ALPHA-AMYLASE VARIANTS THAT HAVE AMINO ACID SUBSTITUTIONS THAT LOWER THE PKA OF THE GENERAL ACID

Abstract

The present invention relates to a recombinant variant of a parental Family 13 β-amylase, characterized in that the variant has at least 92% amino acid sequence identity with the amino acid sequence of SEC. ID NO.: 1 and has an amino acid mutation in an amino acid residue covering the N-terminal side of the β-barrel structure of the β-amylase core, resulting in the variant having an amino acid residue that is different from the naturally occurring amino acid in the original β-amylase, and resulting in a reduction of the apparent pKa value of the overall acid and an increase in the activity of the variant at a pH between approximately 8.5 and 10.5, wherein the mutation is an amino acid substitution at Y364 corresponding to the amino acid sequence of SEC. ID NO.: 1, wherein the amino acid substitution is Y364L.

Description

DETAILED DESCRIPTION OF THE INVENTION Compositions and methods relating to α-amylase variant enzymes are described. Some illustrative applications for amylase variant enzymes include starch liquefaction and saccharification, removing starch stains from laundry, dishwashing, and other applications, processing textiles (e.g., desizing), improving digestibility in animal feed, and in baking and brewing. These and other aspects of the compositions and methods are described in detail below. Before describing the various aspects and modalities of the present compositions and methods, the following definitions and abbreviations are described. Definitions and abbreviations In accordance with this detailed description, the following abbreviations and definitions apply. It should be noted that the singular forms *un*, *una*, and *el* include plural referents, unless the context clearly dictates otherwise. Thus, for example, a reference to an enzyme includes a plurality of enzymes, and a reference to a dosage includes a reference to one or more dosages and their equivalents known to those skilled in the art, and so on. This document is organized into several sections to facilitate reading; however, the reader will appreciate that statements made in one section can be applied to other sections. Therefore, the headings used for the different sections of the description should not be interpreted as limiting. Unless otherwise defined, all scientific and technical terms used herein have the same meaning as understood by a person skilled in the art. The following terms are defined below for clarity. Abbreviations and acronyms The following abbreviations / acronyms have the following meanings unless otherwise specified: n?QQi η / ι ζηζ / α / γ DNA deoxyribonucleic acid EC Enzyme Commission GA glucoamylase GH general hardness HDL high density liquid detergent HDD industrial-grade powder detergent HSG high-foaming granular detergent HFCS high fructose corn syrup IRS residual insoluble starch kDa kilodalton PM molecular weight UPM modified Wohlgemuth unit; 1.6xl0~5mg / UPM = unit of activity NCBI National Center for Information Biotechnology IR yield index ppm parts per million, for example, μg of protein per gram of dry solid FCR relative centrifugal / centripetal force (i.e., gravity x) sp. species w / v weight / volume w / w weight / weight v / v volume / volume % by weight percentage by weight °C degrees Celsius H2O water dH2O or DI deionized water dIH2O deionized water, filtration Milli-Q go gm grams pg micrograms n?QQi η / ι ζηζ / α / γ mg milligrams kg kilograms gL and μA microliters mL and mi milliliters mm millimeters gm micrometer M molar mM millimolar gM micromolar U units sec seconds min(s) minute / minutes h(s) hour / hours ETOH ethanol Normal N MWCO molecular weight limit CAZy carbohydrate-active enzyme database WT wild type Definitions The term amylase or the expression amylolytic enzyme refers to an enzyme that is, among other things, capable of catalyzing the degradation of starch. α-Amylases are hydrolases that cleave the α-D-(1⁴) O-glycosidic bonds in starch. Generally, α-amylases (EC 3.2.1.1; an;aa ι η / ι ζηζ / α / γ D-(1^4)-glucan glucanohydrolase) enzymes are defined as endoactive enzymes that randomly cleave α-D-(1^4) O-glycosidic linkages within the starch molecule, thereby yielding polysaccharides containing three or more D-glucose units with (1-4)-α linkages. In contrast, exoactive amylase enzymes such as β-amylases (EC 3.2.1.2; α-D-(1^4)-glucan-maltohydrolase) and some product-specific amylases such as maltogenic α-amylase (EC 3.2.1.133) cleave the polysaccharide molecule from the non-reducing end of the substrate. β-amylases, α-glucosidases (EC 3.2.1.20; α-D-glucoside-glucohydrolase), glucoamylase (EC 3.2.1.3; α-D-(1^4)-glucan-glucohydrolase), and product-specific amylases such as maltotetraosidases (EC 3.2.1.60) and maltohexasidases (EC 3.2.1.98) can produce maltooligosaccharides of a specific length or syrups enriched with specific maltooligosaccharides. The term starch refers to any material composed of complex polysaccharide carbohydrates from plants, specifically amylose and amylopectin, with the formula (CeH₂oCMₓ), where X can be any number. The term includes plant-based materials such as grains, cereals, herbs, tubers, and roots, and more specifically materials derived from wheat, barley, maize, rye, rice, sorghum, bran, cassava, millet, sorghum, potato, sweet potato, and tapioca. The term starch includes granular starch. The expression nyooi η / ι ζηζ / α / γ granular starch refers to raw starch, that is, uncooked starch, for example, starch that has not undergone gelatinization. As used herein, the term liquefaction or liquefying refers to a process by which starch is converted into less viscous, shorter-chain dextrins. The terms "natural," "precursor," or "reference," with respect to a polypeptide, refer to a naturally occurring polypeptide that does not include any artificial substitutions, insertions, or deletions at one or more amino acid positions. Similarly, the terms "natural," "parental," or "reference," with respect to a polynucleotide, refer to a naturally occurring polynucleotide that does not include any artificial nucleoside changes. However, it is important to note that a polynucleotide encoding a natural, precursor, or reference polypeptide is not limited to a naturally occurring polynucleotide and encompasses any polynucleotide that encodes the natural, precursor, or reference polypeptide. The reference to the wild-type polypeptide is understood to include the mature form of the polypeptide. A mature polypeptide, or a variant thereof, is one in which a signal sequence is absent, for example, cleaved from an immature form of the polypeptide during or after the expression of the nyooi η / ι ζηζ / α / γ polypeptide. The term variant, with respect to a polypeptide, refers to a polypeptide that differs from a specified wild-type, precursor, or reference polypeptide in that it includes one or more natural or artificial substitutions, insertions, or deletions of an amino acid. Similarly, the term variant, with respect to a polynucleotide, refers to a polynucleotide that differs in its nucleotide sequence from a specified wild-type, precursor, or reference polynucleotide. The identity of the wild-type, precursor, or reference polypeptide or polynucleotide will be evident from the context. In the case of the present α-amylases, the term activity refers to α-amylase activity, which can be measured as described herein. The term "core β-barrel structure" refers to amino acid residues that form a central region of parallel β-sheet secondary structure, curving back on themselves so that the first hydrogen strand joins the last strand in a continuous barrel. Specifically, in BspAmy24, the amino acids that form the barrel are residues 8-11, 40-44, 98-103, 232-235, 263-267, 288-290, 325-328, and 364-368. The expression "an amino acid residue covering the N-terminal side of the core β-barrel structure" refers to an amino acid at the end of the core β-barrel structure that is opposite the side adjacent to the active site. The strands comprising the barrel are oriented in parallel, such that the N-terminal ends of the β-strands are on one side of the barrel and the C-terminal ends of the strands are on the other side of the barrel, adjacent to the active site.More specifically, the amino acids covering the N-terminal side of the core β-barrel include most of the N-terminal residues of each strand that participate in hydrogen bonding with adjacent strands, as defined by common algorithms for assigning secondary structure, such as in PyMol and MOE, as well as the residue immediately before or after those residues if they participate in hydrogen bonding of the main structure with an adjacent strand. In particular, the residues covering the N-terminal side of the core β-barrel structure in BspAmy24 include positions 8, 9, 40, 41, 97, 98, 230, 232, 263, 288, 325, 326, and 364. The apparent pKa of the general acid refers to the experimentally determined value for the highest pKa of the enzyme's activity, such that at pH values ​​above the pKa, activity decreases with increasing pH, with a drop of approximately one order of magnitude in activity for each pH unit at values ​​well above the pKa. This observed pKa is expected to correspond to that of the general acid in the reaction mechanism (Rydberg, EH et al. (2002) Mechanistic analyses of catalysis in human pancreatic α-amylases: Detailed kinetic and structural studies of three conserved carboxylic acids. Biochemistry 41:4492-4502); however, it is also recognized that the enzyme's kinetic properties and other ionizations can complicate direct assignment. The term "performance advantage" refers to an improvement in a desirable property of a molecule. Illustrative performance advantages include, but are not limited to, greater hydrolysis of a starch substrate, increased liquefaction yield of grain, cereal, or other starch substrates, increased cleaning performance, greater thermal stability, greater stability against detergents, greater storage stability, greater solubility, an altered pH profile, reduced calcium dependence, increased specific activity, modified substrate specificity, modified substrate binding, modified pH-dependent activity, modified pH-dependent stability, greater oxidative stability, and greater expression. In some cases, the performance advantage is achieved at a relatively low temperature. In some cases, the performance advantage is achieved at a relatively high temperature. Combinatorial variants are variants that comprise two or more mutations, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more substitutions, deletions and / or insertions. nyooi η / ι ζηζ / α / γ The term recombinant, when used in reference to a cell, nucleic acid, protein, or vector, indicates that the object has been modified from its native state. Thus, for example, recombinant cells express genes that are not found in the native (non-recombinant) form of the cell or express native genes at different levels or under different conditions than those found in nature. Recombinant nucleic acids differ from a native sequence by one or more nucleotides and / or are operatively linked to heterologous sequences, e.g., a heterologous promoter in an expression vector. Recombinant proteins may differ from a native sequence by one or more amino acids and / or are fused to heterologous sequences. A vector comprising a nucleic acid encoding an amylase is a recombinant vector. The terms recovered, isolated, and separated refer to a compound, protein (polypeptide), cell, nucleic acid, amino acid, or other specified material or component that is removed from at least one other material or component with which it is naturally associated as found in nature. An isolated polypeptide includes, but is not limited to, a culture medium containing a secreted polypeptide expressed in a heterologous host cell. The term purified refers to material (e.g., an isolated polypeptide or polynucleotide) that is in a relatively pure state, e.g., at least approximately 90% pure, at least approximately 95% pure, at least approximately 98% pure, or even at least approximately 99% pure. The term enriched refers to material (e.g., an isolated polypeptide or polynucleotide) that is approximately 50% pure, at least approximately 60% pure, at least approximately 70% pure, or even at least approximately 70% pure. The terms thermostable and thermostability, when referring to an enzyme, describe the enzyme's ability to retain its activity after exposure to elevated temperatures. The thermostability of an enzyme, such as an amylase enzyme, is measured by its half-life (ti / 2), expressed in minutes, hours, or days, during which half of the enzyme's activity is lost under defined conditions. The half-life can be calculated by measuring the residual α-amylase activity after exposure (i.e., stimulated by) elevated temperatures. A pH range, with reference to an enzyme, refers to the range of pH values ​​in which the enzyme exhibits catalytic activity. The expressions pH stable and pH stability, with reference to an enzyme, relate to the ability of the enzyme to retain its activity over a wide range of pH values ​​for a predetermined period of time (e.g., 15 min, 30 min, 1 hour). The term amino acid sequence is synonymous with polypeptide, protein, and peptide, and these terms are used interchangeably. When amino acid sequences exhibit activity, they may be called enzymes. Conventional one-letter or three-letter codes are used for amino acid residues, and amino acid sequences are presented in the conventional amino-terminal to carboxy-terminal (i.e., N≡C) orientation. The term nucleic acid encompasses DNA, RNA, heteroduplex, and synthetic molecules capable of encoding a polypeptide. Nucleic acids can be single-stranded or double-stranded and may contain chemical modifications. The terms nucleic acid and polynucleotide are used interchangeably. Because the genetic code is redundant, more than one codon can be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences that encode a particular amino acid sequence. Unless otherwise stated, nucleic acid sequences are presented in the 5' to 3' orientation. The term hybridization refers to the process by which a nucleic acid strand forms a duplex, that is, nη / ι ζηζ / α / γ base pairs with a complementary strand, as occurs during transfer hybridization and PCR techniques. Rigorous hybridization conditions are exemplified by hybridization under the following conditions: 65 °C and 0.1X SSC (where 0.15 M NaCl, 30.015 M Na citrate, pH 7.0). Hybridized duplex nucleic acids are characterized by a melting temperature (Tm), at which one half of the hybridized nucleic acid is unpaired with the complementary strand. A synthetic molecule is produced by chemical or enzymatic synthesis in vitro, rather than with an organism. The terms and expressions transformed, stably transformed, and transgenic, used in reference to a cell, refer to the fact that the cell contains a non-native (e.g., heterologous) sequence of nucleic acids integrated into its genome or carried as an episome that is maintained through several generations. The term introduced in the context of the insertion of a nucleic acid sequence into a cell means transfection, transformation, or transduction, as it is known in the technique. A host strain or host cell is an organism into which an expression vector, phage, virus, or other DNA construct has been introduced, containing a polynucleotide that encodes a polypeptide of interest (e.g., n?QQi η / ι ζηζ / α / γ, an amylase). Examples of host strains include cells of microorganisms (e.g., bacteria, filamentous fungi, and yeasts) capable of expressing the polypeptide of interest and / or fermenting saccharides. Host cell expression includes protoplasts created from cells. The term heterologous, in reference to a polynucleotide or protein, refers to a polynucleotide or protein that is not naturally occurring in a host cell. The term endogenous, with reference to a polynucleotide or protein, refers to a polynucleotide or protein naturally present in the host cell. The term expression refers to the process by which a polypeptide is produced based on a nucleic acid sequence. The process includes both transcription and translation. The term specific activity refers to the number of moles of substrate that can be converted into product by the action of an enzyme or enzyme preparation per unit of time under specific conditions. Specific activity is generally expressed as units (U) / mg of protein. As used herein, water hardness is a measure of the minerals (e.g., calcium and magnesium) present in the water. A sample is a piece of material, such as a fabric, to which a stain has been applied. The material can be, for example, fabrics made of cotton, polyester, or mixtures of natural and synthetic fibers. The sample can also be paper, such as filter paper or nitrocellulose, or a piece of hard material such as ceramic, metal, or glass. For amylases, the stain is starch-based, but it can include blood, milk, ink, grass, tea, wine, spinach, gravy, chocolate, egg, cheese, clay, pigment, oil, or mixtures of these compounds. A sub-sample, or micro-sample, is a section of the sample that has been cut with a single-hole punch, or cut with a custom-made multi-hole punch where the multi-hole punch pattern matches standard multi-well microtiter plates, or the section has been removed from the sample in some other way. The sample may be made of textile, paper, metal, or other suitable material. The stain may be fixed to the sub-sample before or after placing it in the well of a 24-, 48-, or 96-well microtiter plate. The sub-sample may also be prepared by applying a stain to a small piece of material. For example, the sub-sample may be a stained piece of fabric 5 / 8" or 0.25" or 5.5 mm in diameter.The custom-made punch was designed to simultaneously deliver 96 samples to all wells of a 96-well plate. The nyooi η / ι ζηζ / α / γ device allows for the delivery of more than one sample per well simply by loading the same 96-well plate multiple times. Multi-hole punches can be conceived to simultaneously deliver samples to any plate format, including, but not limited to, 24-well, 48-well, and 96-well plates. In another conceivable method, the soiled sample platform can be a bead made of metal, plastic, glass, ceramic, or other suitable material coated with the soiled substrate. The coated bead(s) are then placed in wells of 96-, 48-, or 24-well plates, or larger formats, containing a suitable buffer and enzyme.In other conceivable methods, the stained cloth is exposed to the enzyme by placing enzyme solution on the cloth, wetting the sample attached to a holding device, or immersing the sample in a larger solution containing the enzyme. The expression "cultured cellular material comprising an amylase," or a similar expression, refers to a cell lysate or supernatant (including the medium) that includes an amylase as a component. The cellular material may be from a heterologous host that is grown in culture to produce the amylase. The expression "sequence identity percentage" refers to the fact that a particular sequence has at least a certain percentage of amino acid residues identical to those of a specified reference sequence when aligned using the CLUSTAL W algorithm with default parameters. See Thompson et al. (1994) Nucleic Acids Res. 22:4673-4680. The default parameters for the CLUSTAL W algorithm are: nyooi η / ι ζηζ / α / γ Gap opening penalty: 10.0 Gap extension penalty: 0.05 Protein weight matrix: BLOSUM series AUN weight matrix: IUB % divergent sequence delay: 40 Gap separation distance: 8 DNA transition weight: 0.50 Hydrophilic residue list: GPSNDQEKR Negative matrix use: INACTIVATED Rest-specific penalty toggle: ON Hydrophilic penalty toggle: ON End gap separation penalty switching: INACTIVATED Percent identity for multiple sequences It can also be determined using MUSCLE (Edgar, RC (2004) Nuc. Acids Res. 32:1792-97 and Edgar, RC (2004) Bioinformatics 5:113). In all cases, deletions are counted as non-identical residues, compared to a reference sequence. The fused polypeptide sequences are connected, i.e., operationally joined, by means of a peptide bond between two target polypeptide sequences. The term filamentous fungi refers to all filamentous forms of the Eumycotina subdivision, particularly the Pezizomycotina species. The term dry solids content (ds) refers to the total solids in a dense suspension expressed as a percentage of dry weight. The term dense suspension refers to an aqueous mixture containing insoluble solids. The term simultaneous saccharification and fermentation (SSF) refers to a process in the production of biochemicals in which a microbial organism, such as an ethanol-producing microorganism, and at least one enzyme, such as an amylase, are present during the same process step. SSF includes the simultaneous hydrolysis of starch substrates (granular, liquefied, or solubilized) to saccharides, including glucose, and the fermentation of the saccharides to alcohol or another biochemical or biomaterial product in the same reactor vessel. An ethanologenic microorganism refers to a microorganism with the ability to convert a sugar or oligosaccharide into ethanol. The term fermented beverage refers to any beverage produced by a method involving a fermentation process, such as microbial fermentation, for example, bacterial and / or fungal fermentation. Beer is an example of such a fermented beverage, and the term beer is intended to encompass any fermented wort produced by the fermentation / brewing of starchy plant material. Often, beer is produced exclusively from malt or adjuvants, or any combination of malt and adjuvant. The term malt refers to any malted cereal grain such as malted barley or wheat. The term adjuvant refers to any plant material containing starch and / or sugar other than malt, such as barley or wheat malt. Examples of adjuvants include common ground corn, refined ground corn, ground brewer's yeast, rice, sorghum, refined corn starch, barley, barley starch, hulled barley, wheat, wheat starch, roasted cereal, cereal flakes, rye, oats, potato, tapioca, cassava, and syrups such as corn syrup, cane sugar syrup, invert sugar syrup, barley and / or wheat syrups, and the like. The term mash refers to an aqueous suspension of any plant material containing starch and / or sugar, such as milled grain, for example, comprising crushed barley malt, crushed barley and / or other adjunct or a combination thereof, mixed with water to be later separated into wort and spent grains. η^ααι η / ι ζηζ / α / γ The term must refers to the runoff of unfermented liquor after the grain has been removed from milling during mashing. The term approximately refers to ± 15% with respect to the referenced value. Aspects and modalities of the present compositions and methods The following paragraphs describe in detail various aspects and modalities of the present compositions and methods. α-amylase variants that have a reduced pKa of their general acid One way to increase the activity of an α-amylase is to reduce the pKa of its general acid. Reducing the pKa of a general acid results in increased reactivity of the protonated species. The Brønsted equation for the reactivity of a general acid (see, for example, Jencks, W.P. (1986) Catalysis in Chemistry and Enzymology, Dover Publications, New York.) expresses the relationship between pKa and reactivity with a factor α that is characteristic of a given reaction, where α is the rate constant of the protonated species, pKa is the pKa of that general acid, and C is a constant: log(^Hu) = -atpKa) + C While it is well established that reducing the pKa of a general acid can increase the reactivity of fully protonated species, the means and mechanisms for changing the pKa of an amino acid side chain acting as a general acid within an enzyme active site are often very complex. Effects such as nearby charged or hydrophobic environments can alter pKa values ​​(see, for example, Schmidt, DE and Westheimer, FH). (1971) pK of the lysine amino group in the active site of acetoacetate decarboxylase. Biochemistry 10:1249-53 and Ho, MC. et al. (2009) The origin of the electrostatic perturbation in acetoacetate decarboxylase. Nature 459:393-97). In general, it is difficult to predict the impact of protein mutations on pKa values ​​for catalytic side chains in all enzymes, including α-amylases. (Nielsen, JE and Borchert, TV (2000) Protein engineering of bacterial amylases. Biochemistry et Biophysics Acta 1543:253-274.). As described herein, a set of residues covering the N-terminal side of the β-barrel core in α-amylases was unexpectedly found to substantially impact the measured pKa values ​​of the enzyme and increase α-amylase activity at pH values ​​near and below the pKa. The positions of these residues are shown in Figures 2 and 3. Mutations in these amino acids resulted in a reduction of the pKa value expected to correspond to that of the general acid in the reaction. (Rydberg, EH et al. (2002) Mechanistic analyses of catalysis in human pancreatic α-amylases: Detailed kinetic and structural studies of three conserved carboxylic acids. Biochemistry 41:4492-4502). Without limiting itself to any one theory, it is postulated that the mutation of these residues can affect the electrostatic environment of the general acid because their interactions anchor the barrel in which the general acid is positioned for catalysis. Alternative packing arrangements on the N-terminal side of the barrel can serve to tighten or loosen the barrel and thus change the electrostatic environment of the general acid. The model α-amylase used to exemplify the present compositions and methods is an α-amylase from Bacillus sp., designated herein as BspAmy24. The amino acid sequence of α-amylase from BspAmy24 is shown below as SEC. ID NO.: 1: HHNGTNGTMM QYFEWHLPND GQHWNRLRND AANLKNLGIT AVWIPPAWKG nyooi η / ι ζηζ / α / γ TSQNDVGYGA YDLYDLGEFN QKGTIRTKYG TRSQLQSAIA SLQNNGIQVY GDWMNHKGG ADGTEWVQAV EVNPSNRNQE VTGEYTIEAW TKFDFPGRGN THSSFKWRWY HFDGTDWDQS RQLNNRIYKF RGTGKAWDWE VDTENGNYDY LMYADVDMDH PEVINELRRW GVWYTNTLNL DGFRIDAVKH IKYSFTRDWL NHVRSTTGKN NMFAVAEFWK NDLGAIENYL HKTNWNHSVF DVPLHYNLYN ASKSGGNYDM RQILNGTWS KHPIHAVTFV DNHDSQPAEA LESFVEAWFK PLAYALILTR EQGYPSVFYG DYYGIPTHGV AAMKGKIDPI LEARQKYAYG TQHDYLDHHN IIGWTREGNS AHPNSGLATI MSDGPGGSKW MYVGRHKAGQ VWRDITGNRT GTVTINADGW GNFSVNGGSV SIWVNK nyooi η / ι ζηζ / α / γ BspAmy24 is similar to α-amilasa of Bacillus sp. 707 que se denomina α-amilasa Amy 707. The secuencia de Amylasa amino acids707 this month, a continuation, come to the SEC. ID NO.: 2: HHNGTNGTMM QYFEWYLPND GNHWNRLNSD ASNLKSKGIT AVWIPPAWKG ASQNDVGYGA YDLYDLGEFN QKGTVRTKYG TRSQLQAAVT SLKNNGIQVY GDWMNHKGG ADATEMVRAV EVNPNNRNQE VTGEYTIEAW TRFDFPGRGN THSSFKWRWY HFDGVDWDQS RRLNNRIYKF RGHGKAWDWE VDTENGNYDY LMYADIDMDH PEWNELRNW GVWYTNTLGL DGFRIDAVKH IKYSFTRDWI NHVRSATGKN MFAVAEFWKN DLGAIENYLQ KTNWNHSVFD VPLHYNLYNA SKSGGNYDMR NIFNGTWQR HPSHAVTFVD NHDSQPEEAL ESFVEEWFKP LAYALTLTRE QGYPSVFYGD YYGIPTHGVP AMRSKIDPIL EARQKYAYGK QNDYLDHHNI IGWTREGNTA HPNSGLATIM SDGAGGSKWM FVGRNKAGQV WSDITGNRTG TVTINADGWG NFSVNGGSVS IWVNK BspAmy24 también es similar to una α-amilasa de otro Bacillus sp. That denominates α-amilasa AA560, that connects it to amino acids that also contain a continuation, including SEC. ID NO.: 3: HHNGTNGTMM QYFEWYLPND GNHWNRLRSD ASNLKDKGIS AVWIPPAWKG ASQNDVGYGA YDLYDLGEFN QKGTIRTKYG TRNQLQAAVN ALKSNGIQVY GDWMNHKGG ADATEMVRAV EVNPNNRNQE VSGEYTIEAW TKFDFPGRGN THSNFKWRWY HFDGVDWDQS RKLNNRIYKF RGDGKGWDWE VDTENGNYDY LMYADIDMDH PEWNELRNW GVWYTNTLGL DGFRIDAVKH IKYSFTRDWI NHVRSATGKN MFAVAEFWKN DLGAIENYLN KTNWNHSVFD VPLHYNLYNA SKSGGNYDMR QIFNGTWQR HPMHAVTFVD NHDSQPEEAL ESFVEEWFKP LAYALTLTRE QGYPSVFYGD YYGIPTHGVP AMKSKIDPIL EARQKYAYGR QNDYLDHHNI IGWTREGNTA HPNSGLATIM SDGAGGNKWM FVGRNKAGQV WTDITGNRAG TVTINADGWG NFSVNGGSVS IWVNK nyooi η / ι ζηζ / α / γ We use amino acid secuencias from BspAmy24, Amy707 and AA560 to put them in Figura 1. We identify the amino acid secuencias that we use in the MUSCLE to put them in the tabla 1. Table 1. The matrix of identification of the amino acids of BspAmy24, Amy707 and AA560 BspAmy2 4 Amy707 ΆΑ560 BspAmy2 4 (100) 90.3 89.5 Amy707 90.3 (100) 95.5 AA560 89.5 95.5 (100) In some forms, the variant α-amylase has at least 60%, at least 70%, at least 80%, at least a 85%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least one 95%, at least 96%, at least 97%, at least 98%, or even at least 99% amino acid sequence identity with SEC. ID NO.: 1, 2 and / or 3, excluding wild-type BspAmy24, Amy707 and AA560 and known variants thereof. Many bacterial α-amylases are known to share the same fold and often benefit from the same mutations. In this case, the corresponding amino acid positions in other α-amylases can be readily identified by aligning the amino acid sequence with BspAmy24, Amy707, and AA560 using Clustal W with predefined parameters. The α-amylases in which the above mutations are likely to produce a performance benefit include those that have a similar fold and / or have 60% or greater amino acid sequence identity with any of the well-known Bacillus amylases (e.g., from B. licheniformis, B. stearothermophilus, B. amyloliquifaciens, Bacillus sp. SP722, Cytophaga sp., and the like), Carbohydrate Active Enzymes Database (CAZy), family 13 amylases, or any amylase that has been referred to so far by the term termamyl-like descriptive term.The reader will appreciate that when a naturally occurring α-amylase has a mutation listed above (i.e., where the wild-type α-amylase already contained a residue identified as a mutation), then that particular mutation does not apply to that α-amylase. However, other described mutations may function in combination with the naturally occurring residue at that position. Due to their close sequence identity, nyooi η / ι ζηζ / α / γ (including substitutions, insertions, and deletions), which produce a beneficial effect in BspAmy24, are particularly likely to produce a similar effect in the α-amylase of Amy707 and A1560, and vice versa. 2.2 Additional mutations In some forms, in addition to one or more of the mutations described above (e.g., in Section 2.1), the present amylases also include one or more mutations that provide an additional performance or stability benefit. Illustrative performance advantages include, but are not limited to, greater hydrolysis of a starch substrate, increased liquefaction yield of grain, cereal, or other starch substrate, increased cleaning yield, greater thermal stability, greater storage stability, greater solubility, an altered pH profile, reduced calcium dependence, increased specific activity, modified substrate specificity, modified substrate binding, modified pH-dependent activity, modified pH-dependent stability, greater oxidative stability, and increased expression. In some cases, the performance advantage is achieved at a relatively low temperature.In some cases, the performance advantage is realized at a relatively high temperature. In some forms, the present aamylase variants additionally have at least one mutation in the calcium-binding loop based on the work of Suzuki et al. (1989) nyooi η / ι ζηζ / α / γ J. Biol. Cheia. 2 64:18933-938. Example mutations include a deletion or substitution of one or more residues corresponding to positions 181, 182, 183 and / or 184 in any of the SEQ. ID NO.: 1-3. In particular forms, the mutation corresponds to the deletion of 181 and 182 or 183 and 184 (using any of the SEQ. ID NO.: 1-3 for numbering). Homologous residues in other amylases can be determined by structural alignment or primary structure alignment. In some forms, the present amylase variants additionally have at least one known mutation that confers a performance, stability, or solubility benefit over other microbial α-amylases, including, but not limited to, those with a similar fold and / or 60% or greater amino acid sequence identity with any of the Family 13 carbohydrate-active enzyme (CAZy) amylases (SEQ ID NO.: 1-3), or any amylase previously referred to by the descriptive term term "termamyl-like." Amino acid sequence identity can be determined using Clustal W with predefined parameters. The amylases present may include any number of conservative amino acid substitutions. The conservative amino acid substitutions are listed in the n?QQi η / ι ζηζ / α / γ Table 2: njQQi η / ι ζηζ / α / γ Table 2. Conservative amino acid substitutions Amino Acid Code Replace with any of Alanine A D-Ala, Gly, β-Ala, L-Cys, D-Cys Arginine R D-Arg, Lys, D-Lys, homo-Arg, D-homoArg, Met, lie, D-Met, D-Ile, Orn, D-Orgina, D-Asp, D-Asp, D-Asp, D-Asp Glu, D-Glu, Gln, DGln Aspartic acid D D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, DGln Cysteine ​​0 D-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr Glutamine Q D-Gln, Asn, D-Asp, D-Glu, Asp, D-Gln DAsp Glutamic acid E D-Glu, D-Asp, Asp, Asn, D-Asn, Gln, DGln Glycine G Ala, D-Ala, Pro, D-Pro, β-Ala, Acp Isoleucine I D-Ile, Val, D-Val, Leu, D-Leu, Met, DMet Leu, Leu, D-Val, Leu, Leu, D-Val D-Leu, Met, DMet Lysine K D-Lys, Arg, D-Arg, homo-Arg, D-homoArg, Met, D-Met, lie, D-Ile, Orn, D-Orn Methionine M D-Met, S-Me-Cis, lie, D-Ile, Leu, D-Leu, Val, D-Val, F-F, D-F D-Tr, L-Dopa, His, D-His, Trp, D-Trp, Trans-3,4 or 5-phenylproline, cis-3,4 or 5-phenylproline Proline P D-Pro, LI-thioazolidine-4carboxylic acid, Do Ll-oxazolidine-4-Servine acid, Scar-Serine D-Thr, allo-Thr, Met,D-Met, Met(O), D-Met(0), L-Cys, D-Cys Threonine T D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Met(0), D-Met(0), Val, D-Val Tyrosine AND D-Tyr, Phe, D-Phe, L-Dopa, His, D-His Valine V D-Val, Leu, D-Leu, lie, D-Ile, Met, DMet, It will be noted that some of the conservative mutations mentioned above can be produced by genetic manipulation, while others are produced by the introduction of synthetic amino acids into a polypeptide by genetic or other means. The present amylase can also be derived from any of the amylase variants described above by substitution, deletion, or addition of one or more amino acids in the amino acid sequence—for example, fewer than 10, fewer than 9, fewer than 8, fewer than 7, fewer than 6, fewer than 5, fewer than 4, fewer than 3, or even fewer than 2 substitutions, deletions, or additions. The variants should have the same activity as the amylase from which they were derived. Specific deletions include N-terminal and / or C-terminal truncations of one or a few amino acid residues—for example, 1, 2, 3, 4, or 5 amino acid residues. The amylases presented herein may be precursor, immature, or full-length, in which case they include a signal sequence, or mature, in which case they lack a signal sequence. The mature forms of the polypeptides are generally the most useful. Unless otherwise stated, the numbering of amino acid residues used herein refers to the mature forms of the respective amylase polypeptides. The amylase polypeptides presented herein may also be truncated to remove the N-terminal or C-terminal end, provided that the resulting polypeptides retain amylase activity. The present amylase may be a chimeric, hybrid, or swap-domain polypeptide, comprising at least a portion of a first amylase polypeptide and at least a portion of a second amylase polypeptide. The present amylases may also include a heterologous signal sequence, an epitope to enable tracking or purification, or the like. Illustrative heterologous signal sequences are derived from B. licheniformis amylase (LAT), B. subtilis amylase (AmyE or AprE), and Streptomyces CelA. Nucleotides that encode polypeptides of amylase variants In another aspect, nucleic acids encoding a polypeptide of an amylase variant are provided. The nucleic acid may encode a particular amylase polypeptide, or an amylase having a specified degree of amino acid sequence identity with the particular amylase. In some forms, the nucleic acid encodes an amylase that 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 even at least 99% amino acid sequence identity with one or more of the sequences listed in Section ID No. 1-3. It will be appreciated that due to the degeneracy of the genetic code, a plurality of nucleic acids may encode the same polypeptide. Production of variant amylases These variants can be produced in host cells, for example, by secretion or intracellular expression, using well-established techniques. Fermentation, separation, and concentration techniques are well-established, and conventional methods can be used to prepare a solution containing an amylase variant polypeptide. For production-scale recovery, polypeptides of α-amylase variants can be enriched or partially purified as described above by removing cells through polymer flocculation. Alternatively, the enzyme can be enriched or purified by microfiltration followed by concentration via ultrafiltration using readily available membranes and equipment. However, for some applications, the enzyme does not need to be enriched or purified, and the entire broth culture can be used without further treatment. The enzyme can then be processed, for example, into granules. Carbohydrate processing compositions and uses of variant amylases Variant amylases are useful for a variety of carbohydrate processing applications that are well known in the art and are not reiterated herein. nyooi η / ι ζηζ / α / γ These uses include the production of fuel ethanol, syrup production, and the production of other valuable biochemicals. Preparation of starch substrates The methods for preparing starch substrates for use in the processes disclosed herein are well known. Useful starch-type substrates can be obtained from, for example, tubers, roots, stems, legumes, cereals, or whole grains. More specifically, granulated starch can be obtained from corn, cobs, wheat, barley, rye, triticale, bicolor sorghum, sago, millet, cassava, tapioca, sorghum, rice, peas, beans, plantains, or potatoes. Some starch substrates specifically considered are corn starch and wheat starch. Starch from a grain may be crushed or whole and includes corn solids such as kernels, bran, and / or cobs. Starch may also be highly refined raw starch or raw material from refining processes. Gelatinization and liquefaction of starch In general, gelatinization is carried out simultaneously with, or followed by, the contact of a starch substrate with an α-amylase, although additional liquefaction-inducing enzymes may optionally be added. In some embodiments, a dense suspension of the starch substrate prepared as described above is formed with water. Liquefaction may also be carried out below liquefaction temperatures, such as in a cold-cooking or no-cooking process. Saccharification Liquefied starch can be saccharified to obtain a syrup rich in saccharides with lower DP (e.g., DPI + DP2) using variants of α-amylases, optionally in the presence of one or more other enzymes. The exact composition of the saccharification products depends on the combination of enzymes used, as well as the type of granulated starch processed. Saccharification and fermentation can be carried out simultaneously or in overlapping fashion (see below). Isomerization The hydrolyzed soluble starch product obtained by amylase treatment can be converted into a high-fructose starch-based syrup (HESS), such as high-fructose corn syrup (HFCS). This conversion can be achieved using a glucose isomerase, especially a glucose isomerase immobilized on a solid support. Fermentation Soluble starch hydrolysate, especially a glucose-rich syrup, can be fermented by contacting the starch hydrolysate with a fermentation organism. EOF products include metabolites such as citric acid, lactic acid, succinic acid, monosodium glutamate, gluconic acid, sodium gluconate, calcium gluconate, potassium gluconate, itaconic acid and other carboxylic acids, glucono delta-lactone, sodium erythorbate, lysine and other amino acids, omega-3 fatty acids, butanol, isoprene, 1,3-propanediol, and other biomaterials. Ethanogenic microorganisms include yeast, such as Saccharomyces cerevisiae, and bacteria, such as Zymomonas mobilis, which express alcohol dehydrogenase and pyruvate decarboxylase. Improved strains of ethanologenic microorganisms are known in the art. Commercial sources of yeast include ETHANOL RED® (LeSaffre); FERMAX™ (Martrex); THERMOSACC®; TRANSFERM® Yield+; and YP3™ (Lallemand); RED STAR® (Red Star); FERMIOL® (DSM Specialties); SUPERSTART® (Alltech); and SYNERXIA® and SYNERXIA® Thrive (DuPont Industrial Biosciences). Microorganisms that produce other metabolites, such as citric acid and lactic acid, through fermentation are also known in the art. Compositions comprising variant amylases and additional enzymes Variant amylases can be combined with a glucoamylase (EC 3.2.1.3), starting from, for example, Trichoderma, Aspergillus, Talaromyces, Clostridium, Fusarium, Thielavia, Thermomyces, Athelia, Humicola, Penicillium, Artomyces, Gloeophyllum, Pycnoporus, Stecherinum, Trametes, etc. Suitable commercial glucoamylases include AMG 200L; AMG 300 L; SAN™ SUPER and AMG™ E (Novozymes); OPTIDEX® 300 and OPTIDEX L-400 (Danisco US Inc.); AMIGASE™ and AMIGASE™ PLUS (DSM); G-ZYME® G900 (Enzyme Bio-Systems); and G-ZYME® G990 ZR. Other suitable enzymes that can be used with amylase include phytase, protease, pullulase, β-amylase, isoamylase, α-glucosidase, cellulase, xylanase, other hemicellulases, β-glucosidase, transferase, pectinase, lipase, cutinase, esterase, mannanase, redox enzymes, a different α-amylase, or a combination of these. The compositions comprising the present amylases may be aqueous or non-aqueous formulations, granules, powders, gels, dense suspensions, pastes, etc., and may further comprise one or more of any of the additional enzymes listed herein, along with additives, salts, preservatives, water, co-solvents, surfactants, and the like. The compositions may function in combination with endogenous enzymes or other ingredients already present in a dense suspension, water bath, washings, food product, or beverage, etc., for example, endogenous plant enzymes (including those from algae), residual enzymes from a previous processing step, and the like. nyooi η / ι ζηζ / α / γ Compositions and methods for baking and food preparation The present invention also relates to a food composition that includes, without limitation, a food product, animal feed, and / or food / feed additive comprising an amylase, and methods for preparing such a food composition comprising mixing an amylase variant with one or more food ingredients, or uses thereof. Furthermore, the present invention relates to the use of an amylase in the preparation of a food composition, wherein the food composition is baked after the addition of the polypeptide of the invention. Brewing compositions The present amylase variant may be a component of a brewing composition used in a brewing process, i.e., the production of a fermented malt beverage. Non-fermentable carbohydrates make up the majority of the dissolved solids in the final beer. This residue remains due to the inability of malt enzymes to hydrolyze the alpha-1,6 linkages of starch. Non-fermentable carbohydrates contribute approximately 50 calories per 12 ounces of beer. An amylase, combined with a glucoamylase and optionally a pullulase and / or isoamylase, helps convert starch into dextrins and fermentable sugars, thereby reducing residual non-fermentable carbohydrates in the final beer. Textile de-seasoning compositions Compositions and methods for treating fabrics (e.g., for desizing a textile product) using an amylase are also considered. These fabric treatment methods are well known in the art (see, for example, U.S. Patent No. 6,077,316). For instance, the feel and appearance of a fabric can be improved by a method comprising contacting the fabric with an amylase in a solution. The fabric can be treated with the solution under pressure. Cleaning compositions One aspect of the present compositions and methods is a cleaning composition that includes an amylase as a component. An amylase polypeptide can be used as a component in detergent compositions for, for example, handwashing, laundry, dishwashing, and other hard surface cleaning. The compositions include handwashing (manual), industrial liquid (HDL), and industrial dry (HDD) detergent compositions, including single-dose laundry compositions, and handwashing (manual) and automatic dishwasher (ADW) compositions, including single-dose dishwashing compositions. Summary An amylase polypeptide can be a component of a detergent composition, either as the sole enzyme or in combination with other enzymes, including other amylolytic enzymes. Therefore, it can be included in the detergent composition as a dust-free granule, a stabilized liquid, or a protected enzyme. Detergent compositions can be in any useful form, for example, as powders, granules, pastes, bars, or liquids. A liquid detergent can be aqueous, typically containing up to approximately 70% water and from 0% to approximately 30% organic solvent. It can also be found in the form of a compact gel containing only approximately 30% water. Detergent compositions comprise one or more surfactants, each of which can be anionic, nonionic, cationic, or zwitterionic. Detergent compositions may further comprise one or more different enzymes such as proteases, other amylolytic enzymes, mannanase, cutinase, lipase, cellulase, pectatolyase, perhydrolase, xylanase, peroxidase, and / or laccase in any combination. Some specific detergent formulations incorporating α-amylase are described below. Many of these formulations can be supplied in single-dose formats for ease of use. The nyooi η / ι ζηζ / α / γ describe single-dose formulations and packaging, for example, in documents US20090209445A1, US20100081598A1, US7001878B2, EP1504994B1, W02001085888A2, WO2003089562A1, WO2009098659A1, WO2009098660A1, WO2009112992A1, WO2009124160A1, WO2009152031A1, WO2010059483A1, W02010088112A1, WO2010090915A1, WO2010135238A1, W02011094687A1, WO2011094690A1, WO2011127102A1, WO2011163428A1, WO2008000567A1, WO2006045391A1, WO2006007911A1, WO2012027404A1, EP1740690B1, WO2012059336A1, US6730646B1, nyooi η / ι ζηζ / α / γ WO2008087426A1, WO2010116139A1 and WO2012104613A1. Liquid laundry detergent composition for industrial use (HDL) Illustrative HDL laundry detergent compositions include a detergent surfactant (10%-40% w / w), comprising an anionic detergent surfactant (selected from a group of alkyl sulfates, alkyl sulfonates, alkoxylated alkyl sulfates, alkyl phosphates, alkyl phosphonates, alkyl carboxylates, linear or branched or random chain, substituted or unsubstituted, and / or mixtures thereof) and optionally a nonionic surfactant (selected from a group of linear or branched or random chain alkoxylated alkyl alcohols, substituted or unsubstituted, e.g., ethoxylated C8-C18 alkyl alcohols and / or C6-C12 alkyl alkoxylates), wherein the weight ratio of anionic detergent surfactant (having a hydrophilic index (HIc) of from 6.0 to 9) relative to non-ionic detergent surfactant is greater than 1:1.Suitable detergent surfactants also include cationic detergent surfactants (selected from a group of alkylpyridinium compounds, quaternary alkylammonium compounds, quaternary alkylphosphonium compounds, ternary alkylsulfonium compounds and / or mixtures thereof); zwitterionic and / or amphoteric ion detergent surfactants (selected from a group of alkanolamine sulfobetaines); amphoteric surfactants; non-ionic semipolar surfactants and mixtures thereof. The composition may optionally include a surfactant-enhancing polymer consisting of amphiphilic alkoxylated grease-cleaning polymers (selected from a group of alkoxylated polymers having branched hydrophilic and hydrophobic properties, such as alkoxylated polyalkylene imines in the range of 0.05 wt-10 wt) and / or random graft polymers typically comprising a hydrophilic backbone comprising monomers selected from the group consisting of: unsaturated C1-C6 carboxylic acids, ethers, alcohols, aldehydes, benzoins, esters, sugar units, alkoxy units, maleic anhydride, saturated polyalcohols such as glycerol, and mixtures thereof; and hydrophilic side chain(s) selected from the group consisting of: C4 alkyl group n?QQi η / ι ζηζ / α / γ C25, polypropylene, polybutylene, vinyl ester of a saturated C1-C6 monocarboxylic acid, C1-C6 alkyl ester of acrylic or methacrylic acid and mixtures thereof. The composition may include additional polymers such as soil-releasing polymers (including anionic end-terminated polyesters, e.g., SRP1); polymers comprising at least one monomeric unit selected from saccharide, dicarboxylic acid, polyol and combinations thereof, in random or block configurations; ethylene terephthalate-based polymers and their copolymers in random or block configurations, e.g., Repel-o-tex SF, SF-2 and SRP6, Texcare SRA100, SRA300, SRN100, SRN170, SRN240, SRN300 and SRN325, Marloquest SL; anti-redeposition polymers (of a 0.1% by weight to 10% by weight, include carboxylate polymers, such as polymers comprising at least one monomer selected from acrylic acid, maleic acid (or maleic anhydride), fumaric acid, itaconic acid, aconitic acid, mesaconic acid, citraconic acid, methylenemalonic acid, and any mixture thereof; vinylpyrrolidone homopolymer; and / or polyethylene glycol having a molecular weight in the range of 500 to 100,000 Da); cellulosic polymer (including, for example, alkylcellulose; alkylalkoxyalkylcellulose; carboxyalkylcellulose; alkylcarboxyalkylalkylcellulose; examples of which include carboxymethylcellulose, methylcellulose, methylhydroxyethylcellulose, methylcarboxymethylcellulose and mixtures thereof); and polymeric carboxylate (such as, for example, maleate / acrylate random copolymer or polyacrylate homopolymer). The composition may further include a saturated or unsaturated fatty acid, preferably a saturated or unsaturated C12-C24 fatty acid (from 0% by weight to 10% by weight); deposition aids (examples of which include polysaccharides, preferably cellulosic polymers, polydiallyldimethylammonium halides (DADMAC) and DAD MAC copolymers with vinylpyrrolidone, acrylamides, imidazoles, imidazolinium halides and mixtures thereof, in random or block configurations; cationic guar gum; cationic cellulose such as cationic hydroxyethylcellulose; cationic starch; cationic polyacylamides; and mixtures thereof. The composition may also include dye transfer inhibitors, examples of which include manganese phthalocyanine, peroxidases, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, N-vinylpyrrolidone and N-vinylimidazole copolymers, polyvinyloxazolidones and polyvinylimidazoles and / or mixtures thereof; chelating agents, examples of which include ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentamethylenephosphonic acid (DTPMP), hydroxyethanediphosphonic acid (HEDP), ethylenediamine-N,N' disuccinic acid (EDDS), methylglycinediacetic acid (MGDA); diethylenetriaminepentaacetic acid (DTPA), propylenediaminetetraacetic acid (PDTA), 2-hydroxypyridine N-oxide (HPNO); or methylglycinodiacetic acid (MGDA), glutamic acid; N,N-diacetic acid (tetrasodium salt of N,N-dicarboximethylglutamic acid (GLDA); nitrilotriacetic acid (NTA), 4,5-dihydroxy-m-benzenedisulfonic acid; citric acid and any of its salts;N-hydroxyethylethylenediaminotriacetic acid (HEDTA), triethylentetraaminohexaacetic acid (TTHA), N-hydroxyethylaminodiacetic acid (HEIDA), dihydroxyethylglycine (DHEG), ethylenediaminetetrapropionic acid (EDTP) and their derivatives.; The composition preferably included enzymes (generally from approximately 0.01% to approximately 0.03% by weight of active enzyme) selected from proteases, amylases, lipases, cellulases, choline oxidases, peroxidases / oxidases, pectate lyases, mannanases, cutinases, laccases, phospholipases, lysophospholipases, acyltransferases, perhydrolases, arylesterases, and any mixture thereof. The composition may include an enzyme stabilizer (examples of which include polyols such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, a reversible protease inhibitor, boric acid or a boric acid derivative, for example, an aromatic borate ester or a phenylboronic acid derivative such as 4-formylphenylboronic acid). The composition optionally includes silicone- or fatty acid-based foam suppressants; coloring dyes, calcium and magnesium cations, visual signaling ingredients, antifoaming agents (from 0.001% by weight to approximately 4.0% by weight) and / or structuring / thickening agents (from 0.01% by weight to 5% by weight) selected from the group consisting of diglycerides, triglycerides, ethylene glycol distearate, microcrystalline cellulose, cellulose-based materials, microfiber cellulose, biopolymers, xanthan gum, gellan gum and mixtures thereof. The composition can be in any liquid form, for example, a liquid or gel form, or any combination of these. The composition can be in any single-dose form, for example, a sachet. Detergent composition for industrial dry / solid laundry (HDD) Illustrative HDD laundry detergent compositions include a detergent surfactant, which includes anionic detergent surfactants (e.g., alkyl sulfates, alkylsulfonates, alkoxylated alkyl sulfate, alkyl phosphates, alkyl phosphonates, alkyl carboxylates and / or mixtures thereof substituted or unsubstituted, linear, branched or random chain), non-ionic detergent surfactant (e.g., substituted or unsubstituted C8C18 alkyl ethoxylates and / or C6-C12 alkyl phenolalkoxylates, linear, branched or random chain), cationic detergent surfactants (e.g., alkylpyridinium compounds, alkyl quaternary ammonium compounds, alkyl quaternary phosphonium compounds, tertiary sulfonium compounds) alkyls and mixtures thereof), zwitterionic and / or amphoteric detergent surfactants,nonionic semipolar surfactants and mixtures thereof; improvers including phosphate-free improvers (e.g., examples of zeolite-type improvers including zeolite A, zeolite X, zeolite P and zeolite MAP in the range of 0 wt% to less than 10 wt%), phosphate-type improvers (e.g., sodium tripolyphosphate in the range of 0 wt% to less than 10 wt%), citric acid, citrate salts and nitrilotriacetic acid, silicate salt (e.g., sodium or potassium silicate or sodium metasilicate in the range of 0 wt% to less than 10 wt%, or stratified silicate (SDS-6)); carbonate salt (e.g., sodium carbonate and / or sodium bicarbonate in the range of 0 wt% to less than 80 wt%); and bleaching agents including photobleaching agents (e.g., sulfonated zinc phthalocyanines, sulfonated aluminum phthalocyanines,xanthene-type dyes and mixtures thereof) nyooi η / ι ζηζ / α / γ hydrophobic or hydrophilic bleaching activators (e.g., dodecanoyloxybenzenesulfonate, decanoyloxybenzenesulfonate, decanoyloxybenzoic acid or salts thereof, 3,5,5-trimethylhexanoyloxybenzenesulfonate, tetraacetylethylenediamine-TAED, nonanoyloxybenzenesulfonate-NOBS, nitrile quaternary ammonium cations and mixtures thereof), sources of hydrogen peroxide (e.g., inorganic perhydrate salts, examples of which include sodium salt of perborate, percarbonate, persulfate, perphosphate or persilicate mono- or tetrahydrate), preformed hydrophilic and / or hydrophobic peracids (e.g., percarboxylic acids and salts, acids percarbonates and salts, and mixtures thereof), and / or bleaching catalysts (for example, imine-type bleaching enhancers (examples of which include iminium cations and polyions), iminium zwitterions, modified amines, modified amine oxides, N-sulfonylimines,N-phosphonilimines, N-acylimines, thiadiazole dioxides, perfluoroimines, cyclic sugar ketones and mixtures thereof, and bleaching catalysts containing metals (for example, copper, iron, titanium, ruthenium, tungsten, molybdenum or manganese cations together with an auxiliary metal cation such as zinc or aluminum and a sequestering agent such as ethylenediaminetetraacetic acid, ethylenediaminetetra(methylenephosphonic acid) and water-soluble salts thereof). n?QQi η / ι ζηζ / α / γ The composition includes enzymes, for example, proteases, amylases, lipases, cellulases, choline oxidases, peroxidases / oxidases, pectate lyases, mannanases, cutinases, laccases, phospholipases, lysophospholipases, acyltransferase, perhydrolase, arylesterase and any mixture of these. The composition may optionally include additional detergent ingredients including perfume microcapsules, starch-encapsulated perfume note, coloring agents, additional polymers including fabric integrity and cationic polymers, dye ingredients, fabric softening agents, brighteners (e.g., fluorescent brighteners CI), flocculants, chelating agents, alkoxylated polyamines, fabric deposition aids and / or cyclodextrin. Detergent composition for automatic dishwashers (ADW) The illustrative detergent composition for ADW includes non-ionic surfactants, including ethoxylated non-ionic surfactants, alkoxylated alcoholic surfactants, epoxy-capped poly(oxyalkylated) alcohols, or amine oxide surfactants present in amounts of 0 to 10% by weight; Detergency enhancers, in the range of 560%, including phosphate detergency enhancers (e.g., mono-phosphates, di-phosphates, tri-polyphosphates, other oligomeric polyphosphates, sodium tripolyphosphate STPP), and phosphate-free detergency enhancers (e.g., amino acid-based compounds including methylglycinodiacetic acid (MGDA) and salts and derivatives thereof, glutamic-N,N-diacetic acid (GLDA) and salts and derivatives thereof, iminodisuccinic acid (IDS) and salts and derivatives thereof, carboxymethylinulin and salts and derivatives thereof, nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA),β-alaninediacetic acid (β-ADA) and its salts), homopolymers and copolymers of polycarboxylic acids and their partially or completely neutralized salts, monomeric polycarboxylic acids and hydroxycarboxylic acids and salts thereof in the range of approximately 0.5% to approximately 50% by weight, or sulfonated / carboxylated polymers in the range of approximately 0.1% to approximately 50% by weight to provide dimensional stability; drying aids in the range of approximately 0.1% to approximately 10% by weight (e.g., polyesters, especially anionic polyesters, optionally together with additional monomers having 3 to 6 functionalities, typically acidic, alcoholic, or spherical functionalities leading to polycondensation, polycarbonate-, polyurethane- and / or polyurea-polyorganosiloxane compounds or precursor compounds thereof,particularly of the reactive cyclic carbonate and urea type); silicates in the range of approximately 1% to approximately 20% by weight (including sodium or potassium silicates, e.g., sodium disilicate, sodium metasilicate, and crystalline phyllosilicates); inorganic bleach (e.g., perhydrate salts such as perborate, percarbonate, perphosphate, persulfate, and persilicate salts) and organic bleach (e.g., organic peroxyacids including diacyl- and tetraacylperoxides, especially diperoxidodecanedioic acid, diperoxytetradecanedioic acid, and diperoxyhexadecanedioic acid); bleaching activators (i.e., organic peracid precursors in the range of approximately 0.1% to approximately 10% by weight); bleaching catalysts (e.g., manganese triazacyclononane and related complexes; Co, Cu,Mn and Fe bispyridylamine and related complexes; and cobalt(III) pentamine acetate and related complexes; metal care agents in the range of approximately 0.1% to approximately 5% by weight (e.g., benzatriazoles, metal salts and complexes and / or silicates); enzymes in the range of approximately 0.01 to 5.0 mg of active enzyme per gram of automatic dishwasher detergent composition and enzyme stabilizing components (e.g., oligosaccharides, polysaccharides and inorganic divalent metal salts). Additional enzymes Any of the cleaning compositions described herein may include any number of additional enzymes. In general, the enzyme(s) should be compatible with the selected detergent (e.g., with respect to optimum pH, compatibility with other enzymatic and non-enzymatic ingredients, and the like), and the enzyme(s) should be present in effective quantities. The following enzymes are provided as examples. Suitable proteases include those of animal, plant, or microbial origin. This includes chemically modified or genetically engineered mutant proteins, as well as naturally occurring processed proteins. The protease may be a serine protease or a metalloprotease, an alkaline microbial protease, a trypsin-like protease, or a chymotrypsin-like protease. Examples of alkaline proteases are the subtilisins, especially those derived from Bacillus, for example, subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147, and subtilisin 168 (refer, for example, to WO 89 / 06279).Illustrative proteases include, but are not limited to, those described in documents WO95 / 23221, WO 92 / 21760, W02008010925, WO20100566356, WO2011072099, WO201113022, WO2011140364, WO 2012151534, WO2015038792, W02015089441, WO2015089447, WO2015143360, WO2016001449, WO2016001450, WO2016061438, WO2016069544, WO2016069548, W02016069552, WO 2016069557, WO2016069563, WO2016069569, WO2016087617, WO2016087619, WO2016145428, WO2016174234, WO2016183509, WO2016202835, njQQi π / i ζηζ / α / γ. WO2016205755, US 2008 / 0090747, US 5,801,039, US 5,340,735, US 5, 500, 364, US 5,855, 625, RE 34, 606, US 5, 955,340, US 5, 700, 676, US 6,312,936, US 6,482,628, US8530219, US Provisional Sol. #sPCT / CN2 017 / 07 67 4 9 and, in addition, the metalloproteases described in documents WO 2007 / 044993, WO 2009 / 058303, WO 2009 / 058661, WO 2014 / 071410, WO 2014 / 194032, WO 2014 / 194034, WO 2014 / 194054 and WO 2014 / 194117. Illustrative commercial proteases include, but are not limited to, MAXATASE, MAXACAL, MAXAPEM, OPTICLEAN®, OPTIMASE®, PROPERASE®, PURAFECT®, PURAFECT® OXP, PURAMAX®, EXCELLASE®, PREFERENZ™ (e.g., P100, P110, P280), EFFECTENZ™ (e.g., P1000, P1050, P2000), EXCELLENZ™ (e.g., P1000), ULTIMASE® and PURAFAST (Danisco USA); ALCALASE®, ALCALASE® ULTRA, BLAZE®, BLAZE® EVITY®, BLAZE® EVITY® 16L, CORONASE®, SAVINASE®, SAVINASE® ULTRA, SAVINASE® EVITY®, SAVINASE® EVERIS®, PRIMASE, DURAZYM, POLARZYME®, OVOZYME®, KANNASE®, LIQUANASE®, EVERIS®, NEUTRASE®, PROGRESS UNO®, RELASE® and ESPERASE® (Novozymes); BLAP™ and BLAP™ variants (Henkel); LAVERGY™ PRO 104 L (BASF) and KAP® (B. alkalophilus subtilisin) (Kao). Suitable proteases include natural proteases or genetically engineered variants selected or modified specifically to function at relatively low temperatures. Suitable lipases include those of bacterial or fungal origin. Chemically modified, proteolytically modified, or protein-engineered mutants are included. Some examples of useful lipases include, but are not limited to, lipases from Humicola (a synonym for Thermomyces), for example, from H. lanugínosa (T. lanugínosus) (refer, for example, to documents EP 258068 and EP 305216), from H. insolens (refer, for example, to document WO 96 / 13580); a Pseudomonas lipase (for example, from P. alcaligenes or P. pseudoalcaligenes; refer, for example, to EP 218 272), P. cepacia (refer, for example, to EP 331 376), P. stutzerl (refer, for example, to GB 1 372 034), P. fluorescens, Pseudomonas sp. strain SD 705 (refer, for example, to WO 95 / 06720 and WO 96 / 27002), P. wisconsinensis (refer, for example, to WO 96 / 12012); a Bacillus lipase (for example, from B.subtills; refer, for example, to Dartois et al. (1993) Biochemíca et Biophysica Acta 1131:253-360), B. stearothermophílus (refer, for example, to document JP 64 / 744992), or B. pumilus (refer, for example, to document WO 91 / 16422). Additional lipase variants contemplated for use in formulations include those described, for example, in documents: WO 92 / 05249, WO 94 / 01541, WO 95 / 35381, WO 96 / 00292, WO 95 / 30744, WO 94 / 25578, WO 95 / 14783, nyooi η / ι ζηζ / α / γ. WO 95 / 22615, WO 97 / 04079, WO 97 / 07202, EP 407225 and EP 260105. Illustrative commercial lipases include, but are not limited to, MI LIPASE, LUMA FAST and LIPOMAX (Genencor); LIPEX®, LIPOCLEAN®, LIPOLASE® and LIPOLASE® ULTPA (Novozymes); and LIPASE P (Amano Pharmaceutical Co. Ltd). Polyesterases: Suitable polyesterases may be included in the composition, such as those described in, for example, documents WO 01 / 34899, WO 01 / 14629 and US6933140. The present compositions can be combined with other amylases, including other α-amylases. Such a combination is particularly desirable when different α-amylases exhibit different performance characteristics, and combining a plurality of different α-amylases results in a composition that provides the benefits of the different α-amylases. Other amylases include commercially available amylases such as, but not limited to, STAINZYME®, NATALASE®, DURAMYL®, TERMAMYL®, FUNGAMYL®, and BAN™ (Novo Nordisk A / S and Novozymes A / S); PAPIDASE®, POWERASE®, PURASTAR®, and PREFERENZ™ (DuPont Industrial Biosciences). Illustrative α-amylases are described in WO9418314A1, US20080293607, WO2013063460, WO10115028, WO2009061380A2, WO2014099523, WO2015077126A1, WO2013184577, WO2014164777, W09510603, WO9526397, WO9623874, WO9623873, WO9741213, WO9919467, WO0060060, WO0029560, WO9923211, WO9946399, nyooi η / ι ζηζ / α / γ WO0060058, WO0060059, WO9942567, WO0114532, WO02092797, WO0166712, WO0188107, WO0196537, WO0210355, WO2006002643, WO2004055178 and WO9813481. Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein-engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, and Acremonium; for example, fungal cellulases produced from Humicola insolens, Myceliophthora thermophila, and Fusarium oxysporum described, for example, in U.S. Patent Nos. 4,435,307; 5,648,263; 5,691,178; 5,776,757; and WO 89 / 09259. The illustrative cellulases contemplated for use are those that have a color-care benefit for the textile product. Some examples of cellulases are those described, for example, in documents EP 0495257, EP 0531372, WO 96 / 11262, WO 96 / 29397 and WO 98 / 08940. Other examples are cellulase variants such as those described in documents WO 94 / 07998; WO 98 / 12307; WO 95 / 24471; PCT / DK98 / 00299; EP 531315; U.S. Patent Nos. 5,457,046; 5,686,593 and 5,763,254. Illustrative cellulases include those described in documents WO2005054475, WO2005056787, US 7,449,318, US 7,833,773, US 4,435,307; EP 0495257 and US Provisional Solutions Nos. 62 / 296,678 and 62 / 435340. Illustrative commercial cellulases include, but are not limited to, CELLUCLEAN®, CELLUZYME®, CAREZYME®, CAREZYME® nyooi η / ι ζηζ / α / γ PREMIUM, ENDOLASE® and RENOZYME® (Novozymes); REVITALENZ®100, REVITALENZ® 200 / 220 and REVITALENZ® 2 00 0 (Danisco USA) and KAC500 (B) (Kao Corporation). Illustrative mananases include, but are not limited to, those of bacterial or fungal origin, such as those described in documents WO2016007929; USPN 6,566,114, 6,602,842 and 6,440,991; and U.S. provisional applications No. PCT / US2016 / 060850 and PCT / US2016 / 060844. Illustrative mananases include, but are not limited to, those of bacterial or fungal origin, such as those described in documents WO2016007929; USPN 6566114, 6,602,842 and 6,440,991; and U.S. provisional applications No. PCT / US2016 / 060850 and PCT / US2016 / 060844. Suitable peroxidases / oxidases considered for use in the compositions include those of plant, bacterial, or fungal origin. Chemically modified mutants or protein-engineered modifications are included. Examples of useful peroxidases include Coprinus peroxidases, for example, from C. cinereus, and variants thereof as described in WO 93 / 24618, WO 95 / 10602, and WO 98 / 15257. Commercially available peroxidases include, for example, GUARDZYME™ (Novo Nordisk A / S and Novozymes A / S). The detergent composition may also comprise 2,6β-D-fructan hydrolase, which is effective for the removal / cleaning of biofilms present in domestic and / or industrial textile / laundry products. Detergent enzymes can be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all such enzymes. A detergent additive, whether a separate or combined additive, can be formulated, for example, as granules, a liquid, a dense suspension, and the like. Illustrative detergent additive formulations include, but are not limited to, granules, particularly non-dusting granules, liquids, particularly liquids, or stabilized dense suspensions. Detergent products can be found in any convenient form, such as a bar, tablet, powder, granule, paste, or liquid. Liquid detergents can be aqueous, typically containing up to approximately 70% water and from 0% to approximately 30% organic solvent. Compact detergent gels containing approximately 30% or less water are also available. Numerous illustrative detergent formulations to which the present amylases may be added (or in some cases are identified as a component thereof) are described in WO2013063460. These include marketed single-dose detergent formulations / packs such as PUREX® UltraPacks (Henkel), FINISH® Quantum (Reckitt Benckiser), CLOROX™ 2 Packs (Clorox), OxiClean Max Force Power Paks (Church & Dwight), TIDE® Stain Release, CASCADE® ActionPacs and TIDE® Pods (Procter & Gamble), PS. Methods for evaluating amylase activity in detergent compositions Numerous α-amylase cleanup assays are known in the art, including sample and microsample assays. The accompanying examples describe only some of these assays. To further illustrate the compositions and methods, and their advantages, the following specific examples are provided, with the understanding that they are illustrative rather than limiting. All references cited herein are incorporated herein by reference in their entirety for all purposes. To further illustrate the compositions and methods, and their advantages, the following specific examples are provided, it being understood that they are illustrative rather than limiting. EXAMPLES Example 1: Methods Structural modeling of BspAmy24 A homology model of BspAmy24 was constructed as follows: η^ααι η / ι ζηζ / α / γ. The amino acid sequence of BspAmy24 (SEC. ID NO.: 1) was used as a query in MOE (Chemical Computing Group, Montreal, CA) to search the public structure database. The alpha-amylase from Bacillus licheniformas (1BLI) was the most successful public match. The homology model function was used with all default parameters to create a model. An X-ray diffraction crystal structure was also determined for a variant of BspAmy24; this experimental structure closely matched the homology model and supported the analysis performed using the homology model. Cell growth and protein quantification BspAmy24-Vl (which has a deletion of residues R181 and G182) and variants of BspAmy24-Vl were expressed in Bacillus licheniformis cells after following standard cloning procedures to introduce appropriate DNA sequences. The cells were cultured for 68 hours in an expression medium suitable for the expression of proteins secreted from B. licheniformis. The secreted protein was collected by centrifugation followed by filtration through 0.45 µm membranes (EMD Millipore). In some cases, further purification was performed using ion-exchange chromatography with Phenyl Sepharose 6 Fast Flow resin (GE Healthcare). Protein concentration was determined by high-performance liquid chromatography (HPLC) and absorbance at 280 nm. n?QQi η / ι ζηζ / α / γ Enzyme performance assay and measurement of pKa values The activity of α-amylases was measured at different pH values ​​using a microsample assay. In a 96-well microtiter plate containing two microsamples (CS28 starch dye on cotton, Test Materials Centre, Vlaardingen, The Netherlands), diluted enzyme was added to a buffer containing 2 mM CaCl₂ and 0.005% Tween-80, for a total reaction volume of 200 μA. The plate was incubated at 50 °C for 15 minutes with shaking at 1,150 rpm. The supernatant was then removed from the sample plate, and the amount of dye released into the supernatant was optically quantified. Dye release into the supernatant was correlated with enzyme activity.The rate constant was determined at each pH and the plot of the logarithm of the rate constant versus pH was fitted to a single or double ionization model, as follows, where kmax represents the maximum rate constant, pKa, pKai and pfa2 represent pKa values ​​for the enzyme, and pH represents the pH of the reaction:. / L· \ ,7, / ^max \ ^8^ = ^(171577^) Z, — 7 ( ^max i — íog ^QpKal-pH ^QpH-pKa2 J Example 2: Results obtained using variants of α-amylase pKameasured values ​​for four variants njQQi η / ι ζηζ / α / γ For four variants (i.e., T40N, F261Y, S286D, and Y362L), α-amylase activity was measured at various pH values ​​and used to determine apparent pKa values ​​from curve fittings. All variants additionally included the deletion of residues R181 and G182 (alternative deletion of the adjacent residues T183 and G184 would be expected to produce similar results), which is a standard mutation in α-amylases. The enzyme rate constant was determined at each pH value, and the plot of the logarithm of the rate constant versus pH was fitted to a single or double ionization model. The results are shown in Table 3 and Figures 4–7. Amino acid numbering is provided as deletion numbers, based on the amino acid sequence of BspAmy24-Vl, and non-deletion numbering, based on the amino acid sequence of BspAmy24. nyooi η / ι ζηζ / α / γ Table 3. pKa values ​​for four variants Mutations in the deletion numbering Mutations in the non-deletion numbering pKa value obtained from curve fitting - del-R181-G182 10.9 T40N T40N 10.3 F261Y F263Y 10.2 S286D S288D 10.8 Y362L Y364L 9.7 Reason for the activity for seven additional variants For seven additional variants, activity was measured at pH 8.5 and pH 10.5 and the ratio of activity at pH 8.5 to activity at pH 10.5 was determined according to the formula (TS in this context is the del-Rl81-G182 molecule): nyooi η / ι ζηζ / α / γ / pH 85 The data in Table 4 show that these additional mutations have increased activity ratios compared to the wild-type enzyme. Table 4. Reasons for the activity for seven additional variants Mutations in deletion numbering Mutations in non-deletion numbering Activity ratio None del-R181-G182 1.0 T40C T40C 1.2 T40D T40D 4.9 T40E T40E 2.0 F261P F263P 1.4 S286K S288K 1.1 Y362E Y364E 1.4 Y362M Y364M 5.8 It is hereby stated that, as of this date, the best method known to the applicant for putting the aforementioned invention into practice is the one that is clear from the present description of the invention.

Claims

1. A recombinant variant of a parental Family 13 α-amylase, characterized in that it has at least 60% amino acid sequence identity with the amino acid sequence of SEC. ID NO.: 1 and having an amino acid mutation in an amino acid residue covering the N-terminal side of the β-barrel structure of the α-amylase core, resulting in the variant having an amino acid residue that is different from the amino acid that occurs naturally in the original α-amylase, and resulting in a reduction of the apparent pKa value of the overall acid and an increase in the activity of the variant at a pH between approximately 8.5 and 10.

5.

2. The variant α-amylase according to claim 1, characterized in that the ratio of activity at pH 8.5 to activity at pH 10.5 for the variant divided by the ratio of activity at pH 8.5 to activity at pH 10.5 of an otherwise identical α-amylase lacking the amino acid mutation in an amino acid residue covering the N-terminal side of the β-barrel structure of the α-amylase core is greater than 1, greater than 2, greater than 3, or greater than 4.

3. The variant α-amylase according to claim 1 or 2, characterized in that it comprises an amino acid substitution at a selected position of the group consisting of T40, F263, S288 and Y364 corresponding to the amino acid sequence of SEC. ID NO.:

1.

4. The variant α-amylase according to claim 3, characterized in that it comprises an amino acid substitution selected from the group consisting of T40C, T40D, T40E, T40N, F263P, F263Y, S288D, S288K, Y364E, Y364L and Y364M corresponding to the amino acid sequence of SEC. ID NO.:

1.

5. The variant α-amylase according to any of claims 1-4, characterized in that it further comprises: (i) a deletion or substitution of one or more residues corresponding to positions 181, 182, 183 and / or 184 in the amino acid sequence of SEC. ID NO.: 1; (ii) a deletion of residues 181 and 182 or 183 and 184 corresponding to positions 181, 182, 183 and / or 184 in the amino acid sequence of SEC. ID NO.: 1; (iii) any single, multiple or combinatorial mutation previously described in an α-amylase of Family 13; and / or (iv) an N-terminal and / or C-terminal truncation.

6. The variant α-amylase according to any of claims 1-5, characterized in that it has at least 70%, at least 80%, at least 90%, or at least 95% amino acid sequence identity with the amino acid sequence of SEC. ID NO.: 1; encoded by a polynucleotide having at least 60%, at least 70%, at least 80%, or at least 90% nucleic acid sequence identity with a polynucleotide encoding SEC. ID NO.: 1; and / or encoded by a polynucleotide that hybridizes under strict conditions with a polynucleotide encoding SEC. ID NO.: 1, or its complement.

7. A method for modulating the activity of an α-amylase, characterized in that it comprises introducing into a parental Family 13 α-amylase having at least 60% amino acid sequence identity with the amino acid sequence of SEC. ID NO.: 1 a mutation of a residue that affects the electrostatic environment of the general acid positioned for catalysis, wherein the mutation is located on the N-terminal side of the β-barrel of the α-amylase, and wherein the mutation changes the electrostatic environment of the general acid in the resulting variant α-amylase.

8. The method according to claim 7, characterized in that the ratio of the activity of the variant α-amylase at pH 8.5 to the activity at pH 10.5 divided by the ratio of the activity at pH 8.5 to the activity at pH 10.5 of an otherwise identical α-amylase lacking the amino acid mutation at an amino acid residue covering the N-terminal side of the β-barrel structure of the α-amylase core is greater than 1, greater than 2, greater than 3, or greater than 4.

9. The method according to claim 7 or 8, characterized in that the mutation is a substitution at a selected position of the group consisting of T40, F263, S288 and Y364 corresponding to the amino acid sequence of SEC. ID NO.:

1.

10. The method according to claim 9, characterized in that the substitution is selected from the group consisting of T40C, T40D, T40E, T40N, F263P, F263Y, S288D, S288K, Y364E, Y364L and Y364M corresponding to the amino acid sequence of SEC. ID NO.:

1.

11. The method according to any of claims 7-10, characterized in that the variant α-amylase further comprises: (i) a deletion or substitution of one or more residues corresponding to positions 181, 182, 183 and / or 184 in the amino acid sequence of SEC. ID NO.: 1; (ii) a deletion of residues 181 and 182 or 183 and 184 corresponding to positions 181, 182, 183 and / or 184 in the amino acid sequence of SEC. ID NO.: 1; (iii) any single, multiple or combinatorial mutation previously described in an α-amylase of Family 13; and / or (iv) an N-terminal and / or C-terminal truncation.

12. A starch liquefying composition, characterized in that it comprises the α-amylase variant according to any of claims 1-6.

13. A detergent composition, characterized in that it comprises the amylase variant according to any of claims 1-6.

14. A method for converting starch into oligosaccharides, characterized in that it comprises contacting the starch with an effective amount of the amylase variant according to any of claims 1-6.

15. A method for removing a starchy stain or soil from a surface, characterized in that it comprises contacting the surface with an effective amount of the amylase variant according to any of claims 1-6, and allowing the polypeptide to hydrolyze the starch components present in the starchy stain to produce smaller starch-derived molecules that dissolve in the aqueous composition, thereby removing the starchy stain from the surface.