G303R、G7A+W140Y+E189P+K 280Y+E390A、G7A+W140Y+E189P+G303R+E390A、G7A+W140Y+Y242F+E259G+N279S、G7A+W140Y+Y242F+E259G+K280Y、G7A+W140Y+Y242F+E259G+G303R、<h2 style=";text-align:left;direction:ltr">G7A+W140Y+Y242F+E259G+E390A、G7A+W140Y+Y242F+N279S+K280Y、G7A+W140Y+Y242F+N279S+G303R、G7A+W140Y+Y242F+N279S+E390A、G7A+W140Y+ Y242F+K280Y+G303R、G7A+W140Y+Y242F+K280Y+E390A、G7A+W140Y+Y242F+G303R+E390A、G7A+W140Y+E259G+N279S+K280Y、G7A+W140Y+E259G+N279S +G303R、G7A+W140Y+E259G+N279S+E390A、G7A+W140Y+E259G+K280Y+G303R、G7A+W140Y+E259G+K280Y+E390A、G7A+W140Y+E259G+G303R+E390A、G7A +W140Y+N279S+K280Y+G303R、G7A+W140Y+N279S+K280Y+E390A、G7A+W140Y+N279S+G303R+E390A、G7A+W140Y+K280Y+G303R+E390A、G7A+E189P+Y242 F+E259G+N279S、G7A+E189P+Y242F+E259G+K280Y、G7A+E189P+Y242F+E25 9G+G303R、G7A+E189P+Y242F+E259G+E390A、G7A+E189P+Y242F+N279S+K2 80Y, G7A+E189P+Y242F+N279S+G303R, G7A+E189P+Y242F+N279S+E390A, G7A+E189P+Y242F+K280Y+G303R, G7A+E189P+Y242F+K280Y+E390A, G7A+E18 9P+Y242F+G303R+E390A、G7A+E189P+E259G+N279S+K280Y、G7A+E189P+E2 59G+N279S+G303R、G7A+E189P+E259G+N279S+E390A、G7A+E189P+E259G+K 280Y+G303R、G7A+E189P+E259G+K280Y+E390A、G7A+E189P+E259G+G303R+E390A、G7A+E189P+N279S+K280Y+G303R、G7A+E189P+N279S+K280Y+E390A、G7A+E189P+N279S+G303R+E390A、G7A+E189P+K280Y+G303R+E390A、G7A+Y242F+E259G+N279S+K280Y、G7A+Y242F+E259G+N279S+G303R、G7A+Y242F+E259G+N279S+E390A、G7A+Y242F+E259G+K280Y+G303R、G7A+Y242F+E259G+K280Y+E390A、G7、 A+Y242F+E259G+G303R+E390A、G7A+Y242F+N279S+K280Y+G303R、G7A+Y242F+N279S+K280Y+E390A、G7A+Y242F+N279S+G303R+E390A、G7A+Y242F+K280Y+G303R+E390A、G7A+E259G+N279S+K280Y+G303R、G7A+E259G+N279S+K280Y+E390A、G7A+E259G+N279S+G303R+E390A、G7A+E259G+K280Y+G303R+E390A、G7A+N279S+K280Y+G303R+E390A、W140Y+E189P+Y242F+E259G+N279S、W140Y+E189P+Y242F+E259G+K280Y、W140Y+E189P+Y242F+E259G+G303R、W140Y+E189P+Y242F+E259G+E390A、W140Y+E189P+Y242F+N279S+K280Y、W140Y+E189P+Y242F+N279S+G303R、W140Y+E189P+Y242F+N279S+E390A、W140Y+E189P+Y242F+K280Y+G303R、W140Y+E189P+Y242F+K280Y+E390A、W140Y+E189P+Y242F+G303R+E390A、W140Y+E189P+E259G+N279S+K280Y、W140Y+E189P+E259G+N279S+G303R、W140Y+E189P+E259G+N279S+E390A、W140Y+E189P+E259G+K280Y+G303R、W140Y+E189P+E259G+K280Y+E390A、W140Y+E189P+E259G+G303R+E390A、W140Y+E189P+N279S+K280Y+G303R、W140Y+E189P+N279S+K280Y+E390A、W140Y+E189P+N279S+G303R+E390A、W140Y+E189P+K280Y+G303R+E390A、W140Y+Y242F+E259G+N279S+K280Y、W140Y+Y242F+E259G+N279S+G303R、W140Y+Y242F+E259G+N279S+E390A、W140Y+Y242F+E259G+K280Y+G303R、W140Y+Y242F+E259G+K280Y+E390A, W140Y+Y242F+E259G+G303R+E390A, W1 40Y+Y242F+N279S+K280Y+G303R, W140Y+Y242F+N279S+K280Y+E390A, W140 Y+Y242F+N279S+G303R+E390A, W140Y+Y242F+K280Y+G303R+E390A, W140Y+ E259G+N279S+K280Y+G303R, W140Y+E259G+N279S+K280Y+E390A, W140Y+E25 9G+N279S+G303R+E390A, W140Y+E259G+K280Y+G303R+E390A, W140Y+N279S +K280Y+G303R+E390A, E189P+Y242F+E259G+N279S+K280Y, E189P+Y242F+E2 59G+N279S+G303R, E189P+Y242F+E259G+N279S+E390A, E189P+Y242F+E259 G+K280Y+G303R, E189P+Y242F+E259G+K280Y+E390A, E189P+Y242F+E259G+G 303R+E390A, E189P+Y242F+N279S+K280Y+G303R, E189P+Y242F+N279S+K28 0Y+E390A, E189P+Y242F+N279S+G303R+E390A, E189P+Y242F+K280Y+G303R +E390A, E189P+E259G+N279S+K280Y+G303R, E189P+E259G+N279S+K280Y+E 390A, E189P+E259G+N279S+G303R+E390A, E189P+E259G+K280Y+G303R+E390 and a modification at a position corresponding to A, E189P+N279S+K280Y+G303R+E390A, Y242F+E259G+N279S+K280Y+G303R, Y242F+E259G+N279S+K280Y+E390A, Y242F+E259G+N279S+G303R+E390A, Y242F+E259G+K280Y+G303R+E390A, Y242F+N279S+K280Y+G303R+E390A, E259G+N279S+K280Y+G303R+E390A,and having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, wherein the variant has α-amylase activity.

[0377] In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: a) a deletion at positions corresponding to positions R180* and S181*, using SEQ ID NO:1 for numbering, b) using SEQ ID NO: 1 for numbering, positions H1*+G7A+W140Y+E189P+Y242F+E259G, H1*+G7A+W140Y+E189P+Y242F+N279S, H1*+G7A+W140Y+E189P+Y242F+K280Y, H1*+G7A+W140Y+E189P+Y242F+G303R, H1*+G7A+W140Y+E189P+Y242F+E390A, H1*+G7A+W140Y+E189P+E259G+N279S, H1*+G7A+W140Y+E189P+E259G+K280Y Y, H1*+G7A+W140Y+E189P+E259G+G303R, H1*+G7A+W140Y+E189P+E259G+E 390A, H1*+G7A+W140Y+E189P+N279S+K280Y, H1*+G7A+W140Y+E189P+N279S +G303R, H1*+G7A+W140Y+E189P+N279S+E390A, H1*+G7A+W140Y+E189P+K2 80Y+G303R, H1*+G7A+W140Y+E189P+K280Y+E390A, H1*+G7A+W140Y+E189P+ G303R+E390A, H1*+G7A+W140Y+Y242F+E259G+N279S, H1*+G7A+W140Y+Y24 2F+E259G+K280Y, H1*+G7A+W140Y+Y242F+E259G+G303R, H1*+G7A+W140Y+Y 242F+E259G+E390A, H1*+G7A+W140Y+Y242F+N279S+K280Y, H1*+G7A+W140 Y+Y242F+N279S+G303R, H1*+G7A+W140Y+Y242F+N279S+E390A, H1*+G7A+W1 40Y+Y242F+K280Y+G303R, H1*+G7A+W140Y+Y242F+K280Y+E390A, H1*+G7A +W140Y+Y242F+G303R+E390A, H1*+G7A+W140Y+E259G+N279S+K280Y, H1*+G 7A+W140Y+E259G+N279S+G303R, H1*+G7A+W140Y+E259G+N279S+E390A, H1 *+G7A+W140Y+E259G+K280Y+G303R, H1*+G7A+W140Y+E259G+K280Y+E390A,<h2 style=";text-align:left;direction:ltr">H1*+G7A+W140Y+E259G+G303R+E390A、H1*+G7A+W140Y+N279S+K280Y+G303R、H1*+G7A+W140Y+N279S+K280Y+E390A、H1*+G7A+W140Y+N279S+G303R+E3 90A、H1*+G7A+W140Y+K280Y+G303R+E390A、H1*+G7A+E189P+Y242F+E259G+N279S、H1*+G7A+E189P+Y242F+E259G+K280Y、H1*+G7A+E189P+Y242F+E259 G+G303R、H1*+G7A+E189P+Y242F+E259G+E390A、H1*+G7A+E189P+Y242F+N2 79S+K280Y、H1*+G7A+E189P+Y242F+N279S+G303R、H1*+G7A+E189P+Y242F+ N279S+E390A、H1*+G7A+E189P+Y242F+K280Y+G303R、H1*+G7A+E189P+Y242F+K280Y+E390A、H1*+G7A+E189P+Y242F+G303R+E390A、H1*+G7A+E189P+E2 59G+N279S+K280Y、H1*+G7A+E189P+E259G+N279S+G303R、H1*+G7A+E189P+E259G+N279S+E390A、H1*+G7A+E189P+E259G+K280Y+G303R、H1*+G7A+E189 P+E259G+K280Y+E390A、H1*+G7A+E189P+E259G+G303R+E390A、H1*+G7A+E1 89P+N279S+K280Y+G303R、H1*+G7A+E189P+N279S+K280Y+E390A、H1*+G7A+ E189P+N279S+G303R+E390A、H1*+G7A+E189P+K280Y+G303R+E390A、H1*+G7A+Y242F+E259G+N279S+K280Y、H1*+G7A+Y242F+E259G+N279S+G303R、H1*+ G7A+Y242F+E259G+N279S+E390A、H1*+G7A+Y242F+E259G+K280Y+G303R、H1*+G7A+Y242F+E259G+K280Y+E390A、H1*+G7A+Y242F+E259G+G303R+E390A、H1*+G7A+Y242F+N279S+K280Y+G303R、H1*+G7A+Y242F+N279S+K280Y+E390A、H1*+G7A+Y242F+N279S+G303R+E390A、H1*+G7A+Y242F+K280Y+G303R+E390A、H1*+G7A+E259G+N279S+K280Y+G303R、H1*+G7A+E259G+N279S+K280Y+E390A、H1*+G7A+E259G+N279S+G303R+E390A、H1*+G7A+E259G+K280Y+G303R+E390A、H1*+G7A+N279S+K280Y+G303R+E390A、H1*+W140Y+E189P+Y242F+E259G+N279S、H1*+W140Y+E189P+Y242F+E259G+K280Y、H1*+W140Y+E189P+Y242F+E259G+G303R、H1*+W140Y+E189P+Y242F+E259G+E390A、H1*+W140Y+E189P+Y242F+N279S+K280Y、H1*+W140Y+E189P+Y242F+N279S+G303R、H1*+W140Y+E189P+Y242F+N279S+E390A、H1*+W140Y+E189P+Y242F+K280Y+G303R、H1*+W140Y+E189P+Y242F+K280Y+E390A、H1*+W140Y+E189P+Y242F+G303R+E390A、H1*+W140Y+E189P+E259G+N279S+K280Y、H1*+W140Y+E189P+E259G+N279S+G303R、H1*+W140Y+E189P+E259G+N279S+E390A、H1*+W140Y+E189P+E259G+K280Y+G303R、H1*+W140Y+E189P+E259G+K280Y+E390A、H1*+W140Y+E189P+E259G+G303R+E390A、H1*+W140Y+E189P+N279S+K280Y+G303R、H1*+W140Y+E189P+N279S+K280Y+E390A、H1*+W140Y+E189P+N279S+G303R+E390A、H1*+W140Y+E189P+K280Y+G303R+E390A、H1*+W140Y+Y242F+E259G+N279S+K280Y、H1*+W140Y+Y242F+E259G+N279S+G303R、H1*+W140Y+Y242F+E259G+N279S+E390A、H1*+W140Y+Y242F+E259G+K280Y+G303R、H1*+W140Y+Y242F+E259G+K280Y+E390A、H1*+W140Y+Y242F+E259G+G303R+E390A、H1*+W140Y+Y242F+N279S+K280Y+G303R、H1*+W140Y+Y242F+N279S+K280Y+E390A、H1*+W140Y+Y242F+N279S+G303R+E390A、H1*+W140Y+Y242F+K280Y+G303R+E390A、H1*+W140Y+E259G+N279S+K280Y+G303R、H1*+W140Y+E259G+N279S+K280Y+E390A、H1*+W140Y+E259G+N279S+G303R+E390A、H1*+W140Y+E259G+K280Y+G303R+E390A、H1*+W140Y+N279S+K280Y+G303R+E390A、H1*+E189P+Y242F+E259G+N279S+K280Y、H1*+E189P+Y242F+E259G+N279S+G303R、H1*+E189P+Y242F+E259G+N279S+E390A、H1*+E189P+Y242F+E259G+K280Y+G303R、H1*+E189P+Y242F+E259G+K280Y+E390A、H1*+E189P+Y242F+E259G+G303R+E390A、H1*+E189P+Y242F+N279S+K280Y+G303R、H1*+E189P+Y242F+N279S+K280Y+E390A、H1*+E189P+Y242F+N279S+G303R+E390A、H1*+E189P+Y242F+K280Y+G303R+E390A、H1*+E189P+E259G+N279S+K280Y+G303R、H1*+E189P+E259G+N279S+K280Y+E390A、H1*+E189P+E259G+N279S+G303R+E390A、H1*+E189P+E259G+K280Y+G303R+E390A、<h2 style=";text-align:left;direction:ltr">H1*+E189P+N279S+K280Y+G303R+E390A、H1*+Y242F+E259G+N279S+K280Y+G303R、H1*+Y242F+E259G+N279S+K280Y+E390A、H1*+Y242F+E259G+N279S +G303R+E390A、H1*+Y242F+E259G+K280Y+G303R+E390A、H1*+Y242F+N279S+K280Y+G303R+E390A、H1*+E259G+N279S+K280Y+G303R+E390A、G7A+W140 Y+E189P+Y242F+E259G+N279S、G7A+W140Y+E189P+Y242F+E259G+K280Y、G7A+W140Y+E189P+Y242F+E259G+G303R、G7A+W140Y+E189P+Y242F+E259G+E 390A, G7A+W140Y+E189P+Y242F+N279S+K280Y, G7A+W140Y+E189P+Y242F+N279S+G303R, G7A+W140Y+E189P+Y242F+N279S+E390A, G7A+W140Y+E189P+Y 242F+K280Y+G303R、G7A+W140Y+E189P+Y242F+K280Y+E390A、G7A+W140Y+E189P+Y242F+G303R+E390A、G7A+W140Y+E189P+E259G+N279S+K280Y、G7A+ W140Y+E189P+E259G+N279S+G303R、G7A+W140Y+E189P+E259G+N279S+E39 0A、G7A+W140Y+E189P+E259G+K280Y+G303R、G7A+W140Y+E189P+E259G+K28 0Y+E390A、G7A+W140Y+E189P+E259G+G303R+E390A、G7A+W140Y+E189P+N2 79S+K280Y+G303R、G7A+W140Y+E189P+N279S+K280Y+E390A、G7A+W140Y+E1 89P+N279S+G303R+E390A、G7A+W140Y+E189P+K280Y+G303R+E390A、G7A+W140Y+Y242F+E259G+N279S+K280Y、G7A+W140Y+Y242F+E259G+N279S+G303R、G7A+W140Y+Y242F+E259G+N279S+E390A、G7A+W140Y+Y242F+E259G+K280Y+G303R、G7A+W140Y+Y242F+E259G+K280Y+E390A、G7A+W140Y+Y242F+E259G+G303R+E390A、G7A+W140Y+Y242F+N279S+K280Y+G303R、G7A+W140Y+Y242F+N279S+K280Y+E390A、G、 <h2 style=";text-align:left;direction:ltr">7A+W140Y+Y242F+N279S+G303R+E390A、G7A+W140Y+Y242F+K280Y+G303R+E390A、G7A+W140Y+E259G+N279S+K280Y+G303R、G7A+W140Y+E259G+N279S+ K280Y+E390A、G7A+W140Y+E259G+N279S+G303R+E390A、G7A+W140Y+E259G+K280Y+G303R+E390A、G7A+W140Y+N279S+K280Y+G303R+E390A、G7A+E189P +Y242F+E259G+N279S+K280Y、G7A+E189P+Y242F+E259G+N279S+G303R、G7 A+E189P+Y242F+E259G+N279S+E390A、G7A+E189P+Y242F+E259G+K280Y+G3 03R、G7A+E189P+Y242F+E259G+K280Y+E390A、G7A+E189P+Y242F+E259G+G303R+E390A、G7A+E189P+Y242F+N279S+K280Y+G303R、G7A+E189P+Y242F+N2 79S+K280Y+E390A、G7A+E189P+Y242F+N279S+G303R+E390A、G7A+E189P+Y242F+K280Y+G303R+E390A、G7A+E189P+E259G+N279S+K280Y+G303R、G7A+E 189P+E259G+N279S+K280Y+E390A、G7A+E189P+E259G+N279S+G303R+E390A、G7A+E189P+E259G+K280Y+G303R+E390A、G7A+E189P+N279S+K280Y+G303R +E390A、G7A+Y242F+E259G+N279S+K280Y+G303R、G7A+Y242F+E259G+N279S+K280Y+E390A、G7A+Y242F+E259G+N279S+G303R+E390A、G7A+Y242F+E259 G+K280Y+G303R+E390A、G7A+Y242F+N279S+K280Y+G303R+E390A、G7A+E259 G+N279S+K280Y+G303R+E390A、W140Y+E189P+Y242F+E259G+N279S+K280Y、W140Y+E189P+Y242F+E259G+N279S+G303R, W140Y+E189P+Y242F+E259G+N 279S+E390A, W140Y+E189P+Y242F+E259G+K280Y+G303R, W140Y+E189P+Y24 2F+E259G+K280Y+E390A, W140Y+E189P+Y242F+E259G+G303R+E390A, W140Y +E189P+Y242F+N279S+K280Y+G303R, W140Y+E189P+Y242F+N279S+K280Y+E 390A, W140Y+E189P+Y242F+N279S+G303R+E390A, W140Y+E189P+Y242F+K28 0Y+G303R+E390A, W140Y+E189P+E259G+N279S+K280Y+G303R, W140Y+E189P +E259G+N279S+K280Y+E390A, W140Y+E189P+E259G+N279S+G303R+E390A, W 140Y+E189P+E259G+K280Y+G303R+E390A, W140Y+E189P+N279S+K280Y+G30 3R+E390A, W140Y+Y242F+E259G+N279S+K280Y+G303R, W140Y+Y242F+E259 G+N279S+K280Y+E390A, W140Y+Y242F+E259G+N279S+G303R+E390A, W140Y+ Y242F+E259G+K280Y+G303R+E390A, W140Y+Y242F+N279S+K280Y+G303R+E3 90A, W140Y+E259G+N279S+K280Y+G303R+E390A, E189P+Y242F+E259G+N279 and modifications at positions corresponding to S+K280Y+G303R, E189P+Y242F+E259G+N279S+K280Y+E390A, E189P+Y242F+E259G+N279S+G303R+E390A, E189P+Y242F+E259G+K280Y+G303R+E390A, E189P+Y242F+E259G+K280Y+G303R+E390A, E189P+Y242F+N279S+K280Y+G303R+E390A, E189P+E259G+N279S+K280Y+G303R+E390A, Y242F+E259G+N279S+K280Y+G303R+E390A,With respect to variants of SEQ ID NO:1, the variants have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, and the variants have α-amylase activity.

[0378] In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: a) a deletion at positions corresponding to positions R180* and S181*, using SEQ ID NO:1 for numbering, b) using SEQ ID NO:1 for numbering, positions H1*+G7A+W140Y+E189P+Y242F+E259G+N279S, H1*+G7A+W140Y+E189P+Y242F+E259G+K280Y, H1*+G7A+W140Y+E189P+Y242F+E259G+G303R, H1*+G7A+W140Y+E189P+Y242F+E259G+E390A, H1*+G7A+W140Y+E189P+Y242F+N279S+K280Y, H1*+G7A+W140Y+E189P+Y242F+N279 S+G303R, H1*+G7A+W140Y+E189P+Y242F+N279S+E390A, H1*+G7A+W140Y+ E189P+Y242F+K280Y+G303R, H1*+G7A+W140Y+E189P+Y242F+K280Y+E390A ,H1*+G7A+W140Y+E189P+Y242F+G303R+E390A,H1*+G7A+W140Y+E189P+E2 59G+N279S+K280Y, H1*+G7A+W140Y+E189P+E259G+N279S+G303R,H1*+G7A +W140Y+E189P+E259G+N279S+E390A, H1*+G7A+W140Y+E189P+E259G+K28 0Y+G303R,H1*+G7A+W140Y+E189P+E259G+K280Y+E390A,H1*+G7A+W140Y+ E189P+E259G+G303R+E390A, H1*+G7A+W140Y+E189P+N279S+K280Y+G303R , H1*+G7A+W140Y+E189P+N279S+K280Y+E390A, H1*+G7A+W140Y+E189P+N2 79S+G303R+E390A, H1*+G7A+W140Y+E189P+K280Y+G303R+E390A, H1*+G7A +W140Y+Y242F+E259G+N279S+K280Y, H1*+G7A+W140Y+Y242F+E259G+N279 S+G303R, H1*+G7A+W140Y+Y242F+E259G+N279S+E390A, H1*+G7A+W140Y+Y 242F+E259G+K280Y+G303R, H1*+G7A+W140Y+Y242F+E259G+K280Y+E390A,<h2 style=";text-align:left;direction:ltr">H1*+G7A+W140Y+Y242F+E259G+G303R+E390A、H1*+G7A+W140Y+Y242F+N27 9S+K280Y+G303R、H1*+G7A+W140Y+Y242F+N279S+K280Y+E390A、H1*+G7A+W 140Y+Y242F+N279S+G303R+E390A、H1*+G7A+W140Y+Y242F+K280Y+G303R+E 390A、H1*+G7A+W140Y+E259G+N279S+K280Y+G303R、H1*+G7A+W140Y+E259G +N279S+K280Y+E390A、H1*+G7A+W140Y+E259G+N279S+G303R+E390A、H1*+ G7A+W140Y+E259G+K280Y+G303R+E390A、H1*+G7A+W140Y+N279S+K280Y+G3 03R+E390A、H1*+G7A+E189P+Y242F+E259G+N279S+K280Y、H1*+G7A+E189P+Y242F+E259G+N279S+G303R、H1*+G7A+E189P+Y242F+E259G+N279S+E390A、 H1*+G7A+E189P+Y242F+E259G+K280Y+G303R、H1*+G7A+E189P+Y242F+E259G+K280Y+E390A、H1*+G7A+E189P+Y242F+E259G+G303R+E390A、H1*+G7A+E 189P+Y242F+N279S+K280Y+G303R、H1*+G7A+E189P+Y242F+N279S+K280Y+E390A、H1*+G7A+E189P+Y242F+N279S+G303R+E390A、H1*+G7A+E189P+Y242F +K280Y+G303R+E390A、H1*+G7A+E189P+E259G+N279S+K280Y+G303R、H1*+ G7A+E189P+E259G+N279S+K280Y+E390A、H1*+G7A+E189P+E259G+N279S+G3 03R+E390A、H1*+G7A+E189P+E259G+K280Y+G303R+E390A、H1*+G7A+E189P+N279S+K280Y+G303R+E390A、H1*+G7A+Y242F+E259G+N279S+K280Y+G303R、H1*+G7A+Y242F+E259G+N279S+K280Y+E390A、H1*+G7A+Y242F+E259G+N279S+G303R+E390A、H1*+G7A+Y242F+E259G+K280Y+G303R+E390A、H1*+G7A+Y242F+N279S+K280Y+G303R+E390A、H1*+G7A+E259G+N279S+K280Y+G303R+E390A、H1*+W140Y+E189P+Y242F+E259G+N279S+K280Y、H1*+W140Y+E189P+Y242F+E259G+N279S+G303R、H1*+W140Y+E189P+Y242F+E259G+N279S+E390A、H1*+W140Y+E189P+Y242F+E259G+K280Y+G303R、H1*+W140Y+E189P+Y242F+E259G+K280Y+E390A、H1*+W140Y+E189P+Y242F+E259G+G303R+E390A、H1*+W140Y+E189P+Y242F+N279S+K280Y+G303R、H1*+W140Y+E189P+Y242F+N279S+K280Y+E390A、H1*+W140Y+E189P+Y242F+N279S+G303R+E390A、H1*+W140Y+E189P+Y242F+K280Y+G303R+E390A、H1*+W140Y+E189P+E259G+N279S+K280Y+G303R、H1*+W140Y+E189P+E259G+N279S+K280Y+E390A、H1*+W140Y+E189P+E259G+N279S+G303R+E390A、H1*+W140Y+E189P+E259G+K280Y+G303R+E390A、H1*+W140Y+E189P+N279S+K280Y+G303R+E390A、H1*+W140Y+Y242F+E259G+N279S+K280Y+G303R、H1*+W140Y+Y242F+E259G+N279S+K280Y+E390A、H1*+W140Y+Y242F+E259G+N279S+G303R+E390A、H1*+W140Y+Y242F+E259G+K280Y+G303R+E390A、H1*+W140Y+Y242F+N279S+K280Y+G303R+E390A、<h2 style=";text-align:left;direction:ltr">H1*+W140Y+E259G+N279S+K280Y+G303R+E390A、H1*+E189P+Y242F+E259G+N279S+K280Y+G303R、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> H1*+E189P+Y242F+E259G+N279S+K280Y+E390A、H1*+E189P+Y242F+E259G+N279S+G303R+E390A、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> H1*+E189P+Y242F+E259G+K280Y+G303R+E390A、H1*+E189P+Y242F+N279S+K280Y+G303R+E390A、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> H1*+E189P+E259G+N279S+K280Y+G303R+E390A、H1*+Y242F+E259G+N279S+K280Y+G303R+E390A、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> G7A+W140Y+E189P+Y242F+E259G+N279S+K280Y、G7A+W140Y+E189P+Y242F+E259G+N279S+G303R、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> G7A+W140Y+E189P+Y242F+E259G+N279S+E390A、G7A+W140Y+E189P+Y242F+E259G+K280Y+G303R、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> G7A+W140Y+E189P+Y242F+E259G+K280Y+E390A、G7A+W140Y+E189P+Y242F+E259G+G303R+E390A、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> G7A+W140Y+E189P+Y242F+N279S+K280Y+G303R、G7A+W140Y+E189P+Y242F+N279S+K280Y+E390A、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> G7A+W140Y+E189P+Y242F+N279S+G303R+E390A、G7A+W140Y+E189P+Y242F+K280Y+G303R+E390A、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> G7A+W140Y+E189P+E259G+N279S+K280Y+G303R、G7A+W140Y+E189P+E259G+N279S+K280Y+E390A、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">G7A+W140Y+E189P+E259G+N279S+G303R+E390A、G7A+W140Y+E189P+E259G+K280Y+G303R+E390A、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> G7A+W140Y+E189P+N279S+K280Y+G303R+E390A、G7A+W140Y+Y242F+E259G+N279S+K280Y+G303R、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> G7A+W140Y+Y242F+E259G+N279S+K280Y+E390A、G7A+W140Y+Y242F+E259G+N279S+G303R+E390A、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> G7A+W140Y+Y242F+E259G+K280Y+G303R+E390A、G7A+W140Y+Y242F+N279S+K280Y+G303R+E390A、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> G7A+W140Y+E259G+N279S+K280Y+G303R+E390A、G7A+E189P+Y242F+E259G+N279S+K280Y+G303R、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> G7A+E189P+Y242F+E259G+N279S+K280Y+E390A、G7A+E189P+Y242F+E259G+N279S+G303R+E390A、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> G7A+E189P+Y242F+E259G+K280Y+G303R+E390A、G7A+E189P+Y242F+N279S+K280Y+G303R+E390A、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> G7A+E189P+E259G+N279S+K280Y+G303R+E390A、G7A+Y242F+E259G+N279S+K280Y+G303R+E390A、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> W140Y+E189P+Y242F+E259G+N279S+K280Y+G303R、W140Y+E189P+Y242F+E259G+N279S+K280Y+E390A、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> W140Y+E189P+Y242F+E259G+N279S+G303R+E390A、W140Y+E189P+Y242F+E259G+K280Y+G303R+E390A、<h2 style=";text-align:left;direction:ltr"> W140Y+E189P+Y242F+N279S+K280Y+G303R+E390A, W140Y+E189P+E259G+N279S+K280Y+G303R+E390A, and modifications at positions corresponding to W140Y+Y242F+E259G+N279S+K280Y+G303R+E390A, E189P+Y242F+E259G+N279S+K280Y+G303R+E390A, wherein the variant is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, at least 400%, at least 400%, at least 400%, at least 410%, at least 420%, at least 430%, at least 440%, at least 450%, at least 460%, at least 470%, at least 480%, at least 490%, at least 500%, at least 510%, at least 520%, at least 530%, at least 540%, at least 550%, at least 560%, at least 570%, at least 580%, at least 590%, at least 600%, at least 600%, at least at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity but less than 100%, and the variant has α-amylase activity.

[0379] In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: a) a deletion at positions corresponding to positions R180* and S181*, using SEQ ID NO:1 for numbering, b) using SEQ ID NO: 1 for numbering, positions H1*+G7A+W140Y+E189P+Y242F+E259G+N279S+K280Y, H1*+G7A+W140Y+E189P+Y242F+E259G+N279S+G303R, H1*+G7A+W140Y+E189P+Y242F+E259G+N279S+E390A, H1*+G7A+W140Y+E189P+Y242F+E259G+K280Y+G303R, H1*+G7A+W140Y+E189P+Y242F+E259G+K280Y+E390A, H1*+ G7A+W140Y+E189P+Y242F+E259G+G303R+E390A, H1*+G7A+W140Y+E189P+Y2 42F+N279S+K280Y+G303R, H1*+G7A+W140Y+E189P+Y242F+N279S+K280Y+E3 90A, H1*+G7A+W140Y+E189P+Y242F+N279S+G303R+E390A, H1*+G7A+W140Y+ E189P+Y242F+K280Y+G303R+E390A, H1*+G7A+W140Y+E189P+E259G+N279S+ K280Y+G303R, H1*+G7A+W140Y+E189P+E259G+N279S+K280Y+E390A, H1*+G7 A+W140Y+E189P+E259G+N279S+G303R+E390A, H1*+G7A+W140Y+E189P+E259 G+K280Y+G303R+E390A, H1*+G7A+W140Y+E189P+N279S+K280Y+G303R+E390 A, H1*+G7A+W140Y+Y242F+E259G+N279S+K280Y+G303R, H1*+G7A+W140Y+Y2 42F+E259G+N279S+K280Y+E390A, H1*+G7A+W140Y+Y242F+E259G+N279S+G3 03R+E390A, H1*+G7A+W140Y+Y242F+E259G+K280Y+G303R+E390A, H1*+G7A+ W140Y+Y242F+N279S+K280Y+G303R+E390A, H1*+G7A+W140Y+E259G+N279S+ K280Y+G303R+E390A, H1*+G7A+E189P+Y242F+E259G+N279S+K280Y+G303R,<h2 style=";text-align:left;direction:ltr">H1*+G7A+E189P+Y242F+E259G+N279S+K280Y+E390A、H1*+G7A+E189P+Y242F+E259G+N279S+G303R+E390A、H1*+G7A+E189P+Y242F+E259G+K280Y+G303R +E390A、H1*+G7A+E189P+Y242F+N279S+K280Y+G303R+E390A、H1*+G7A+E189P+E259G+N279S+K280Y+G303R+E390A、H1*+G7A+Y242F+E259G+N279S+K280 Y+G303R+E390A、H1*+W140Y+E189P+Y242F+E259G+N279S+K280Y+G303R、H1*+W140Y+E189P+Y242F+E259G+N279S+K280Y+E390A、H1*+W140Y+E189P+Y24 2F+E259G+N279S+G303R+E390A、H1*+W140Y+E189P+Y242F+E259G+K280Y+G 303R+E390A、H1*+W140Y+E189P+Y242F+N279S+K280Y+G303R+E390A、H1*+W1 40Y+E189P+E259G+N279S+K280Y+G303R+E390A、H1*+W140Y+Y242F+E259G+ N279S+K280Y+G303R+E390A、H1*+E189P+Y242F+E259G+N279S+K280Y+G303R +E390A、G7A+W140Y+E189P+Y242F+E259G+N279S+K280Y+G303R、G7A+W140Y+E189P+Y242F+E259G+N279S+K280Y+E390A、G7A+W140Y+E189P+Y242F+E259 G+N279S+G303R+E390A、G7A+W140Y+E189P+Y242F+E259G+K280Y+G303R+E3 90A、G7A+W140Y+E189P+Y242F+N279S+K280Y+G303R+E390A、G7A+W140Y+E18 9P+E259G+N279S+K280Y+G303R+E390A、G7A+W140Y+Y242F+E259G+N279S+K280Y+G303R+E390A、G7A+E189P+Y242F+E259G+N279S+K280Y+G303R+E390A、and modifications at positions corresponding to W140Y+E189P+Y242F+E259G+N279S+K280Y+G303R+E390A, wherein the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, and wherein the variant has α-amylase activity. ,

[0380] In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: a) a deletion at positions corresponding to positions R180* and S181*, using SEQ ID NO:1 for numbering, b) using SEQ ID NO: 1 for numbering, positions H1*+G7A+W140Y+E189P+Y242F+E259G+N279S+K280Y+G303R, H1*+G7A+W140Y+E189P+Y242F+E259G+N279S+K280Y+E390A, H1*+G7A+W140Y+E189P+Y242F+E259G+N279S+G303R+E390A, H1*+G7A+W140Y+E189P+Y242F+E259G+K280Y+G303R +E390A, H1*+G7A+W140Y+E189P+Y242F+N279S+K280Y+G303R+E390A, H1*+G7A+W140Y+E189P+E259G+N279S+K280Y+G303R+E390A, H 1*+G7A+W140Y+Y242F+E259G+N279S+K280Y+G303R+E390A, H1*+G7A+E189P+Y242F+E259G+N279S+K280Y+G303R+E390A, H1*+W140Y +E189P+Y242F+E259G+N279S+K280Y+G303R+E390A, G7A+W140Y+E189P+Y242F+E259G+N279S+K280Y+G303R+E390A, wherein the variant comprises a modification at a position corresponding to The variant has 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity but less than 100%, and the variant has α-amylase activity.

[0381] In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: a) a deletion at positions corresponding to positions R180* and S181*, using SEQ ID NO:1 for numbering, H1*+G7A+W140Y+E189P+Y242F+E259G+N279S+K280Y+G303R+E390A, using SEQ ID NO: 1 for numbering, the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity, and the variant has α-amylase activity.

[0382] In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: a) a deletion at positions corresponding to positions R180* and S181*, using SEQ ID NO:1 for numbering, b) Using SEQ ID NO:1 for numbering, positions H1*+D118T, H1*+W140Y, H1*+T182S, H1*+A185D, H1*+E189P, H1*+Y242F, H1*+E259G, H1*+F266Y, H1*+N279S, H1*+K280Y, H1*+G303R, H1*+E390A, H1*+G475K, H1*+A185Q, H1*+N194F, D118T+W140Y, D118T+T182S, D118T+A185D, D118T+E189P, D118T+Y242F, D118T+E259G, D1 18T+F266Y, D118T+N279S, D118T+K280Y, D118T+G303R, D118T+E390A, D118 T+G475K, D118T+A185Q, D118T+N194F, W140Y+T182S, W140Y+A185D, W140Y+ E189P, W140Y+Y242F, W140Y+E259G, W140Y+F266Y, W140Y+N279S, W140Y+K2 80Y, W140Y+G303R, W140Y+E390A, W140Y+G475K, W140Y+A185Q, W140Y+N194 F, T182S+A185D, T182S+E189P, T182S+Y242F, T182S+E259G, T182S+F266Y, T182S+N279S, T182S+K280Y, T182S+G303R, T182S+E390A, T182S+G475K, T1 82S+A185Q, T182S+N194F, A185D+E189P, A185D+Y242F, A185D+E259G, A185 D+F266Y, A185D+N279S, A185D+K280Y, A185D+G303R, A185D+E390A, A185D+ G475K, A185D, +A185Q, A185D, +N194F, E189P+Y242F, E189P+E259G, E189P+ F266Y, E189P+N279S, E189P+K280Y, E189P+G303R, E189P+E390A, E189P+G4 75K, E189P+A185Q, E189P+N194F, Y242F+E259G, Y242F+F266Y, Y242F+N279 S, Y242F+K280Y, Y242F+G303R, Y242F+E390A, Y242F+G475K, Y242F+A185Q,Y242F+N194F, E259G+F266Y, E259G+N279S, E259G+K280Y, E259G+G303R, E259G+E390A, E259G+G475K, E259G+A185Q, E 259G+N194F, F266Y+N279S, F266Y+K280Y, F266Y+G303R, F266Y+E390A, F266Y+G475K, F266Y+A185Q, F266Y+N194F, N27 9+K280Y, N279S+G303R, N279S+E390A, N279S+G475K, N279S+A185Q, N279S+N194F, K280Y+G303R, K280Y+E390A, K280Y+ G475K, K280Y+A185Q, K280Y+N194F, G303R+E390A, G303R+G475K, G303R+A185Q, G303R+N194F, E390A+G475K, E390A+A1 and a modification at a position corresponding to 85Q, E390A+N194F, G475K+A185Q, G475K+N194F, A185Q+N194F, the variant having a similar structure to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 ... , at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, and the variant has α-amylase activity.

[0383] In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: a) a deletion at positions corresponding to positions R180* and S181*, using SEQ ID NO:1 for numbering, b) Using SEQ ID NO:1 for numbering, positions H1*+D118T+W140Y, H1*+D118T+T182S, H1*+D118T+A185D, H1*+D118T+E189P, H1*+D118T+Y242F, H1*+D118T+E259G, H1*+D118T+F266Y, H1*+D118T+N279S, H1*+D118T+K280Y, H1*+D118T+G303R, H1*+D118T+E390A, H1*+D118T+G475K, H1*+D118T+A185Q, H1*+D118T+N194F, H1 *+W140Y+T182S, H1*+W140Y+A185D, H1*+W140Y+E189P, H1*+W140Y+Y242F, H1*+W140Y+E259G, H1*+W140Y+F266Y, H1*+W140Y+N279S, H1*+W140Y+K280Y , H1*+W140Y+G303R, H1*+W140Y+E390A, H1*+W140Y+G475K, H1*+W140Y+A18 5Q, H1*+W140Y+N194F, H1*+T182S+A185D, H1*+T182S+E189P, H1*+T182S+Y2 42F, H1*+T182S+E259G, H1*+T182S+F266Y, H1*+T182S+N279S, H1*+T182S+ K280Y, H1*+T182S+G303R, H1*+T182S+E390A, H1*+T182S+G475K, H1*+T182S +A185Q, H1*+T182S+N194F, H1*+A185D+E189P, H1*+A185D+Y242F, H1*+A18 5D+E259G, H1*+A185D+F266Y, H1*+A185D+N279S, H1*+A185D+K280Y, H1*+A1 85D+G303R, H1*+A185D+E390A, H1*+A185D+G475K, H1*+A185D+A185Q, H1*+ A185D+N194F, H1*+E189P+Y242F, H1*+E189P+E259G, H1*+E189P+F266Y, H1* +E189P+N279S, H1*+E189P+K280Y, H1*+E189P+G303R, H1*+E189P+E390A, H 1*+E189P+G475K, H1*+E189P+A185Q, H1*+E189P+N194F, H1*+Y242F+E259G,H1*+Y242F+F266Y、H1*+Y242F+N279S、H1*+Y242F+K280Y、H1*+Y242F+G303R、H1*+Y242F+E390A、H1*+Y242F+G475K、H1*+Y242F+A185Q、H1*+Y242F+N194F、H1*+E259G+F266Y、H1*+E259G+N279S、H1*+E259G+K280Y、H1*+E259G+G303R、H1*+E259G+E390A、H1*+E259G+G475K、H1*+E259G+A185Q、H1*+E259G+N194F、H1*+F266Y+N279S、H1*+F266Y+K280Y、H1*+F266Y+G303R、H1*+F266Y+E390A、H1*+F266Y+G475K、H1*+F266Y+A185Q、H1*+F266Y+N194F、H1*+N279S+K280Y、H1*+N279S+G303R、H1*+N279S+E390A、H1*+N279S+G475K、H1*+N279S+A185Q、H1*+N279S+N194F、H1*+K280Y+G303R、H1*+K280Y+E390A、H1*+K280Y+G475K、H1*+K280Y+A185Q、H1*+K280Y+N194F、H1*+G303R+E390A、H1*+G303R+G475K、H1*+G303R+A185Q、H1*+G303R+N194F、H1*+E390A+G475K、H1*+E390A+A185Q、H1*+E390A+N194F、H1*+G475K+A185Q、H1*+G475K+N194F、H1*+A185Q+N194F、D118T+W140Y+T182S、D118T+W140Y+A185D、D118T+W140Y+E189P、D118T+W140Y+Y242F、D118T+W140Y+E259G、D118T+W140Y+F266Y、D118T+W140Y+N279S、D118T+W140Y+K280Y、D118T+W140Y+G303R、D118T+W140Y+E390A、D118T+W140Y+G475K、D118T+W140Y+A185Q、D118T+W140Y+N194F、D118T+T182S+A185D、D118T+T182S+E189P、D118T+T182S+Y242F、D118T+T182S+E259G、D118T+T182S+F266Y、D118T+T182S+N279S、D118T+T182S+K280Y、D118T+T182S+G303R、D118T+T182S+E390A、D118T+T182S+G475K、D118T+T182S+A185Q、D118T+T182S+N194F、D118T+A185D+E189P、D118T+A185D+Y242F、D118T+A185D+E259G、D118T+A185D+F266Y、D118T+A185D+N279S、D118T+A185D+K280Y、D118T+A185D+G303R、D118T+A185D+E390A、D118T+A185D+G475K、D118T+A185D+A185Q、D118T+A185D+N194F、D118T+E189P+Y242F、D118T+E189P+E259G、D118T+E189P+F266Y、D118T+E189P+N279S、D118T+E189P+K280Y、D118T+E189P+G303R、D118T+E189P+E390A、D118T+E189P+G475K、D118T+E189P+A185Q、D118T+E189P+N194F、D118T+Y242F+E259G、D118T+Y242F+F266Y、D118T+Y242F+N279S、D118T+Y242F+K280Y、D118T+Y242F+G303R、D118T+Y242F+E390A、D118T+Y242F+G475K、D118T+Y242F+A185Q、D118T+Y242F+N194F、D118T+E259G+F266Y、D118T+E259G+N279S、D118T+E259G+K280Y、D118T+E259G+G303R、D118T+E259G+E390A、D118T+E259G+G475K、D118T+E259G+A185Q、D118T+E259G+N194F、D118T+F266Y+N279S、D118T+F266Y+K280Y、D118T+F266Y+G303R、D118T+F266Y+E390A、D118T+F266Y+G475K、D118T+F266Y+A185Q、D118T+F266Y+N194F、D118T+N279S+K280Y、D118T+N279S+G303R、D118T+N279S+E390A、D118T+N279S+G475K、D118T+N279S+A185Q、D118T+N279S+N194F、D118T+K280Y+G303R、D118T+K280Y+E390A、D118T+K280Y+G475K、D118T+K280Y+A185Q、D118T+K280Y+N194F、D118T+G303R+E390A、D118T+G303R+G475K、D118T+G303R+A185Q、D118T+G303R+N194F、D118T+E390A+G475K、D118T+E390A+A185Q、D118T+E390A+N194F、D118T+G475K+A185Q、D118T+G475K+N194F、D118T+A185Q+N194F、W140Y+T182S+A185D、W140Y+T182S+E189P、W140Y+T182S+Y242F、W140Y+T182S+E259G、W140Y+T182S+F266Y、W140Y+T182S+N279S、W140Y+T182S+K280Y、W140Y+T182S+G303R、W140Y+T182S+E390A、W140Y+T182S+G475K、W140Y+T182S+A185Q、W140Y+T182S+N194F、W140Y+A185D+E189P、W140Y+A185D+Y242F、W140Y+A185D+E259G、W140Y+A185D+F266Y、W140Y+A185D+N279S、W140Y+A185D+K280Y、W140Y+A185D+G303R、W140Y+A185D+E390A、W140Y+A185D+G475K、W140Y+A185D+A185Q、W140Y+A185D+N194F、W140Y+E189P+Y242F、W140Y+E189P+E259G、W140Y+E189P+F266Y、W140Y+E189P+N279S、W140Y+E189P+K280Y、W140Y+E189P+G303R、W140Y+E189P+E390A、W140Y+E189P+G475K、W140Y+E189P+A185Q、W140Y+E189P+N194F、W140Y+Y242F+E259G、W140Y+Y242F+F266Y、W140Y+Y242F+N279S、W140Y+Y242F+K280Y、W140Y+Y242F+G303R、W140Y+Y242F+E390A、W140Y+Y242F+G475K、W140Y+Y242F+A185Q、W140Y+Y242F+N194F、W140Y+E259G+F266Y、W140Y+E259G+N279S、W140Y+E259G+K280Y、W140Y+E259G+G303R、W140...

Claims

1. 1. A cleaning composition comprising: (i) an α-amylase variant of a parent α-amylase having an amino acid sequence of SEQ ID NO:1, said variant comprising modifications at positions corresponding to positions H1*, G7A, R180*+S181*, Y242F, E259G, G303R, E390A, and G475K, using SEQ ID NO:1 for numbering, wherein said variant has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to SEQ ID NO:1, and wherein said variant has improved performance when compared to said parent α-amylase, such as improved cleaning performance relative to a specific activity measurement of an improvement factor (IF)>1.0; and (ii) a cleaning adjunct.

2. The α-amylase variants are numbered as follows, using SEQ ID NO:1 for numbering: positions H2, N3, R26, A31, R37, T40, A41, N54, V56, A60, L63, K72, G73, K93, N94, Q98, V103, V104, M105, N106, A109, G110, A111, G113, T114, F116, V117, D118, D123, N126, N128, T131, S132, T133, T134, T135, T136, T137, T138, T139, T140, T141, T142, T143, T144, T145, T146, T147, T148, T149, T150, T151, T152, T153, T154, T155, T156, T157, T158, T159, T160, T161, T162, T163, T164, T165, T166, T167, T168, T169, T170, T171, T172, T173, T174, T175, T176, T177, T178, T179, T180, T181, T182, T183, T184, T185, T186, T187, T188, T189, T190, T191, T192, T193, T194, T195, T196, T197, T198, T200, T2 4, Y135, Q136, I137, Q138, W140, K142, D144, P146, T151, Y152, F155, W159, Y160, T165, W167, E169, K172, L173, N174, I176, Y177, K178, A185, W188, E189, E193, N194, N196, F202, A203, L205, T224, N225, S243, F244, V252, T25 6, K258, M260, F261, F266, N276, N279, K280, W283, S286, S302, T315, H320, P321, M322, H323, W346, L354, E359, Q360, G361, Y362, V365, Y371, A383, N394, F395, D405, I409, R414, H420, N422, T427, G455, K457, P458, N470, S47 2, V473, N474, and G476, wherein the variant has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to SEQ ID NO:

1.

3. The α-amylase variants have the following structure: * , N3D, N3G, N3S, R26Y, A31S, R37A, R37N, R37V, T40G, T40N, A41D, N54S, V56T, A 60V, A60P, L63F, K72R, G73L, K93R, N94D, Q98L, V103A, V103I, V103L, V104M, M 105I, M105L, N106D, A109G, G110K, A111G, A111L, G113Q, G113S, G113A, T114K , F116L, F116N, F116Q, F116W, V117G, D118Q, D118T, D123N, N126D, N128Y, T131 I, T131V, S132D, S132L, T134D, Y135E, Q136T, Q136K, I137F, Q138V, W140S, W1 40M, W140Y, K142E, K142G, K142P, D144N, P146S, P146H, T151A, Y152L, F155W, W 159H, W159L, Y160P, T165V, W167A, W167D, W167E, W167F, W167P, W167S, E169I , E169Q, E169V, K172G, K172Q, L173F, N174K, N174S, I176V, Y177D, K178A, T182 * , T182D, T182E, T182G, T182I, T182L, T182M, T182S, G183 * , G183P, A185D, A185E, A185K, A185N, A185Q, A185R, W188C, E189P, E193I, N194F, F202Y, F202Y, A203F, L205F, L 205N, L205Q, L205R, L205V, L205Y, T224F, T224Y, N225S, S243Q, F244I, F244L, V252I, T256A, T256D, K258V, M260 D, M260W, F261K, F261P, F261S, F266Y, N276F, N276W, N276Y, N279S, N280F, N280W, N280Y, W283N, S286K, S286L, S302T, S302Y, T315A, H320T, P321H, P321Y, M322G, M322Y, H323A, H323N, H323W, W346N, L354V, E359W, Q360M, Q36 0W, Q360Y, G361M, Y362V, V365I, Y371S, Y371N, A383D, N394W, N394D, F395Y, D405G, I409L, R414M, H420P, H420A , H420Q, H420Y, N422V, T427L, G455R, K457N, K457R, R458S, N470A, N470M, S472R, V473C, N474F, N474L, G476H, G4 3. The cleaning composition of claim 1 or claim 2, further comprising a modification at at least one position corresponding to G476A, G476B, and G476C, wherein the variant has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to SEQ ID NO:

1.

4. The mutant is, using SEQ ID NO:1 for numbering, R180 * +T182 * , R180 * +G183 * , S181 * +T182 * , S181 * +G183 * , and T182 * +G183 * 2. The cleaning composition of claim 1, comprising a deletion at one or more positions selected from the group consisting of:

5. The variants include (a) substitutions and / or deletions at two positions in the parent α-amylase, which positions correspond to positions 182 and 183, using SEQ ID NO:1 for numbering, and (b) substitutions and / or deletions at positions H2 and H3, using SEQ ID NO:1 for numbering, respectively. * 、N3D、N3G、N3S、R26Y、A31S、R37A、R37N、R37V、T40G、T40N、A41D、N54S、V56T、A60V、A60P、L63F、K72R、G73L、K93R、N94D、Q98L、V103A、V103I、V103L、V104M、M105I、M105L、N106D、A109G、G110K、A111G、A111L、G113Q、G113S、G113A、T114K、F116L、F116N、F116Q、F116W、V117G、D118Q、D118T、D123N、N126D、N128Y、T131I、T131V、S132D、S132L、T134D、Y135E、Q136T、Q136K、I137F、Q138V、W140S、W140M、W140Y、K142E、K142G、K142P、D144N、P146S、P146H、T151A、Y152L、F155W、W159H、W159L、Y160P、T165V、W167A、W167D、W167E、W167F、W167P、W167S、E169I、E169Q、E169V、K172G、K172Q、L173F、N174K、N174S、I176V、Y177D、K178A、A185D、A185E、A185K、A185N、A185Q、A185R、W188C、E189P、E193I、N194F、F202Y、F202Y、A203F、L205F、L205N、L205Q、L205R、L205V、L205Y、T224F、T224Y、N225S、S243Q、F244I、F244L、V252I、T256A、T256D、K258V、M260D、M260W、F261K、F261P、F261S、F266Y、N276F、N276W、N276Y、N279S、N280F、N280W、N280Y、W283N、S286K、S286L、S302T、S302Y、T315A、H320T、P321H、P321Y、M322G、M322Y、H323A、H323N、H323W、W346N、L354V、E359W、Q360M、Q360W、Q360Y、G361M、Y362V、V365I、Y371S、Y371N、A383D、N394W、N394D、F395Y、D405G、I409L、R414M、H420P、H420A、H420Q、H420Y、N422V、T427L、G455R、and modifications at one or more positions corresponding to K457N, K457R, R458S, N470A, N470M, S472R, V473C, N474F, N474L, G476H, G476E, and G476A, wherein the variants have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to SEQ ID NO:

1.

6. The variants, using SEQ ID NO:1 for numbering, are H1 * +G7A+R180 * +S181 * +Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +D118T+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +D118T+W140Y+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +D118T+W140Y+N194F+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * 2. The cleaning composition of claim 1, comprising one or more modifications selected from the group consisting of: + D118T + W140Y + N194F + Y242F + E259G + K280Y + N279S + G303R + E390A + G475K, wherein the variants have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to SEQ ID NO:1, and wherein the variants have α-amylase activity.

7. The variants, using SEQ ID NO:1 for numbering, are H1 * +G7A+R180 * +S181 * +Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +T182S+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+A60P+R180 * +S181 * +Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+M105L+R180 * +S181 * +Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+M105I+R180 * +S181 * +Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +T182D+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +T182E+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +T182G+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +T182M+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +A185K+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +A185D+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +A185Q+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +A185N+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +A185R+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +A185E+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +E189P+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +L205Y+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +L205F+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +Y242F+S243Q+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +Y242F+F244L+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +Y242F+F244I+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +Y242F+E259G+F266Y+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +N194F+Y242F+E259G+N279S+G303R+E390A+G475K、 <h2 style=";text-align:left;direction:ltr">11<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> +G7A+R1880<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> 10181<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> 10 ... H1 * +G7A+R180 * +S181 * +N194F+L205Y+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +N194F+L205F+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +N196F+L205Y+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +E189P+Y242F+E259G+N276F+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +E189P+Y242F+E259G+N276Y+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +E189P+Y242F+E259G+N276W+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +E189P+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +E189P+Y242F+E259G+N279S+K280W+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +E189P+L205F+Y242F+E259G+N276F+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +E189P+L205F+Y242F+E259G+N276Y+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +E189P+L205F+Y242F+E259G+N276W+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +E189P+L205F+Y242F+E259G+N279S+K280F+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +E189P+L205F+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +E189P+L205F+Y242F+E259G+N279S+K280W+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +E189P+L205Y+Y242F+E259G+N276F+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +E189P+L205Y+Y242F+E259G+N276Y+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +E189P+L205Y+Y242F+E259G+N276W+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +E189P+L205Y+Y242F+E259G+N279S+K280F+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +E189P+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +E189P+L205Y+Y242F+E259G+N279S+K280W+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +Y242F+E259G+N279S+G303R+M322G+E359W+Q360W+E390A+G475K、 H1 * +G7A+R180 * +S181 * +Y242F+E259G+N279S+G303R+M322G+Q360W+E390A+G475K、 H1 * +G7A+R180 * +S181 * +Y242F+E259G+N279S+G303R+E359W+Q360W+E390A+G475K、 H1 * +G7A+T40N+Q98L+R180 * +S181 * +Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +Y242F+E259G+F261S+N279S+S286K+G303R+Y362V+E390A+G475K、 <h2 style=";text-align:left;direction:ltr">11<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> +G7A+R1880<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> 10181<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> +Y242F+E259G+N279S+W283N+S286K+G303R+H320T+H323A+E390A+G475K、 H1 * +G7A+R180 * +S181 * +Y242F+E259G+N279S+G303R+P321H+M322Y+Q360Y+E390A+G475K、 <h2 style=";text-align:left;direction:ltr">11<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> 10 ...<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> 10181<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> 2566256256 H1 * +G7A+G113A+F116W+D123N+T131V+Q136K+E169Q+K172Q+R180 * +S181 * +Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+G113S+F116W+D123N+R180 * +S181 * +Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+F116L+D123N+N126D+T131I+R180 * +S181 * +F202Y+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+N106D+A109G+F116W+D123N+N126D+T131I+T165V+N174S+R180 * +S181 * +T182I+F202Y+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +Y242F+E259G+N279S+G303R+M322G+E390A+G475K、 H1 * +G7A+R180 * +S181 * +Y242F+E259G+N279S+G303R+M322G+Q360W+E390A+G475K、 H1 * +G7A+T40N+R180 * +S181 * +Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+T40N+Q98L+R180 * +S181 * +Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +Y242F+E259G+N279S+G303R+Y362V+E390A+G475K、 H1 * +G7A+I137F+R180 * +S181 * +Y242F+E259G+F261S+N279S+G303R+Y362V+E390A+G475K、 <h2 style=";text-align:left;direction:ltr">11<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> +G7A+R1880<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> 10181<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> 256 ... <h2 style=";text-align:left;direction:ltr">11<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> +G7A+R1880<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> 10181<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> 256 ... <h2 style=";text-align:left;direction:ltr">11<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> +G7A+R1880<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> 10181<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> 256 ... H1 * +G7A+R180 * +S181 * +Y242F+E259G+N279S+G303R+P321H+Q360Y+E390A+G475K、 H1 * +G7A+R180 * +S181 * +Y242F+E259G+N279S+G303R+M322Y+Q360Y+E390A+G475K、 H1 * +G7A+R180 * +S181 * +Y242F+E259G+F261S+N279S+S286K+G303R+Y362V+E390A+G475K、 H1 * +G7A+R180 * +S181 * +Y242F+E259G+N279S+S286L+G303R+E390A+G475K、 H1 * +G7A+G110K+R180 * +S181 * +Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+W140S+R180 * +S181 * +Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+W167P+R180 * +S181 * +Y242F+E259G+N279S+G303R+E390A+G475K、 <h2 style=";text-align:left;direction:ltr">11<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> +G7A+R1880<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> 10181<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> 256 ... <h2 style=";text-align:left;direction:ltr">11<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> +G7A+R1880<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> 10181<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> 256 ... H1 * +G7A+R180 * +S181 * +Y242F+E259G+N279S+G303R+H320T+E390A+G475K、 H1 * +G7A+R180 * +S181 * +Y242F+E259G+N279S+G303R+P321H+E390A+G475K、 <h2 style=";text-align:left;direction:ltr">11<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> +G7A+R1880<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> 10181<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> 256 ... H1 * +G7A+R180 * +S181 * +Y242F+E259G+N279S+G303R+Q360Y+E390A+G475K、 H1 * +G7A+Q98L+R180 * +S181 * +Y242F+E259G+F261K+N279S+G303R+Y362V+E390A+G475K、 H1 * +G7A+Q98L+R180 * +S181 * +Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +Y242F+E259G+F261S+N279S+G303R+Y362V+E390A+G475K、 H1 * +G7A+R180 * +S181 * +Y242F+E259G+N279S+S286L+G303R+H320T+E390A+G475K、 H1 * +G7A+R180 * +S181 * +Y242F+E259G+F261S+N279S+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182D+N194F+L205Y+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182D+N194F+L205V+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182D+N194F+L205Y+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+F116N+W140Y+R180 * +S181 * +T182D+N194F+L205Y+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+F116N+W140Y+R180 * +S181 * +E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182D+E189P+N194F+L205Y+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+T134D+W140Y+R180 * +S181 * +E189P+N194F+L205V+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+F116N+W140Y+R180 * +S181 * +T182D+E189P+N194F+L205Y+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182S+N194F+L205Y+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182S+N194F+L205V+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+N194F+L205Y+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+F116N+W140Y+R180 * +S181 * +T182S+N194F+L205Y+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205V+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+F116N+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D123N+W140Y+R180 * +S181 * +N194F+L205V+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+D123N+W140Y+I176V+R180 * +S181 * +N194F+L205V+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+G113S+F116W+D123N+W140Y+R180 * +S181 * +N194F+L205V+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+F116L+D123N+N126D+T131I+W140Y+R180 * +S181 * +N194F+F202Y+L205V+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+N106D+A109G+W140Y+R180 * +S181 * +N194F+F202Y+L205V+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+D123N+W140Y+R180 * +S181 * +N194F+L205Y+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+D123N+W140Y+I176V+R180 * +S181 * +N194F+L205Y+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+G113S+F116W+D123N+W140Y+R180 * +S181 * +N194F+L205Y+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+F116L+D123N+N126D+T131I+W140Y+R180 * +S181 * +N194F+F202Y+L205Y+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+Q98L+G110K+P146S+R180 * +S181 * +Y242F+E259G+N279S+G303R+A383D+E390A+G475K、 H1 * +N3S+G7A+Q98L+G110K+R180 * +S181 * +Y242F+V252I+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+Q98L+G110K+S132D+Y135E+R180 * +S181 * +Y242F+E259G+N279S+G303R+T315A+E390A+G475K、 H1 * +N3S+G7A+N94D+Q98L+W140Y+R180 * +S181 * +N194F+L205Y+Y242F+V252I+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+Q98L+G110K+R180 * +S181 * +Y242F+T256A+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+Q98L+G110K+R180 * +S181 * +Y242F+E259G+N279S+G303R+A383D+E390A+G475K、 H1 * +N3S+G7A+N94D+Q98L+G110K+R180 * +S181 * +Y242F+V252I+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+Q98L+G110K+R180 * +S181 * +Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+Q98L+G110K+R180 * +S181 * +Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +Y242F+E259G+N279S+S286L+G303R+Y362V+E390A+G475K、 <h2 style=";text-align:left;direction:ltr">11<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> +G7A+R1880<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> 10181<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> +Y242F+E259G+N279S+W283N+S286K+G303R+E390A+G475K、 <h2 style=";text-align:left;direction:ltr">11<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> +G7A+R1880<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> 10181<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> 256 ... <h2 style=";text-align:left;direction:ltr">11<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> +G7A+R1880<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> 10181<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> 256 ... H1 * +G7A+R180 * +S181 * +Y242F+E259G+N279S+S286K+G303R+H320T+E390A+G475K、 H1 * +G7A+R180 * +S181 * +Y242F+E259G+N279S+S286K+G303R+Y362V+E390A+G475K、 H1 * +G7A+Q98L+R180 * +S181 * +Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+R180 * +S181 * +Y242F+E259G+F261K+N279S+G303R+E390A+G475K、 <h2 style=";text-align:left;direction:ltr">11<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> +G7A+R1880<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> 10181<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> 256 ... H1 * +G7A+F116N+W140Y+R180 * +S181 * +T182D+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+F116N+W140Y+R180 * +S181 * +T182D+E189P+N194F+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+F116N+W140Y+R180 * +S181 * +T182S+E189P+N194F+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+F116N+W140Y+R180 * +S181 * +T182D+N194F+L205Y+Y242F+E259G+F266Y+N279S+G303R+E390A+G475K、 H1 * +G7A+F116N+W140Y+R180 * +S181 * +A185Q+N194F+L205Y+Y242F+E259G+F266Y+N279S+G303R+E390A+G475K、 H1 * +G7A+F116N+W140Y+R180 * +S181 * +T182D+A185Q+N194F+L205Y+Y242F+E259G+F266Y+N279S+G303R+E390A+G475K、 H1 * +G7A+F116N+W140Y+R180 * +S181 * +N194F+L205Y+Y242F+E259G+F266Y+N279S+G303R+E390A+G475K、 H1 * +G7A+F116N+W140Y+R180 * +S181 * +A185Q+N194F+L205Y+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+F116N+W140Y+R180 * +S181 * +T182D+A185Q+N194F+L205Y+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +E189P+N194F+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182D+N194F+L205Y+Y242F+E259G+F266Y+N279S+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +A185Q+N194F+L205Y+Y242F+E259G+F266Y+N279S+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182D+A185Q+N194F+L205Y+Y242F+E259G+F266Y+N279S+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +N194F+L205Y+Y242F+E259G+F266Y+N279S+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +A185Q+N194F+L205Y+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182D+A185Q+N194F+L205Y+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +E189P+N194F+L205V+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182D+E189P+N194F+L205V+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182D+E189P+N194F+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182S+E189P+N194F+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182D+N194F+L205V+Y242F+E259G+F266Y+N279S+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +A185Q+N194F+L205V+Y242F+E259G+F266Y+N279S+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182D+A185Q+N194F+L205V+Y242F+E259G+F266Y+N279S+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +N194F+L205V+Y242F+E259G+F266Y+N279S+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +A185Q+N194F+L205V+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182D+A185Q+N194F+L205V+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182D+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182D+E189P+N194F+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182S+E189P+N194F+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182D+N194F+L205Y+Y242F+E259G+F266Y+N279S+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +A185Q+N194F+L205Y+Y242F+E259G+F266Y+N279S+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182D+A185Q+N194F+L205Y+Y242F+E259G+F266Y+N279S+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +A185Q+N194F+L205Y+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182D+A185Q+N194F+L205Y+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+F116N+W140Y+R180 * +S181 * +E189P+N194F+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182D+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182D+E189P+N194F+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +E189P+N194F+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +E189P+N194F+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +N194F+L205Y+Y242F+E259G+F266Y+N279S+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +N194F+L205Y+Y242F+E259G+N279S+S302T+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +N194F+L205Y+Y242F+E259G+N279S+G303R+E390A+N474F+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +N194F+L205Y+Y242F+E259G+N279S+G303R+E390A+N422V+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +N194F+L205Y+Y242F+E259G+F261P+N279S+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +N194F+L205Y+Y242F+E259G+N279S+G303R+E390A+N470A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +N194F+L205Y+Y242F+E259G+N279S+G303R+E390A+H420P+G475K、 H1 * +G7A+R26Y+W140Y+R180 * +S181 * +N194F+L205Y+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +N194F+L205Y+Y242F+E259G+N279S+G303R+Y371S+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182D+A185Q+N194F+Y242F+S243Q+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182D+Y242F+S243Q+E259G+F266Y+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +A185Q+N194F+Y242F+S243Q+E259G+F266Y+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182D+A185Q+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182D+E189P+Y242F+S243Q+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +A185Q+Y242F+E259G+F266Y+N279S+K280Y+G303R+E390A+T427L+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +A185Q+E189P+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182D+A185Q+E189P+N194F+Y242F+S243Q+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182D+A185Q+E189P+N194F+L205Y+Y242F+S243Q+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182D+E189P+L205Y+Y242F+E259G+F266Y+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +A185Q+E189P+N194F+Y242F+E259G+F266Y+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +A185Q+E189P+N194F+Y242F+S243Q+E259G+F266Y+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +A185Q+E189P+N194F+L205Y+Y242F+S243Q+E259G+F266Y+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182D+A185Q+E189P+N194F+L205Y+Y242F+E259G+F266Y+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182D+A185Q+E189P+N194F+Y242F+E259G+F266Y+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +A185Q+Y242F+S243Q+E259G+F266Y+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182D+A185Q+Y242F+S243Q+E259G+F266Y+N279S+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +Y242F+S243Q+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182D+E189P+N194F+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +A185Q+N194F+Y242F+S243Q+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182D+N194F+Y242F+S243Q+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +A185Q+E189P+N194F+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +E189P+Y242F+S243Q+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182D+Y242F+S243Q+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +E189P+L205Y+Y242F+S243Q+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +Y242F+E259G+F266Y+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182D+A185Q+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182D+A185Q+N194F+Y242F+S243Q+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182D+N194F+Y242F+S243Q+E259G+F266Y+N279S+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182D+E189P+N194F+Y242F+S243Q+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +E189P+N194F+Y242F+E259G+F266Y+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +A185Q+E189P+N194F+L205Y+Y242F+S243Q+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182D+A185Q+N194F+Y242F+E259G+F266Y+N279S+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +A185Q+N194F+Y242F+E259G+F266Y+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +A185Q+E189P+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +Y242F+S243Q+E259G+F266Y+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182D+E189P+L205Y+Y242F+S243Q+E259G+N279S+K280Y+G303R+E390A+I409L+G475K、 H1 * +G7A+A41D+W140Y+R180 * +S181 * +N194F+L205Y+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+T40G+W140Y+R180 * +S181 * +N194F+L205Y+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+R37N+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+H420A+G475K、 H1 * +N3D+G7A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+H420A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+H420P+G475K、 H1 * +N3D+G7A+R37N+D118T+Q138V+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +N3D+G7A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+H420P+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+F261P+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+P321H+E390A+G475K、 H1 * +G7A+D118T+Y135E+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+M260D+N279S+K280Y+G303R+W346N+E390A+G475K、 H1 * +G7A+D118T+S132L+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+M260D+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+E189P+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+N279S+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205V+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205N+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Q+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+A185Q+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+F266Y+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +A185Q+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+A185Q+E189P+N194F+L205Y+Y242F+E259G+F266Y+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+A185Q+E189P+N194F+L205N+Y242F+E259G+F266Y+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+A185D+E189P+N194F+Y242F+E259G+F266Y+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+A185D+E189P+Y242F+S243Q+E259G+F266Y+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+A185Q+E189P+Y242F+E259G+F266Y+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+A185Q+E189P+L205Y+Y242F+E259G+F266Y+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+A185D+E189P+L205Y+Y242F+E259G+F266Y+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+M260D+N279S+K280Y+G303R+Y362V+E390A+G475K、 H1 * +G7A+D118T+Y135E+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+F261P+N279S+K280Y+G303R+Y362V+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+M260D+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+Y135E+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+T224F+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+F261P+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+M260D+N279S+K280Y+G303R+Y371N+E390A+G475K、 H1 * +G7A+D118T+Y135E+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+P321Y+E390A+G475K、 H1 * +G7A+D118T+S132D+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+P321Y+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+M260D+N279S+K280Y+G303R+E390A+H420A+G475K、 H1 * +G7A+D118T+Q138V+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+H420Q+G475K、 H1 * +G7A+R37A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+H420A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+H420P+G475K、 H1 * +G7A+R37N+D118T+S132D+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+N470M+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+H420A+G475K、 H1 * +N3D+G7A+R37N+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+H420A+G475K、 H1 * +N3D+G7A+R37N+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+H420A+G475K、 H1 * +N3D+G7A+R37A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +N3D+G7A+R37N+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+H420A+G475K、 H1 * +N3D+G7A+R37A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+H420A+G475K、 H1 * +G7A+R37N+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+M260D+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+R37A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+M260D+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+A185Q+E189P+N194F+Y242F+E259G+F266Y+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+A185Q+E189P+N194F+L205Q+Y242F+E259G+F266Y+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+A185Q+E189P+Y242F+S243Q+E259G+F266Y+N279S+K280Y+G303R+E390A+G475K、 H1 * +N3D+G7A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +N3D+G7A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+H420A+G475K、 H1 * +N3D+G7A+D118T+S132D+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+H420A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+N470M+G475K、 H1 * +G7A+R37A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+M260D+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+R37N+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+M260D+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+M260D+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+R37A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+M260W+N279S+K280Y+G303R+E390A+N470M+G475K、 H1 * +G7A+R37N+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+M260D+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+M260D+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+R37N+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+M260W+N279S+K280Y+G303R+E390A+H420A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+M260D+N279S+K280Y+G303R+E390A+N470M+G475K、 H1 * +G7A+R37A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+H420A+G475K、 H1 * +G7A+R37A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+M260D+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+R37A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+M260D+N279S+K280Y+G303R+E390A+N470M+G475K、 H1 * +G7A+R37A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+F261P+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+R37A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+T224Y+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +N3D+G7A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+M260D+N279S+K280Y+G303R+E390A+G475K、 H1 * +N3D+G7A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+T224Y+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +N3D+G7A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+T224Y+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +N3D+G7A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+T224Y+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +N3G+G7A+R37N+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+T224F+Y242F+E259G+N279S+K280Y+G303R+E390A+H420A+G475K、 H1 * +G7A+Q98L+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+R37N+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+T256D+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+R37A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+R37N+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+H323W+Q360M+E390A+G475K、 H1 * +N3D+G7A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+H323W+E390A+G475K、 H1 * +N3D+G7A+D118T+W140Y+R180 * +S181 * +T182S+E189P+N194F+L205Y+Y242F+E259G+N279S+K280Y+G303R+H323W+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+A185D+E189P+Y242F+E259G+F266Y+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+S132L+W140Y+R180 * +S181 * +T182D+E189P+N194F+L205Y+Y242F+E259G+M260D+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+S132L+W140Y+R180 * +S181 * +T182S+E189P+N194F+Y242F+E259G+M260D+F266Y+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+S132L+W140Y+R180 * +S181 * +T182S+A185Q+E189P+N194F+L205Y+Y242F+E259G+M260D+F266Y+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182D+A185Q+E189P+N194F+Y242F+E259G+F266Y+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182D+A185Q+Y242F+S243Q+E259G+F266Y+N279S+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182D+N194F+Y242F+S243Q+E259G+F266Y+N279S+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+A185Q+E189P+N196I+Y242F+E259G+F266Y+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +T182S+A185D+E189P+Y242F+E259G+F266Y+N279S+K280Y+G303R+E390A+H420Y+G475K、 H1 * +G7A+D118T+W140Y+R180 * +S181 * +A185Q+E189P+N194F+L205Y+Y242F+E259G+F266Y+N279S+K280Y+G303R+E390A+G475K、 H1 * +G7A+S132L+W140Y+R180 * +S181 * 2. The cleaning composition of claim 1, comprising one or more modifications selected from the group consisting of: + E189P + N194F + Y242F + E259G + M260D + N279S + K280Y + G303R + E390A + G475K, wherein the variant has α-amylase activity.

8. 8. A cleaning composition according to any one of claims 1 to 7, wherein the cleaning adjunct comprises an additional enzyme, preferably selected from the group consisting of proteases, amylases, lipases, catinases, cellulases, nucleases, endoglucanases, xyloglucanases, pectinases, pectin lyases, xanthanases, xanthan lyases, peroxidases, haloperoxygenases, hexosaminidases, catalases and mannases, or any mixtures thereof.

9. A cleaning composition according to any one of claims 1 to 8 which is a liquid or solid detergent composition, preferably a laundry composition or a dishwashing composition, preferably a liquid laundry composition.

10. 1. A method of making a cleaning composition comprising the steps of: (i) obtaining an α-amylase variant of a parent α-amylase having the amino acid sequence of SEQ ID NO:1 by introducing modifications at positions corresponding to positions H1*, G7A, R180*+S181*, Y242F, E259G, G303R, E390A and G475K, using SEQ ID NO:1 for numbering, wherein the variant has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to SEQ ID NO:1; and (ii) mixing the α-amylase variant with a cleaning adjunct.

11. 1. A method of improving the cleaning performance of a cleaning composition, comprising incorporating into said cleaning composition an α-amylase variant of a parent α-amylase having the amino acid sequence of SEQ ID NO:1, said method comprising introducing modifications at positions corresponding to positions H1*, G7A, R180*+S181*, Y242F, E259G, G303R, E390A and G475K, using SEQ ID NO:1 for numbering, wherein said variant has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to SEQ ID NO:1, and wherein said variant has α-amylase activity and improved cleaning performance compared to said parent α-amylase.

12. Use of a composition according to any one of claims 1 to 8 in a cleaning process, preferably in laundry or hard surface cleaning such as dishwashing and industrial cleaning, most preferably for cleaning laundry.

13. A method for treating a surface, preferably a fabric, comprising the steps of: (i) forming an aqueous wash solution comprising water, an α-amylase variant of a parent α-amylase having the amino acid sequence of SEQ ID NO:1, said variant comprising modifications at positions corresponding to positions H1*, G7A, R180*+S181*, Y242F, E259G, G303R, E390A, and G475K, using SEQ ID NO:1 for numbering, said variant having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO:1, and said variant having improved performance when compared to said parent α-amylase, such as improved wash performance relative to a specific activity measurement of an improvement factor (IF)>1.0, and a wash adjunct; (ii) treating the surface with the aqueous cleaning solution; (iii) rinsing the surface.

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