Maltopentaose / maltohexaose forming alpha-amylase variants

Abstract

Compositions and methods related to maltopentaose / maltohexaose forming α-amylases are disclosed. The α-amylase variants are useful, for example, for starch liquefaction and saccharification, for cleaning starchy stains in laundry, dishwashing, and other applications, for fabric treatment (e.g., desizing), for improving digestibility in animal feed, and for baking and brewing.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application No. 63 / 290,085, filed December 16, 2021, which is incorporated by reference in its entirety herein.

[0002] Disclosed are compositions and methods related to maltopentaose / maltohexaose forming α-amylase variants that are useful, for example, for cleaning starchy stains, liquefying and saccharifying starch, desizing fabrics, baking and brewing. [Background technology]

[0003] Starch consists of a mixture of amylose (15-30 w / w%) and amylopectin (70-85 w / w%). Amylose consists of linear chains of α-1,4-linked glucose units with a molecular weight (MW) of about 60,000 to about 800,000. Amylopectin is a branched polymer containing α-1,6 branch points every 24-30 glucose units and its MW can reach 100 million.

[0004] Alpha-amylases hydrolyze starch, glycogen, and related polysaccharides by randomly cleaving internal alpha-1,4-glucosidic bonds. Alpha-amylases, particularly from Bacilli, have been used for a variety of different purposes, including starch liquefaction and saccharification, textile desizing, starch modification in the pulp and paper industry, brewing, baking, and production of syrups for the food industry, production of feedstock for fermentation processes, and animal feed to increase digestibility. These enzymes can also be used to remove starchy soils and stains during dishwashing and laundry washing.

[0005] The products produced by starch hydrolysis by α-amylases differ in terms of the number of consecutive glucose molecules. Most commercially available α-amylases produce products ranging from glucose (G1) to maltoheptaose (G7). For reasons that are not entirely clear, α-amylases that produce significant amounts of maltopentaose and maltohexaose appear to be particularly useful for certain commercial applications, including incorporation into detergent cleaning compositions. Numerous publications have described variants of maltopentaose / maltohexaose-producing α-amylases and others. Nevertheless, there continues to be a need for ever more robust and higher-performing engineered α-amylase molecules. Summary of the Invention [Means for solving the problem]

[0006] The present compositions and methods relate to maltopentaose / maltohexaose forming amylase polypeptide variants and methods of their use. Aspects and embodiments of the compositions and methods of the invention are summarized in the individually numbered paragraphs below. 1. In one aspect, a recombinant, non-naturally occurring variant of a parent alpha-amylase is provided, which alpha-amylase variant has 80% identity to SEQ ID NO:5 and has amino acid substitutions at positions 51 and 125 relative to SEQ ID NO:5. 2. In some embodiments of the alpha-amylase variant of paragraph 1, the amino acid substitutions are T51V and S125R with respect to SEQ ID NO:5. 3. In some embodiments, the alpha-amylase variant of paragraph 1 or paragraph 2 further comprises an amino acid substitution at position 172, 227, or 231 relative to SEQ ID NO:5. 4. In some embodiments, the alpha-amylase variant of paragraph 1 or paragraph 2 further has an amino acid substitution N172Q, N227R, or F231L with respect to SEQ ID NO:5. 5. In another aspect, a recombinant, non-naturally occurring variant of a parent alpha-amylase is provided, the alpha-amylase variant having 80% identity to SEQ ID NO:5 and amino acid substitutions with respect to SEQ ID NO:5: (a)N29Q+T51V+S125R+N227R+S253L+G272E+K319R+S418A (b) T51V+S125R+F231L; (c) T51V+S125R+N172Q+N227R; (d) N029Q+T051V+T244I+S253L+K268R+K319R+S418A; or (e) E415G has. 6. In another aspect, there is provided a detergent composition comprising an α-amylase variant according to any one of claims 1 to 5. 7. Some embodiments of the detergent composition of paragraph 6 further comprise a subtilisin protease variant from Bacillus gibsonii having amino acid substitutions X39E, X99R, X126A, X127E, and X128G. 8. In another aspect, there is provided a method for converting starch to oligosaccharides, the method comprising contacting starch with an effective amount of an α-amylase variant of any one of paragraphs 1 to 5. 9. In another aspect, there is provided a method for removing starchy stains or soils from a surface comprising contacting the surface with an effective amount of an α-amylase variant of any one of clauses 1-5 and allowing the polypeptide to hydrolyze starch components present in the starchy stain to produce smaller starch derived molecules that are soluble in an aqueous composition, thereby removing the starchy stain from the surface. 10. In another aspect, there is provided a nucleic acid encoding an α-amylase variant according to any one of clauses 1 to 5. 11. In another aspect, there is provided a host cell comprising the nucleic acid of paragraph 10.

[0007] These and other aspects and embodiments of the present compositions and methods will become apparent from the following description and accompanying examples. [Brief description of the drawings]

[0008] [Figure 1] MUSCLE alignment of the amino acid sequences of AA2560 α-amylase (sequence number 1), AA707 α-amylase (sequence number 2), AA560 α-amylase (sequence number 3), AAI10 α-amylase (sequence number 4), and a variant of AA2560 α-amylase (sequence number 5) described in WO 2021 / 080948.

[0009] [Diagram 2] The positions of amino acids 51 and 125 in α-amylase AA2560 are indicated. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Compositions and methods related to variants of amylase enzymes that form maltopentaose / maltohexaose are described. The variants were discovered by various experimental approaches as detailed in the accompanying examples. Exemplary uses of the variants of amylase enzymes are for cleaning starchy stains in dishwashing, laundry, and other applications, for starch liquefaction and saccharification, for fabric treatment (e.g., desizing), for improving digestibility in animal feed, and for baking and brewing. These and other aspects of the compositions and methods are described in detail below.

[0011] Before describing the various aspects and embodiments of the compositions and methods of the present invention, the following definitions and abbreviations are provided.

[0012] 1. Definitions and Abbreviations In this detailed description, the following abbreviations and definitions apply. It should be noted that the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an enzyme" includes a plurality of such enzymes, reference to "the dosage" includes one or more dosages and equivalents thereof known to those skilled in the art, and so forth.

[0013] This specification is organized into sections for ease of reading. However, the reader will understand that statements made in one section may be applicable to other sections. Thus, the headings used in the different sections of this disclosure should not be construed as limiting.

[0014] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For clarity, the following terms are defined below.

[0015] 1.1. Abbreviations and Acronyms The following abbreviations / acronyms have the following meanings unless otherwise specified: ℃ Celsius temperature ADW Automatic Dishwashing dH2O or DI deionized water dIH2O Deionized water, Milli-Q filtered DNA deoxyribonucleic acid EC Enzyme Number g or gm grams GA glucoamylase H2O Water HDD Heavy Duty Powder Detergent HDL High Density Liquid Detergent hr(s) time HSG High foaming granular detergent kDa kilodalton kg Kilogram M Molar concentration mg milligram min(s) minutes mL and ml milliliters mm millimeters mM millimolar concentration MW molecular weight MWU Modified Wohlgemuth Unit; 1.6×10 -5 mg / MWU = activity units figure of merit ppm parts per million, e.g., μg protein per gram of dry solids sec second sp. species U Units v / v volume / volume w / v weight / volume w / w weight / weight wt% weight% μg microgram μL and μl microliter μm micrometer μM micromolar

[0016] 1.2.Definition The term "α-amylase" or "amylase enzyme" or amylase in general refers to an enzyme capable of catalyzing, inter alia, the degradation of starch. α-amylase is a hydrolase that cleaves α-D-(1→4) O-glycosidic bonds in starch. In general, α-amylase (EC 3.2.1.1; α-D-(1→4)-glucan glucanohydrolase) is defined as an endo-acting enzyme that randomly cleaves α-D-(1→4) O-glycosidic bonds in starch molecules to produce polysaccharides containing three or more (1-4)-α-linked D-glucose units. On the other hand, exo-acting amylolytic enzymes such as β-amylase (EC 3.2.1.2; α-D-(1→4)-glucan maltohydrolase) and some product-specific α-amylase-like maltogenic α-amylases (EC 3.2.1.133) cleave polysaccharide molecules from the non-reducing end of the substrate. β-amylase, α-glucosidase (EC 3.2.1.20; α-D-glucoside glucohydrolase), amylase glucoamylase (EC 3.2.1.3; α-D-(1→4)-glucan glucohydrolase), and product-specific amylases like maltotetraosidase (EC 3.2.1.60) and maltohexaosidase (EC 3.2.1.98) can produce malto-oligosaccharides of specific lengths or enriched syrups of specific malto-oligosaccharides. Some bacterial α-amylases produce primarily maltotetraose (G4), maltopentaose (G5), or maltohexaose (G6) from starch and related α-1,4-glucans, whereas most α-amylases further convert these to glucose and / or maltose as end products. TM G6 amylases (also called maltohexaose-producing α-amylases (EC 3.2.1.98)), such as the AA560 amylase derived from Bacillus sp. 707 amylase (parent), are technically exo-acting, but have a similar structure compared to the α-amylases and, in some cases, appear to respond to some of the same beneficial mutations.

[0017] "Enzyme units" herein refer to the amount of product formed per hour under the specified conditions of the assay. For example, a "glucoamylase activity unit" (GAU) is defined as the amount of enzyme that will produce 1 g of glucose per hour from a soluble starch substrate (4% DS) at 60°C and pH 4.2. A "soluble starch unit" (SSU) is the amount of enzyme that will produce 1 mg of glucose per minute from a soluble starch substrate (4% DS) at 50°C and pH 4.5. DS refers to "dry solids."

[0018] The term "starch" means a compound of the formula (CH 10 O5) x "starch" refers to any material composed of complex polysaccharide carbohydrates of plants, composed of amylose and amylopectin, having the formula: (wherein X can be any number). The term includes materials obtained from plant-based materials, such as cereals, cereals, grasses, tubers and roots, more specifically wheat, barley, corn, rye, rice, sorghum, bran, cassava, millet, milo, potato, sweet potato and tapioca. The term "starch" includes granular starch. The term "granular starch" refers to raw, i.e. uncooked starch, e.g., starch that has not been gelatinized.

[0019] As used herein, the terms "liquefaction" or "liquefy" refer to the process by which starch is converted into less viscous and shorter chain dextrins.

[0020] The terms "wild-type," "parent," or "reference" with respect to a polypeptide refer to a naturally occurring polypeptide that does not contain any artificial substitutions, insertions, or deletions at one or more amino acid positions. Similarly, the terms "wild-type," "parent," or "reference" with respect to a polynucleotide refer to a naturally occurring polynucleotide that does not contain any artificial nucleoside changes. However, it should be noted that a polynucleotide encoding a wild-type, parent, or reference polypeptide is not limited to naturally occurring polynucleotides, but encompasses any polynucleotide that encodes a wild-type, parent, or reference polypeptide.

[0021] Reference to a wild-type polypeptide is understood to include a mature form of the polypeptide. A "mature" polypeptide or variant thereof is one in which the signal sequence is not present, e.g., cleaved from the immature form of the polypeptide during or after expression of the polypeptide.

[0022] The term "variant" with respect to a polypeptide refers to a polypeptide that differs from a particular wild-type, parent, or reference polypeptide in that it contains one or more naturally occurring or artificial substitutions, insertions, or deletions of amino acids. Similarly, the term "variant" with respect to a polynucleotide refers to a polynucleotide that differs in nucleotide sequence from a particular wild-type, parent, or reference polynucleotide. The identity of the wild-type, parent, or reference polypeptide or polynucleotide will be clear from the context.

[0023] In the case of the present α-amylases, "activity" refers to α-amylase activity, which can be measured as described herein.

[0024] The term "performance benefit" refers to an improvement in a desirable property of a molecule. Exemplary performance benefits include, but are not limited to, increased hydrolysis of starch substrates, increased grain, cereal or other starch substrate liquefaction performance, increased cleaning performance, increased thermal stability, increased detergent stability, increased storage stability, increased solubility, altered pH profile, reduced calcium dependency, increased specific activity, altered substrate specificity, altered substrate binding, altered pH-dependent activity, altered pH-dependent stability, increased oxidative stability and increased expression. In some cases, the performance benefit is realized at a relatively low temperature. In some cases, the performance benefit is realized at a relatively high temperature.

[0025] The terms "protease" and "proteinase" refer to enzyme proteins that have the ability to carry out "proteolysis" or "proteolytic cleavage," which refers to the hydrolysis of the peptide bonds that link amino acids together within the peptide or polypeptide chains that form proteins. This activity of proteases as protein-digesting enzymes is referred to as "proteolytic activity."

[0026] The term "serine protease" refers to an enzyme that cleaves peptide bonds in proteins, in which serine functions as the nucleophilic amino acid at the enzyme active site. Serine proteases are classified into two broad categories based on their structure: chymotrypsin-like (trypsin-like) or subtilisin-like. The serine proteases, especially subtilisin-like, are the most commonly used in laundry and dishwashing detergents.

[0027] A "combination variant" is a variant that contains two or more mutations, for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or more substitutions, deletions and / or insertions.

[0028] The term "recombinant" when used with respect to a subject cell, nucleic acid, protein, or vector indicates that the subject has been modified from its native state. Thus, for example, a recombinant cell expresses a gene not found in the native (non-recombinant) form of the cell, or expresses a native gene at a level or under conditions different from that 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 is fused to a heterologous sequence. A vector containing a nucleic acid encoding an amylase is a recombinant vector.

[0029] The terms "recovered," "isolated," and "separated" refer to a compound, protein (polypeptide), cell, nucleic acid, amino acid, or other particular substance or component that is removed from at least one other substance or component with which it is naturally associated as found in nature. Such "isolated" polypeptides include, but are not limited to, cell culture broth containing secreted polypeptides expressed in a heterologous host cell.

[0030] The term "purified" refers to a material (e.g., an isolated polypeptide or polynucleotide) that is in a substantially pure state, e.g., at least about 90% pure, at least about 95% pure, at least about 98% pure, or even at least about 99% pure.

[0031] The term "enriched" refers to a material (eg, an isolated polypeptide or polynucleotide) that is about 50% pure, at least about 60% pure, at least about 70% pure, or even at least about 70% pure.

[0032] The terms "thermostable" and "thermostability" in reference to an enzyme refer to the ability of the enzyme to retain activity after exposure to high temperature. The thermostability of an enzyme, such as an amylase enzyme, can be measured by its half-life (t1 / 2), given in minutes, hours or days during which half of the enzyme activity is lost under defined conditions. The half-life can be calculated by measuring the residual α-amylase activity after exposure to high temperature (i.e., challenge with high temperature).

[0033] "pH range" in reference to an enzyme refers to the range of pH values ​​over which the enzyme exhibits catalytic activity.

[0034] The terms "pH stable" and "pH stability" in reference to an enzyme refer to the ability of the enzyme to retain activity over a wide pH range for a given period of time (eg, 15 minutes, 30 minutes, 1 hour).

[0035] The term "amino acid sequence" is synonymous with, and is used interchangeably with, the terms "polypeptide," "protein," and "peptide." When such amino acid sequences exhibit activity, they can be referred to as "enzymes." Conventional one-letter or three-letter codes for amino acid residues are used, and amino acid sequences are displayed in the standard amino to carboxy terminal direction (i.e., N→C).

[0036] The term "nucleic acid" includes DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids can be single- or double-stranded and can contain chemical modifications. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon can be used to code for a particular amino acid, and the compositions and methods of the invention encompass nucleotide sequences that code for a particular amino acid sequence. Unless otherwise specified, nucleic acid sequences are presented in a 5'-to-3' orientation.

[0037] A "synthetic" molecule is produced not by an organism, but by in vitro chemical or enzymatic synthesis.

[0038] The term "introduced" in the context of inserting a nucleic acid sequence into a cell means "gene transfer," "transformation," or "transduction," as known in the art.

[0039] A "host strain" or "host cell" is an organism into which an expression vector, phage, virus, or other DNA construct containing a polynucleotide encoding a polypeptide of interest (e.g., an amylase) has been introduced. Exemplary host strains are microbial cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing a polypeptide of interest and / or fermenting sugars. The term "host cell" includes protoplasts made from cells.

[0040] The term "heterologous" with reference to a polynucleotide or protein refers to a polynucleotide or protein that does not naturally occur in a host cell.

[0041] The term "endogenous" with reference to a polynucleotide or protein refers to a polynucleotide or protein that occurs naturally in a host cell.

[0042] The term "expression" refers to the process by which a polypeptide is produced based on a nucleic acid sequence, including both transcription and translation.

[0043] A "signal sequence" is a sequence of amino acids attached to the N-terminal portion of a protein that facilitates secretion of the protein outside the cell. The mature form of an extracellular protein lacks the signal sequence, which is cleaved off during the secretion process.

[0044] "Biologically active" refers to a sequence that has a particular biological activity, such as, for example, enzymatic activity.

[0045] The term "specific activity" refers to the number of moles of substrate that can be converted to product by an enzyme or enzyme preparation per unit time under specified conditions. Specific activity is generally expressed as units (U) / mg of protein.

[0046] As used herein, "water hardness" is a measure of the minerals (eg, calcium and magnesium) present in water.

[0047] "Cultured cellular material containing amylase" or similar terms refers to a cell lysate or supernatant (including culture medium) that contains amylase as a component. The cellular material may be from a heterologous host grown in culture for the purpose of producing amylase.

[0048] "Percent sequence identity" means that a particular sequence has at least a certain percentage of amino acid residues that are identical to the amino acid residues in a specified reference sequence when aligned using a software program such as the MUSCLE algorithm with default parameters. See, e.g., Edgar, RC (2004) Nucleic Acids Research 32:1792-97.

[0049] Deletions are counted as non-identical residues when compared to the reference sequence.

[0050] The term "dry solids content" (ds) refers to the total solids of the slurry on a dry weight percent basis. The term "slurry" refers to an aqueous mixture containing insoluble solids.

[0051] The phrase "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 in the same process step. SSF involves the simultaneous hydrolysis of a starch substrate (granular, liquefied, or solubilized) to sugars, including glucose, and the fermentation of the sugars to alcohol or other biochemicals or biological materials in the same reaction vessel.

[0052] "Ethanologenic microorganism" refers to a microorganism with the ability to convert sugars or oligosaccharides into ethanol.

[0053] The term "fermented beverage" refers to any beverage produced by a process that includes a fermentation process, such as microbial fermentation, e.g., bacterial and / or fungal fermentation.

[0054] The term "malt" refers to any malted grain, for example malted barley or malted wheat.

[0055] The term "mash" refers to an aqueous slurry of milling grains, including any starch- and / or sugar-containing vegetable matter, such as, for example, milled barley malt, milled barley, and / or other additives, or combinations thereof, admixed with water, which is subsequently separated into wort and spent grain.

[0056] The term "wort" refers to the unfermented liquor that flows out after the milling grains have been extracted during the mashing process.

[0057] The term "about" refers to ±15% relative to the referenced value.

[0058] 2. Maltopentaose / maltohexaose-forming α-amylase variants Described is a combination variant of a maltopentaose / maltohexaose forming α-amylase that exhibits advanced performance in automatic dishwashing (ADW) applications. This variant is referred to herein as AA2560 and is most closely related to the α-amylase from a species of Bacillus, previously recognized as BspAmy24 (SEQ ID NO: 1) in WO 2018 / 184004. The mature amino acid sequence of the AA2560 α-amylase is shown below as SEQ ID NO: 1: [ka]

[0059] A closely related maltopentaose / maltohexaose forming α-amylase is from Bacillus sp. 707, referred to herein as "AA707". The mature amino acid sequence of AA707α- is shown below as SEQ ID NO:2: [ka]

[0060] Another closely related maltopentaose / maltohexaose forming α-amylase is from a Bacillus species designated AA560. The mature amino acid sequence of AA560 is shown below as SEQ ID NO:3: [ka]

[0061] Based on amino acid sequence identity, another putative maltopentaose / maltohexaose forming α-amylase is from another Bacillus species and is referred to herein as AAI10. The mature amino acid sequence of the AAI10 α-amylase is shown below as SEQ ID NO:4: [ka]

[0062] A MUSCLE alignment of these four α-amylases is shown in Figure 1. The amino acid sequence identities are summarized in Table 1. AA707, AA560, and AAI10 all have over 80% amino acid identity to AA2560. [Table 1]

[0063] A variant of AA2560 α-amylase, which is described in WO 2021 / 080948 as exhibiting superior cleaning performance, is shown below as SEQ ID NO:5: [ka]

[0064] This variant has the mutations T40N, S91R, Y100F, W116R, Q172N, ΔR181, ΔG182, S244Q, and H281S relative to the AA2560 α-amylase, using the wild-type AA2560 α-amylase (SEQ ID NO: 1) for numbering.

[0065] Using the aforementioned AA2560 α-amylase variants as a starting point, further AA2560 α-amylase variants were designed that exhibited further improved cleaning performance. Most of the new variants contain two mutations, T51V and S125R. Mutations at these positions result in the loss of hydroxyl groups in the starch-binding groove of the molecule. In the structural model of the enzyme, the hydroxyl groups of T51 and S125 are solvent exposed and available for hydrogen bonding in the starch-binding groove (Figure 1).

[0066] Without being limited to any theory, we propose that the combination of the T51V and S125R mutations may work together to reduce non-productive bound starch in the active site by removing hydroxyl groups that may otherwise be exposed for hydrogen bonding in the starch binding groove. The loss of these hydroxyl groups may prevent the binding of starch in a structure that is incompatible with the optimal placement of the molecule and general acid / base side chains with respect to the nucleophile for catalysis. Based on this theory, other substitutions that remove the hydroxyl groups at these positions may also provide similar cleaning benefits, and therefore the substitutions can be more generally described as T51X and S125X (where X is neither S nor T).

[0067] Another feature of the variants of the invention continues to be a mutation at position 91 and / or at least one mutation at the bottom (base) of the α-amylase TIM barrel structure. The barrel bottom residues have a solvent accessible surface area greater than zero and are within or adjacent to the core β-barrel structure on the side of the barrel opposite the active site and on the side containing the N-terminus of each strand. The relevant residues are positions 6, 7, 40, 96, 98, 100, 229, 230, 231, 262, 263, 285, 286, 287, 288, 322, 323, 324, 325, 362, 363, and 364, with reference to SEQ ID NO: 1 for numbering. In all cases, these residues line the base of the TIM barrel structure, which represents a key architectural feature of α-amylases and many other enzymes. An exemplary mutation at residue 91 is the substitution of a polar residue with a charged residue, particularly a positively charged residue, such as arginine (ie, X91R), and in the case of AA2560, this is the particular substitution S91R.

[0068] Variants may further feature mutations within the loop, including surface-exposed residues 167, 169, 171, 172, and 176, referring to SEQ ID NO:1 for numbering. Variants may further feature mutations at positions 116 and 281, which are believed to affect solubility.

[0069] The variants may further be characterized by stabilizing mutations at positions 190 and / or 244, with reference to SEQ ID NO: 1 for numbering. Such mutations have been well characterized and are present in commercially available α-amylases used for both cleaning, grain processing, and fiber processing. Exemplary mutations at these residues are the substitutions X190P and X244A, E or Q, in particular E190P, S244A, S244E, and S244Q. Mutations at positions 275 and 279 in combination with a mutation at position 190 are also of interest.

[0070] The variants can further be characterized by mutations at positions 1, 7, 118, 195, 202, 206, 321, 245, and 459, referring to SEQ ID NO:1 for numbering, which are contained in current, commercially available α-amylases or proposed for such uses.

[0071] The variant further comprises a deletion within the X1G / S1X2G2 motif adjacent to the calcium binding loop corresponding to R181, G182, T183, and G184, using SEQ ID NO: 1 for numbering. In some embodiments, the variant comprises a deletion in the adjacent pair of amino acid residues corresponding to R181 and G182, or T183 and G184. The deletion within the amino acid residues corresponding to R181 and G182 can be referred to as "ΔRG", whereas the deletion within the residue at position 183 (usually T, D, or H) and G184 can be referred to as "ΔTG", "ΔDG", "ΔHG", etc., as appropriate. Both pairs of deletions appear to produce the same effect in α-amylase.

[0072] Variants may further include the mutations described above for use in other α-amylases that have a similar fold and / or have 60% or greater amino acid sequence identity with (i) any of the known Bacillus α-amylases, for example from B. lichenifomis (i.e., BLA and LAT), B. stearothermophilus (i.e., BSG), and B. amyloliquifaciens (i.e., P00692, BACAM, and BAA), or hybrids thereof; (ii) any α-amylase classified as a Carbohydrate Associated Enzyme Database (CAZy) Family 13 α-amylase; or (iii) any amylase referred to hereinafter by the descriptive term "Termamyl-like." Exemplary α-amylases include, but are not limited to, those from Bacillus sp. SG-1, Bacillus sp. 707, and the α-amylases designated A7-7, SP722, DSM90 14, and KSM AP1378. Similarly, any of the combinations of mutations described herein can provide performance benefits in these α-amylases, whether or not they are described as maltopentaose / maltohexaose producing α-amylases.

[0073] Particularly contemplated combination variants are listed below with reference to, and using SEQ ID NO:5 for numbering: Note that the variant of SEQ ID NO:5 already has the deletions ΔR181 and ΔG182, and thus each position after position 183 is reduced by two when referenced to SEQ ID NO:1. T51V+S125R+F231L T51V+S125R+N172Q+N227R

[0074] In related embodiments, particularly contemplated combination variants are listed below with reference to, and using, SEQ ID NO:5 for numbering. N29Q+T51V+S125R+N227R+S253L+G272E+K319R+S418A N29Q+T51V+T244I+S253L+K268R+K319R+S418A E415G

[0075] It will be understood that if an α-amylase naturally has a mutation listed above (i.e., if the wild-type α-amylase already contains the residue identified as being mutated), then that particular mutation does not apply to that α-amylase, but the other described mutations may function in combination with the naturally occurring residue at that position.

[0076] The α-amylase variants may also include substitutions, deletions, or additions of one or a few amino acids in the amino acid sequence, for example, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, or even less than 2 substitutions, deletions, or additions. Such variants are expected to have similar activity to the α-amylase from which they are derived. The α-amylase variants may also include small deletions and / or extensions of one or a few residues at their N- or C-terminus. Such small changes are unlikely to invalidate the inventive concept described herein.

[0077] The amylases of the invention can be "precursor," "immature," or "full-length" (in which case a signal sequence is included), or "mature" (in which case the signal sequence is lacking). The mature forms of the polypeptides are generally the most useful. Unless otherwise indicated, the numbering of amino acid residues used herein refers to the respective mature form of the amylase polypeptide.

[0078] In some embodiments, the α-amylase variant has at least 60%, 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 even at least 99%, but less than 100%, amino acid sequence identity with SEQ ID NO:1, 2, 3, 4, or 5.

[0079] 2.5. Nucleotides encoding amylase polypeptide variants In another aspect, nucleic acids encoding α-amylase polypeptide variants are provided, which can encode a particular amylase polypeptide or an α-amylase having a specified degree of amino acid sequence identity with the particular α-amylase.

[0080] In some embodiments, the nucleic acid encodes an α-amylase having at least 60%, 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 even at least 99%, but less than 100%, amino acid sequence identity with SEQ ID NO: 1, 2, 3, 4, or 5. It will be understood that due to the degeneracy of the genetic code, multiple nucleic acids may encode the same polypeptide.

[0081] In some embodiments, the nucleic acid hybridizes under stringent or highly stringent conditions to a nucleic acid encoding (or complementary to a nucleic acid encoding) an α-amylase having 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 even at least 99%, but less than 100%, amino acid sequence identity to SEQ ID NO:1, 2, 3, 4, or 5.

[0082] 3. Production of α-amylase variants The α-amylase variants of the invention can be produced in host cells, for example by secretion or intracellular expression, using methods well known in the art. Fermentation, separation, and concentration techniques are well known in the art, and concentrated α-amylase polypeptide variant-containing solutions can be prepared using conventional methods.

[0083] For production-scale recovery, the α-amylase polypeptide variants can be concentrated or partially purified as generally described above by removing cells via flocculation with a polymer. Alternatively, the enzymes can be concentrated or purified by microfiltration followed by concentration by ultrafiltration using available membranes and equipment. However, in some applications, the enzymes do not need to be concentrated or purified and the whole broth culture can be dissolved and used without further processing. The enzymes can then be processed, for example, into granules.

[0084] 4. Cleaning compositions containing α-amylase variants One aspect of the compositions and methods of the present invention includes cleaning compositions that include α-amylase variants as components, for example, for automatic and manual dishwashing (ADW), laundry cleaning, and other hard surface cleaning.

[0085] Overview Preferably, the α-amylase variants are incorporated into detergent formulations at or below concentrations conventionally used for known α-amylases. Because the described α-amylase variants are superior in performance to anything previously available, they are expected to perform better at standard dosages and similarly at lower dosages compared to existing α-amylases. Below, specific forms and formulations of detergent compositions for inclusion of the α-amylases of the present invention are described.

[0086] 4.2. Automatic Dishwashing (ADW) Detergent Compositions Exemplary ADW detergent compositions include nonionic surfactants, including ethoxylated nonionic surfactants, alcohol alkoxylated surfactants, epoxy-capped poly(oxyalkylated) alcohol, or amine oxide surfactants, present in amounts of 0-10% by weight; phosphate builders (e.g., monophosphates, diphosphates, tripolyphosphates, other oligomeric polyphosphates, sodium tripolyphosphate - STPP) and phosphate-free builders (e.g., methyl-glycine-diacetate (MGDA) in the range of 0.5% to 50% by weight). and their salts and derivatives, glutamic acid-N,N-diacetic acid (GLDA) and its salts and derivatives, iminodisuccinic acid (IDS) and its salts and derivatives, carboxymethyl inulin and its salts and derivatives, nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), β-alanine diacetic acid (β-ADA) and its salts, homopolymers and copolymers of poly-carboxylic acids and their partially or fully neutralized salts, monomeric polycarboxylic acids and hydroxycarboxylic acids and their salts). also in the range of 5-60% builders; in the range of about 0.1% to about 50% by weight of sulfonated / carboxylated polymers to provide dimensional stability; in the range of about 0.1% to about 10% by weight of drying aids (e.g. polyesters, especially anionic polyesters, polycarbonates-, polyurethanes- and / or polyureas-polyorganosiloxane compounds or their especially of the reactive cyclic carbonate and urea type, optionally together with further monomers having 3-6 functional groups - typically acid, alcohol or ester functional groups to promote polycondensation). precursor compounds), silicates (including sodium or potassium silicates, such as sodium disilicate, sodium metasilicate, and crystalline phyllosilicates) in the range of about 1% to about 20% by weight; inorganic bleaches (e.g., perhydrate salts, such as perborates, percarbonates, perphosphates, persulfates, and persilicates) and organic bleaches (e.g., organic peroxyacids, including diacyl and tetraacyl peroxides, particularly diperoxydodecanedioic acid, diperoxytetradecanedioic acid, and diperoxyhexadecanedioic acid); bleach activators (i.e., about 0.organic peracid precursors in the range of 1% to about 10% by weight; bleach catalysts (e.g., triazacyclononane manganese and related complexes, Co, Cu, Mn and Fe bispyridylamine and related complexes, and pentamine cobalt (III) acetate and related complexes); metal care agents (e.g., benzatriazoles, metal salts and complexes and / or silicates) in the range of about 0.1% to 5% by weight; enzymes (e.g., proteases, α-amylases, lipases, cellulases, choline oxidases, peroxidases / oxidases, pectate lyases, mannanases, cutinases, laccases, phospholipases, lysophospholipases, acyltransferases, perhydrolases, arylesterases and mixtures thereof) in the range of about 0.01 to 5.0 mg of active enzyme per gram of automatic dishwashing detergent composition; and enzyme stabilizer ingredients (e.g., oligosaccharides, polysaccharides and inorganic divalent metal salts).

[0087] Certain exemplary ADW compositions in which at least some of the present variants have been tested are shown in Table 2. [Table 2]

[0088] 4.3. Heavy Duty Liquid (HDL) Laundry Detergent Compositions Exemplary HDL laundry detergent compositions comprise detersive surfactants (10 wt / wt% to 40 wt / wt%), including anionic detersive surfactants (selected from the group of linear, branched or random chain, substituted or unsubstituted alkyl sulfates, alkyl sulfonates, alkoxylated alkyl sulfates, alkyl phosphates, alkyl phosphonates, alkyl carboxylates and / or mixtures thereof), and, optionally, nonionic surfactants (selected from the group of linear, branched or random chain, substituted or unsubstituted alkoxylated alkyl alcohols, such as C8-C18 ethoxylated alkyl alcohols and / or C6-C12 alkylphenol alkoxylates), wherein the weight ratio of anionic detersive surfactants (having a hydrophilicity index (HIc) of 6-9) to nonionic detersive surfactants is greater than 1:1. Suitable detersive surfactants also include cationic detersive surfactants (selected from the group of alkyl pyridinium compounds, alkyl quaternary ammonium compounds, alkyl quaternary phosphonium compounds, alkyl tertiary sulfonium compounds and / or mixtures thereof); zwitterionic and / or amphoteric detersive surfactants (selected from the group of alkanolamine sulfobetaines); amphoteric surfactants; semi-polar nonionic surfactants, and mixtures thereof.

[0089] The composition may optionally comprise a surfactant enhancing polymer consisting of amphiphilic alkoxylated grease cleaning polymers (selected from the group of branched alkoxylated polymers having hydrophilic and hydrophobic properties, such as alkoxylated polyalkyleneimines in the range of 0.05 wt% to 10 wt%) and / or random graft polymers (typically consisting of a hydrophilic backbone comprising monomers selected from the group consisting of unsaturated C1-C6 carboxylic acids, ethers, alcohols, aldehydes, ketones, esters, sugar units, alkoxy units, saturated polyalcohols such as maleic anhydride, glycerol, and mixtures thereof, and hydrophobic side chains selected from the group consisting of C4-C25 alkyl groups, polypropylene, polybutylene, vinyl esters of saturated C1-C6 mono-carboxylic acids, C1-C6 alkyl esters of acrylic or methacrylic acid, and mixtures thereof).

[0090] The composition may be, for example, a soil repellent polymer (non-ionic end-capped polyesters, e.g., SRP1, polymers comprising at least one monomer unit selected from sugars, dicarboxylic acids, polyols and combinations thereof in a random or block structure, ethylene terephthalate-based polymers and copolymers thereof in a random or block structure, e.g., Repel-o-tex SF, SF-2 and SRP6, Texcare SRA100, SRA300, SRN100, SRN170, SRN240, SRN300 and SRN325, Marloquest SL), anti-redeposition polymers (0.1 wt% to 10 wt%; including carboxylic acid 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 mixtures thereof, vinylpyrrolidone homopolymer, and / or polyethylene glycol, with molecular weights in the range of 500 to 100,000 Da); cellulose polymers (including polymers selected from alkyl celluloses, alkyl alkoxyalkyl celluloses, carboxyalkyl celluloses, alkyl carboxyalkyl celluloses (examples of which include carboxymethyl cellulose, methyl cellulose, methyl hydroxyethyl cellulose, methyl carboxymethyl cellulose, and mixtures thereof)), and carboxylate polymers (e.g., maleic acid / acrylic acid random copolymers or polyacrylate homopolymers).

[0091] The composition may further comprise saturated or unsaturated fatty acids, preferably saturated or unsaturated C12-C24 fatty acids (0 wt% to 10 wt%); deposition aids (examples of which include polysaccharides, preferably cellulose polymers, polydiallyldimethylammonium halides (DADMAC) and random or block structured copolymers of DAD MAC with vinylpyrrolidone, acrylamide, imidazole, imidazolinium halides and mixtures thereof, cationic guar gum, cationic celluloses such as cationic hydroxyethylcellulose, cationic starch, cationic polyacrylamide and mixtures thereof).

[0092] The compositions may contain dye transfer inhibitors (examples of which include manganese phthalocyanine, peroxidase, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidone and polyvinylimidazole 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), propylenediamine The present invention may further include an aqueous solution of glycine, such as glycine diacetate (MGDA), glutamic acid N,N-diacetate (N,N-dicarboxymethylglutamic acid tetrasodium salt (GLDA)), nitrilotriacetic acid (NTA), 4,5-dihydroxy-m-benzenedisulfonic acid, citric acid and any salts thereof, N-hydroxyethylethylenediaminetriacetic acid (HEDTA), triethylenetetraaminehexaacetic acid (TTHA), N-hydroxyethyliminodiacetic acid (HEIDA), dihydroxyethylglycine (DHEG), ethylenediaminetetrapropionic acid (EDTP) and derivatives thereof.

[0093] The composition preferably comprises an enzyme (generally about 0.01 wt% to 0.03 wt% active enzyme) selected from protease, α-amylase, lipase, cellulase, choline oxidase, peroxidase / oxidase, pectate lyase, mannanase, cutinase, laccase, phospholipase, lysophospholipase, acyltransferase, perhydrolase, arylesterase, and any mixture thereof. The composition may comprise an enzyme stabilizer (examples of which include polyols such as propylene glycol or glycerol, sugars or sugar alcohols, lactic acid, reversible protease inhibitors, boric acid or boric acid derivatives, such as aromatic boric acid esters or phenyl boric acid derivatives, such as 4-formylphenyl boric acid).

[0094] The composition can optionally include silicone-based or fatty acid-based suds suppressors; hueing dyes, calcium and magnesium cations, visual signal components, antifoam agents (0.001 wt % to about 4.0 wt %), and / or structurants / thickeners (0.01 wt % to about 5 wt %; selected from the group consisting of di- and triglycerides, ethylene glycol distearate, microcrystalline cellulose, cellulosic materials, microfiber cellulose, biopolymers, xanthan gum, gellan gum, and mixtures thereof).

[0095] The composition can be in any liquid form, such as a liquid or gel form or any combination thereof. The composition can be in any unit dose form, such as a pouch.

[0096] 4.4. Heavy Duty Dry / Solids (HDD) Laundry Detergent Compositions Exemplary HDD laundry detergent compositions include detersive surfactants including anionic detersive surfactants (e.g., linear or branched or random chain substituted or unsubstituted alkyl sulfates, alkyl sulfonates, alkyl alkoxylated sulfates, alkyl phosphates, alkyl phosphonates, alkyl carboxylates, and / or mixtures thereof), nonionic detersive surfactants (e.g., linear or branched or random chain substituted or unsubstituted C8-C18 alkyl ethoxylates and / or C6-C12 alkyl phenol alkoxylates), cationic detersive surfactants (e.g., alkyl pyridinium compounds, alkyl quaternary ammonium compounds, alkyl quaternary phosphonium compounds, alkyl tertiary sulfonium compounds, and mixtures thereof), zwitterionic and / or amphoteric detersive surfactants (e.g., alkanolamine sulfobetaines), amphoteric surfactants, semi-polar nonionic surfactants, and mixtures thereof); phosphate-free builders (e.g., examples of which include zeolite A, zeolite X, zeolite carboxylates, and mixtures thereof, in the range of 0 wt % to less than 10 wt %. builders including zeolite builders, including zeolite P and zeolite MAP; phosphate builders (e.g., sodium tripolyphosphate in the range of 0 wt% to less than 10 wt%); citric acid, citrates and nitrilotriacetic acid; silicates (e.g., sodium or potassium silicate or sodium metasilicate in the range of 0 wt% to less than 10 wt%, or layered silicate (SKS-6)); carbonates (e.g., sodium carbonate and / or sodium bicarbonate in the range of 0 wt% to less than 80 wt%); and photobleaching agents. bleaching agents (e.g., sulfonated zinc phthalocyanine, sulfonated aluminum phthalocyanine, xanthene dyes and mixtures thereof), hydrophobic or hydrophilic bleach activators (e.g., dodecanoyloxybenzenesulfonate, decanoyloxybenzenesulfonate, decanoyloxybenzoic acid or its salts, 3,5,5-trimethylhexanoyloxybenzenesulfonate, tetraacetylethylenediamine-TAED, nonanoyloxybenzenesulfonate-NOBS, nitrile quats and mixtures thereof);A source of hydrogen peroxide (e.g., inorganic perhydrate salts, examples of which include sodium salts monohydrate or tetrahydrate of perborates, percarbonates, persulfates, perphosphates, or persilicates), preformed hydrophilic and / or hydrophobic peracids (e.g., percarboxylic acids and salts, percarbonates and salts, perimidic acids and salts, peroxymonosulfates and salts, and mixtures thereof), and / or bleach catalysts (e.g., imine bleach boosters (examples of which include iminium cations and polyions), iminium zwitterions, modified amines ... bleaching agents including amine oxides, N-sulfonylimines, N-phosphonylimines, N-acylimines, thiadiazole dioxides, perfluoroimines, cyclic sugar ketones and mixtures thereof, and metal-containing bleaching catalysts (e.g., copper, iron, titanium, ruthenium, tungsten, molybdenum, or manganese cations, along with auxiliary metal cations such as zinc or aluminum, and sequestering agents such as ethylenediaminetetraacetic acid, ethylenediaminetetra(methylenephosphonic acid), and the water-soluble salts thereof);

[0097] The composition preferably comprises an enzyme such as a protease, α-amylase, lipase, cellulase, choline oxidase, peroxidase / oxidase, pectate lyase, mannanase, cutinase, laccase, phospholipase, lysophospholipase, acyltransferase, perhydrolase, arylesterase and mixtures thereof.

[0098] The compositions may optionally include additional detergent ingredients including perfume microcapsules, starch encapsulated perfume accords, hueing agents, additional polymers including fabric integrity and cationic polymers, dye lock components, fabric softeners, brighteners (e.g., CI optical brighteners), flocculants, chelating agents, alkoxylated polyamines, fabric deposition aids, and / or cyclodextrins.

[0099] 4.5. Additional Enzymes Any of the cleaning compositions described herein may include any number of additional enzymes. In general, the enzyme should be compatible with the selected detergent (e.g., in terms of pH optimum, compatibility with other enzyme and non-enzyme ingredients, etc.) and the enzyme should be present in an effective amount. Examples include the following enzymes:

[0100] Protease: Suitable proteases include proteases of animal, vegetable, or microbial origin. Naturally processed proteins as well as chemically modified or protein engineered variants are included. 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 subtilisins, particularly proteases from the genus Bacillus (e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147, Bacillus gibsonii, Bacillus spp. ... gibsonii), and subtilisin 168 (see, e.g., WO 1989 / 06279, WO 20200242858). Exemplary proteases include, but are not limited to, those described in WO 1995 / 23221, WO 1992 / 21760, WO 2008 / 010925, WO 2010 / 0566356, WO 2011 / 072099, WO 2011 / 13022, WO 2011 / 140364, WO 2012 / 151534, WO 2015 / 038792, WO 2015 / 089441, WO 201 WO 2015 / 143360, WO 2016 / 001449, WO 2016 / 001450, WO 2016 / 061438, WO 2016 / 069544, WO 2016 / 069548, WO 2016 / 069552 No. 2016 / 069557, No. 2016 / 069563, No. 2016 / 069569, No. 2016 / 087617, No. 2016 / 087619, No. 2016 / 145428, No. 2016 / 174234,WO 2016 / 183509, WO 2016 / 202835, WO 2016 / 205755, WO 2008 / 0090747, WO 2018 / 118950, WO 2018 / 169750, WO 2018 / 118917, U.S. Pat. No. 5,801,039, U.S. Pat. No. 5,340,735, U.S. Pat. No. 5,500,364, U.S. Pat. No. 5,855,625, RE 34606, U.S. Pat. No. 5,955,340, U.S. Pat. No. 5,700,676, U.S. Pat. Nos. 6,312,936, 6,482,628, and 8,530,219, as well as the metalloproteases described in 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.

[0101] Exemplary commercially available proteases include, but are not limited to, MAXATASE, MAXACAL, MAXAPEM, OPTICLEAN®, OPTIMASE®, PROPERASE®, PURAFECT®, PURAFECT® OXP, PURAMAX®, EXCELLASE®, PREFERENZ™ Proteases (e.g., P100, P110, P280, P300), EFFECTENZ™ Proteases (e.g., P1000, P1050, P2000), EXCELENZ™ Proteases (e.g., P1000), ULTIMASE®, and PURAFAST (Danisco US; 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 104L (BASF), and KAP® (B. alkalophilus subticillin) (Kao). Suitable proteases include naturally occurring proteases or engineered variants that have been specifically selected or genetically modified to function at significantly lower temperatures.

[0102] In certain embodiments of the compositions and methods of the invention, the described α-amylase variants have the amino acid substitutions X39E, X99R, X126A, X127E, and X128G, and are selected from the group consisting of N74D-M211L-N253P, R179Q-M211L-N253P, N74D-N253P, N85R-G160Q-R179Q-M211L-N212S-N253P, R179Q-N253P, G160Q-R179Q-M211L-N212S-N253P, R179Q-M211L, G160Q-R179Q-M211L-N253P, G160Q- R179Q-N212S-N253P, N74D-M211L, M211L-N242D, G160Q-R179Q-M211L-N21 2S, N74D-R179Q-M211L-N253P, G160Q-R179Q-M211L, G160Q-R179Q-N253P, N74D-Q200L-M211L, N74D-G160Q-N212S-N253P, N74D-G160Q-M211L-N253P , G160Q-R179Q, G160Q-R179Q-N212S, N74D-G160Q-N253P, N74D-G160Q-R179 Q-M211L-N212S-N253P, N74D-N085R-G160Q-R179Q-M211L, N74D-G160Q-M2 11L-N212S-N253P, N74D-N085R-N116R-Q200L-Q256E, N74D-G160Q-R179Q- N212S-N253P, N74D-G160Q-M211L-N212S, N74D-G160Q, N74D-G160Q-R179Q -M211L-N253P, N74D-R179Q-M211L, N74D-G160Q-N212S, N74D-G160Q-M211L , N74D-G160Q-R179Q-N253P, N74D, N74D-G160Q-R179Q-M211L-N212S, N74D -N085R-M211L-N212S, N74D-G160Q-R179Q-N212S, N74D-G160Q-R179Q-M21 1L, N74D-M211L-Q256E, N74D-G160Q-R179Q, R179Q-M211L-N212S-N253P, R 179Q-M211L-N212S, N74D-N085R-R179Q-M211L-N212S, N74D-M211L-N212S,N74D-R179Q-M211L-N212S, N74D-M211L-N242D, N74D-Q200L-M211L-Q256E, N74D-Q200L-M211L-N242D-Q256E, N74D-Q200L, N 74D-M211N-N212Q, N74D-M211N-N212Q-Q256E, N74D-M211N-Q256E, N74D-M211Q, N74D-M211Q-N212Q, N74D-M211Q-N212Q-Q25 6E, N74D-M211Q-Q256E, N74D-N198A-M211Q, N74D-N198A-M211Q-N212Q, N74D-N198A-M211Q-Q256E, N74D-N198G-M211Q, N74D -N198G-M211Q-N212Q, N74D-N198G-M211Q-Q256E, N74D-N198K-M211Q-N212Q, N74D-N198L-M211Q-N212Q, N74D-N198Q-M211Q -N212Q, N74D-N198R-M211Q-N212Q, N74D-N198T-M211Q-N212Q, N74D-N198V-M211Q-N212Q, N74D-N212Q-Q256E, N74D-Q256E, N74D-R207Q, N74D-R207Q-M211N, N74D-R207Q-M211N-N212Q, N74D-R207Q-M211N-N212Q-Q256E, N74D-R207Q-M211N-Q256E, N 74D-R207Q-M211Q, N74D-R207Q-M211Q-N212Q, N74D-R207Q-M211Q-N212Q-Q256E, N74D-R207Q-N212Q, N74D-R207Q-N212Q-Q256E, N74D-R207Q-Q256E, N74D-N198S-M211Q, and N74D-N198L-M211Q. wherein the amino acid positions are numbered according to their correspondence with the amino acid sequence of SEQ ID NO:5, and wherein the variant has at least 90% identity with the amino acid sequence of SEQ ID NO:6. These amino acid sequences are shown below.

[0103] Amino acid sequence of BG46 protease (SEQ ID NO:6): [ka]

[0104] Amino acid sequence of BG46 with substitutions S39E, S99R, S126A, D127E, and F128G (SEQ ID NO:7): [ka]

[0105] Lipase: Suitable lipases include lipases of bacterial or fungal origin, including chemically modified, proteolytically modified, or proteolytically engineered variants. Examples of useful lipases include, but are not limited to, lipases from the genus Humicola (syn. Thermomyces), such as from H. lanuginosa (T. lanuginosus) (see, e.g., EP 258068 and EP 305216), from H. insolens (see, e.g., WO 96 / 13580); Pseudomonas lipases (e.g., P. alcaligenes or P. pseudoalcaligenes (see, e.g., EP 218272); lipases from P. cepacia (see, e.g., EP 331376), P. stutzeri (see, e.g., GB 1,372,034), P. fluorescens, Pseudomonas species, strain SD705 (see, e.g., WO 95 / 06720 and WO 96 / 27002), P. wisconsinensis (see, e.g., WO 96 / 12012); Bacillus lipases (e.g., B. subtilis (e.g., Dartois et al. (1993) Biochemica et Biophysica Acta 1131:253-360), B. stearothermophilus (e.g., JP 64 / 744992), or B. pumilus (e.g., WO 91 / 16422).Additional lipase variants contemplated for use in the formulation include, for example, those described in WO 92 / 05249, WO 94 / 01541, WO 95 / 35381, WO 96 / 00292, WO 95 / 30744, WO 94 / 25578, WO 95 / 14783, WO 95 / 22615, WO 97 / 04079, WO 97 / 07202, EP 407225, and EP 260105.

[0106] Representative commercially available lipases include, but are not limited to, M1 lipase, LUMA FAST, and LIPOMAX (Genencor); LIPEX®, LIPOCLEAN®, LIPOLASE®, and LIPOLASE® ULTRA (Novozymes); and Lipase P (Amano Pharmaceutical Co. Ltd).

[0107] Polyesterase: The composition may include a suitable polyesterase, such as those described in WO 01 / 34899, WO 01 / 14629, and US Pat. No. 6,933,140.

[0108] amylase: The compositions can be combined with other amylases, including other α-amylases. Such combinations are particularly desirable when different α-amylases exhibit different performance characteristics, resulting in a composition that provides the benefits of different α-amylases. Other α-amylases include commercially available α-amylases such as STAINZYME®, NATALASE®, DURAMYL®, TERMAMYL®, FUNGAMYL®, and BAN™ (Novo Nordisk A / S and Novozymes A / S); RAPIDASE®, POWERASE®, PURASTAR®, and PREFERENZ™ (DuPont Industrial Biosciences.).Exemplary α-amylases are described in WO 94 / 18314 A1, WO 2008 / 0293607, WO 2013 / 063460, WO 10 / 115028, WO 2009 / 061380 A2, WO 2014 / 099523, WO 2015 / 077126 A1, and the like. Brochure, International Publication No. 2013 / 184577, Brochure, International Publication No. 2014 / 164777, Brochure, International Publication No. 95 / 10603, Brochure, International Publication No. 95 / 26397, Brochure, International Publication No. 96 / 23874, Brochure, International Publication No. 96 / 23873, Brochure, International Publication No. 97 / 41213, Brochure, International Publication No. 99 / 19467 and WO 00 / 60060, WO 00 / 29560, WO 99 / 23211, WO 99 / 46399, WO 00 / 60058, WO 00 / 60059, WO 99 / 42567, WO 01 / 14532, WO 02 / 092797, WO 01 / 66712, WO 01 / 88107, WO 01 / 96537, WO 02 / 10355, WO 2006 / 002643, WO 2004 / 055178, and WO 98 / 13481.

[0109] Cellulase: Cellulases can be added to the composition. Suitable cellulases include cellulases of bacterial or fungal origin, including chemically modified or protein modified variants. Suitable cellulases include, for example, cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, and Acremonium, such as fungal cellulases from Humicola insolens, Myceliophthora thermophila, and Fusarium oxysporum, as disclosed in U.S. Patent Nos. 4,435,307; 5,648,263; 5,691,178; 5,776,757; and WO 89 / 09259. Representative cellulases contemplated for use are cellulases that have color protection benefits for fabrics. Examples of such cellulases are, for example, those described in EP 0495257, EP 0531372, WO 96 / 11262, WO 96 / 29397, and WO 98 / 08940. Other examples are cellulase variants such as those described in WO 94 / 07998; WO 98 / 12307; WO 95 / 24471; PCT / DK98 / 00299; EP 531315; U.S. Pat. Nos. 5,457,046; 5,686,593; and 5,763,254. Representative cellulases include those described in WO2005054475, WO2005056787, U.S. Pat. No. 7,449,318, U.S. Pat. No. 7,833,773, U.S. Pat. No. 4,435,307; EP0495257; and U.S. Provisional Patent Applications Nos. 62 / 296,678 and 62 / 435340.Representative commercially available cellulases include, but are not limited to, CELLUCLEAN®, CELLUZYME®, CAREZYME®, CAREZYME® PREMIUM, ENDOLASE®, and RENOZYME® (Novozymes), REVITALENZ® 100, REVITALENZ® 200 / 220, and REVITALENZ® 2000 (Danisco US); and KAC-500(B) (Kao Corporation).

[0110] Mannanase: Exemplary mannanases include, but are not limited to, those of bacterial or fungal origin, such as those described in, for example, WO 2016007929; U.S. Patent Nos. 6,566,114, 6,602,842, and 6,440,991; and International Application Nos. PCT / US2016 / 060850 and PCT / US2016 / 060844. Exemplary mannanases include, but are not limited to, those of bacterial or fungal origin, such as those described in, for example, WO 2016007929; U.S. Patent Nos. 6,566,114, 6,602,842, and 6,440,991; and International Application Nos. PCT / US2016 / 060850 and PCT / US2016 / 060844.

[0111] Peroxidase / Oxidase: Suitable peroxidases / oxidases contemplated for use in the composition include those of plant, bacterial, or fungal origin. Chemically modified or protein modified variants are included. Examples of useful peroxidases include peroxidases from the genus Coprinus, for example C. cinereus, and variants thereof, such as those 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).

[0112] The detergent composition may also include 2,6-β-D-fructan hydrolase, which is effective for removing / cleaning biofilms present on household and / or industrial fabrics / laundry.

[0113] Detergent enzymes can be included in the detergent composition by adding a separate additive containing one or more enzymes, or by adding a combined additive containing all of the enzymes. The detergent additives, either separate or combined, can be prepared, for example, as granules, liquids, slurries, etc. Exemplary detergent additive preparations include, but are not limited to, granules, especially non-dusting granules, liquids, especially stabilized liquids or slurries.

[0114] Perhydrolase: Perhydrolases include, for example, those described in WO 2005 / 056782, WO 2007 / 106293, WO 2008 / 063400, WO 2008 / 106214, and WO 2008 / 106215.

[0115] Nuclease: Suitable nucleases include, but are not limited to, those described in WO 2015 / 181287, WO 2015 / 155350, WO 2016 / 162556, WO 2017 / 162836, WO 2017 / 060475 (e.g., SEQ ID NO: 21), WO 2018 / 184816, WO 2018 / 177936, WO 2018 / 177938, WO 2018 / 185269, WO 2018 / 185270, WO 2018 / 185271, WO 2018 / 185272, WO 2018 / 185273, WO 2018 / 185274, WO 2018 / 185275, WO 2018 / 185276, WO 2018 / 185277, WO 2018 / 185278, WO 2018 / 185279 ... Examples of the compounds include those described in International Publication No. WO 2018 / 185285, International Publication No. WO 2018 / 177203, International Publication No. WO 2018 / 184817, International Publication No. WO 2019 / 084349, International Publication No. WO 2019 / 084350, International Publication No. WO 2019 / 081721, International Publication No. WO 2018 / 076800, International Publication No. WO 2018 / 185267, International Publication No. WO 2018 / 185280, and International Publication No. WO 2018 / 206553.

[0116] Other nucleases that can be used in combination with the α-amylase variants of the present invention include those described in Nijland, R. et al. (2010) PLoS ONE 5-e15668 and Whitchurch, CB et al. (2002) Science 295:1487.

[0117] 4.6. Form of cleaning composition The detergent compositions may be in any convenient form, such as a bar, tablet, powder, granule, paste, or liquid. Liquid detergents may be aqueous, typically containing up to about 70% water and 0% to about 30% organic solvent. Compact detergent gels containing up to about 30% water are also contemplated. The α-amylase variants of the present invention are compatible with detergent compositions in known forms and formulations, the specific forms and formulations being described herein.

[0118] A number of exemplary detergent formulations to which the α-amylases herein can be added (or in some cases identified as being a component thereof) are described in WO2013063460. These include commercially available unit dose detergent formulations / packages such as PUREX® UltraPacks (Henkel), FINISH® Quantum (Reckitt Benckiser), CLOROX™ 2 Packs (Clorox), OxiClean Max Force Power Paks (Church&Dwight), TIDE® Stain Release, TIDE® Pods, CASCADE® Action Pacs, CASCADE® Platimun, and CASCADE® and Pure essential (Procter & Gamble).Unit dose formulations and packages may be prepared in any manner, including those described in, for example, U.S. Patent Publication No. 20090209445A1, U.S. Patent Publication No. 20100081598A1, U.S. Patent No. 7001878B2, EP 1504994B1, WO 2001085888A2, WO 2003089562A1, WO 2009098659A1, International Publication No. 2009098660A1, International Publication No. 2009112992A1, International Publication No. 2009124160A1, International Publication No. 2009152031A1, International Publication No. 2010059483A1, International Publication No. 2010088112A1, International Publication No. 2010090915A1, International Publication No. 201 International Publication No. 0135238A1, International Publication No. 2011094687A1, International Publication No. 2011094690A1, International Publication No. 2011127102A1, International Publication No. 2011163428A1, International Publication No. 2008000567A1, International Publication No. 2006045391A1, International Publication No. 2006007911 A1, WO 2012027404A1, EP1740690B1, WO 2012059336A1, US6730646B1, WO 2008087426A1, WO 2010116139A1, and WO 2012104613A1.

[0119] 5. Carbohydrate Processing Using α-Amylase Variants The present α-amylase variants may be useful in a variety of industrial carbohydrate processing applications. For example, the α-amylase variants may be useful in starch conversion processes, particularly in saccharification processes of starch undergoing liquefaction. The desired end product may be any product that can be produced by enzymatic conversion of a starch substrate. For example, the desired product may be a syrup rich in glucose and maltose that can be used in other processes, such as the preparation of HFCS, or can be converted into a number of useful products, such as ascorbic acid intermediates (e.g., gluconate; 2-keto-L-gulonic acid; 5-keto-gluconic acid; and 2,5-diketogluconate); 1,3-propanediol; aromatic amino acids (e.g., tyrosine, phenylalanine, and tryptophan); organic acids (e.g., lactate, pyruvate, succinate, isocitrate, and oxaloacetate); amino acids (e.g., serine and glycine); antibiotics; antimicrobials; enzymes; vitamins; and hormones.

[0120] The starch conversion process may be carried out prior to or simultaneously with a fermentation process designed to produce fuel alcohol or potable alcohol (i.e., drinkable alcohol). Those skilled in the art will recognize the various fermentation conditions that may be used in the production of these end products. The α-amylase variants are also useful in food preparation compositions and methods. These various uses of the α-amylase variants are described in more detail below.

[0121] 5.1. Preparation of starch substrate Methods for preparing starch substrates for use in the processes disclosed herein are well known. Useful starch substrates can be obtained from tubers, roots, stems, legumes, cereals, or whole grains. More specifically, granular starch can be obtained from corn, corncob, wheat, barley, rye, triticale, milo, sago, millet, cassava, tapioca, sorghum, rice, pea, bean, banana, or potato. Particularly contemplated starch substrates are corn starch and wheat starch. Starch from cereals can be milled or whole, including corn solids such as kernel, bran, and / or cob. Starch can also be highly refined raw starch or feedstock from starch refining processes.

[0122] 5.2. Starch Gelatinization and Liquefaction Gelatinization is generally performed simultaneously with or prior to contacting the starch substrate with α-amylase, although additional liquefaction-inducing enzymes may be optionally added. In some embodiments, the starch substrate prepared as described above is slurried with water. To optimize α-amylase stability and activity, the pH of the slurry is typically adjusted to about pH 4.5-6.5, and about 1 mM calcium (about 40 ppm free calcium ions) may also be added depending on the properties of the α-amylase variant used. Any α-amylase remaining in the slurry after liquefaction can be inactivated via several methods, including lowering the pH in a subsequent reaction step, or by removing calcium from the slurry if the enzyme is calcium-dependent. The starch + α-amylase slurry can be continuously pumped through a jet cooker that is steam heated to 105°C. The slurry is then cooled to room temperature.

[0123] 5.3. Saccharification The liquefied starch can be saccharified using α-amylase variants, optionally in the presence of other enzymes, to produce a syrup rich in lower DP (e.g. DP1+DP2) sugars. The exact composition of the product of saccharification depends on the enzyme combination used and the type of granular starch being processed.

[0124] Liquefaction is generally carried out as a continuous process, whereas saccharification is often carried out as a batch process. Saccharification is typically most effective at a temperature of about 60-65°C and a pH of about 4.0-4.5, e.g., pH 4.3, requiring cooling of the liquefied starch and pH adjustment. Saccharification is usually carried out in a stirred tank, which can take several hours to fill or empty. Enzymes are typically added at a fixed ratio to dry solids as the tank is filled, or as a single amount at the beginning of the filling stage. Saccharification reactions to produce syrups are typically carried out for about 24-72 hours, e.g., 24-48 hours. Once the maximum or desired DE is reached, the reaction is stopped, e.g., by heating to 85°C for 5 minutes. Further incubation will result in a lower DE (eventually up to about 90 DE) as accumulated glucose repolymerizes to isomaltose and / or other depolymerization products via enzymatic depolymerization reactions and / or by thermodynamic equilibrium approaches.

[0125] 5.4. Isomerization The soluble starch hydrolysate produced by treatment with α-amylase can be converted to high fructose starch-based syrup (HFSS), e.g., high fructose corn syrup (HFCS). This can be accomplished using glucose isomerase, particularly glucose isomerase immobilized on a solid support. The pH is raised to about 6.0 to about 8.0 (depending on the isomerase), e.g., pH 7.5, and Ca is added. 2+is removed by ion exchange. Suitable isomerases include SWEETZYME®, IT (Novozymes A / S); G-ZYME® IMGI, as well as G-ZYME® G993, KETOMAX®, G-ZYME® G993, G-ZYME® G993 Liquid, and GENSWEET® IGI. After isomerization, the mixture typically contains about 40-45% fructose, e.g., 42% fructose.

[0126] Fermentation Soluble starch hydrolysates, particularly high glucose syrups, can be fermented by contacting the starch hydrolysate with a fermenting organism, typically at a temperature of about 32° C., e.g., 30° C. to 35° C. for alcohol-producing yeasts. The temperature and pH of the fermentation will depend on the fermenting organism. EOF products include metabolic products 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-Δ-lactone, sodium erythorbate, lysine and other amino acids, Ω3 fatty acids, butanol, isoprene, 1,3-propanediol, and other biomaterials.

[0127] 5.6. Combination of α-amylase variants with other enzymes The alpha-amylase variant can be combined with a glucoamylase (EC 3.2.1.3). Exemplary glucoamylases are obtained from the genera Trichoderma, Aspergillus, Talaromyces, Clostridium, Fusarium, Thielavia, Thermomyces, Athelia, Humicola, Penicillium, Artomyces, Gloeophyllum, Pycnoporus, Steccherinum, Trametes, and the like. Suitable commercially available 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.

[0128] Other suitable enzymes that can be used in conjunction with the α-amylase variants include phytases, proteases, pullulanases, β-amylases, isoamylases, α-glucosidases, cellulases, xylanases, other hemicellulases, β-glucosidases, transferases, pectinases, lipases, cutinases, esterases, oxidoreductases, another α-amylase, or combinations thereof.

[0129] Compositions comprising the α-amylase of the present invention may be aqueous or non-aqueous formulations, granules, powders, gels, slurries, pastes, etc., which may further include any one or more of the additional enzymes listed herein, along with buffers, salts, preservatives, water, co-solvents, surfactants, etc.

[0130] 6. Compositions and uses for desizing fabrics Also contemplated are compositions and methods for treating textiles (e.g., desizing fabrics) using amylase. Methods for treating textiles are well known in the art (see, e.g., U.S. Pat. No. 6,077,316). For example, the feel and appearance of textiles can be improved by a method that includes contacting the textile with α-amylase in a solution. The textile can be treated with the solution under pressure.

[0131] Alpha-amylase can be applied during or after weaving of the textile, or during the desizing stage, or during one or more additional textile processing steps. During weaving of a textile, the yarns are subjected to significant mechanical strain. Before weaving on the loom, the warp yarns are often coated with sizing starch or starch derivatives to increase their tensile strength and prevent breakage. Alpha-amylase can be applied during or after weaving to remove these sizing starches or starch derivatives. After weaving, amylase can be used to remove the sizing coating before further processing of the textile to ensure a homogeneous and wash-resistant result.

[0132] Alpha-amylases can be used alone or with other desizing chemicals and / or desizing enzymes as detergent additives to desize textiles, including textiles containing cotton, for example in aqueous compositions. Alpha-amylases can also be used in compositions and methods for creating a stonewashed look on indigo dyed denim textiles and garments. To produce garments, textiles can be cut and sewn into garments or garments, which are then finished. In particular, various enzymatic finishing methods have been developed for producing denim jeans. Finishing denim garments usually begins with an enzymatic desizing process during which the garment is subjected to the action of amylolytic enzymes to impart softness to the textile and to make the cotton susceptible to subsequent enzymatic finishing steps. Alpha-amylases can be used in denim garment finishing (e.g., "bio-stone processing"), enzymatic desizing, and textile softening and / or finishing processes.

[0133] 7. Compositions and methods for baking and food preparation The compositions and methods of the present invention also relate to food compositions (including but not limited to food products, animal feeds, and / or food / feed additives comprising the α-amylase variants) and methods for preparing such food compositions comprising mixing the α-amylase variants with one or more food ingredients, or uses thereof. Additionally, the compositions and methods of the present invention relate to bakery compositions, including but not limited to bread flours, doughs, baking additives, and / or baked bread products.

[0134] 9. Brewing composition The α-amylase variants of the present invention can be components of a brewing composition used in the brewing process, i.e. in producing a fermented malt beverage. Non-fermentable carbohydrates form the majority of the dissolved solids in the final beer. This residue remains due to the inability of malt amylase to hydrolyze the α-1,6-linkages of starch. α-amylase, optionally in combination with glucoamylase and optionally pullulanase and / or isoamylase, converts starch to dextrins and fermentable sugars, helping to reduce the residual non-fermentable carbohydrates in the final beer.

[0135] All references cited herein are incorporated by reference in their entirety for all purposes. To further illustrate the present compositions and methods, and their advantages, the following specific examples are provided with the understanding that they are illustrative and not limiting. EXAMPLES

[0136] Example 1. AA2560 α-amylase variant Protein expression, purification, and quantification: The AA2560 α-amylase combinatorial variant, based on the AA2560 α-amylase variant described in WO 2021 / 080948 (herein SEQ ID NO: 5), was made as a synthetic gene and introduced into suitable Bacillus licheniformis cells using standard procedures. All mutations were confirmed by DNA sequencing. Cells were grown for 72 hours in a medium suitable for protein expression and secretion in the B. licheniformis host. Secreted proteins were harvested by centrifugation. Purification was achieved through hydrophobic interaction chromatography using Phenyl Sepharose 6 Fast Flow resin (GE Healthcare). The purified protein was stabilized in a standard formulation buffer containing HEPES, calcium chloride, and propyl propylene glycol as buffers at pH 8. Protein concentrations were determined by a combination of amino acid analysis, high performance liquid chromatography (HPLC) and absorbance at 280 nm.

[0137] Enzyme performance analysis: The activity of α-amylase was determined by removal of dyed starch stains from white melamine tiles in a detergent background. Mixed corn / rice colored starch tiles and mixed corn / rice starch tiles with food coloring (catalog number DM277) purchased from the Center for Test materials were used to determine the cleaning activity of α-amylase. These tiles were attached to a 96-well plate containing amylase solution diluted to the working range with aqueous buffer and added to a pre-made detergent solution of WFKB detergent (WFK Testgewebe GmbH, Brueggen, Germany) (total volume was 300 μL). The pre-imaged melamine tile with the colored starch stain was then attached on top of the 96-well plate such that agitation of this assembly resulted in splashing of the enzyme-containing detergent on the starch-stained surface. The washing reaction was incubated for 50 min with shaking at 250 rpm. o C for 15 hours. After the washing reaction, the melamine tiles were then rinsed briefly in water, dried and imaged again. The activity of α-amylase is calculated as the difference in RGB (color) values ​​of the pre-wash and post-wash images. The whiter the post-wash image, the higher the enzyme activity. The performance index (PI) is calculated as follows:

number

[0138] All variants in Table 3 outperform the variant of SEQ ID NO:5.

Claims

1. A recombinant, non-naturally occurring variant of a parent alpha-amylase, which has 80% identity with SEQ ID NO:5 and has amino acid substitutions at positions 51 and 125 relative to SEQ ID NO:

5.

2. The alpha-amylase variant of claim 1, wherein the amino acid substitutions are T51V and S125R with respect to SEQ ID NO:

5.

3. The alpha-amylase variant of claim 1 or 2, further comprising an amino acid substitution at position 172, 227, or 231 relative to SEQ ID NO:

5.

4. The alpha-amylase variant of claim 1 or 2, further comprising the amino acid substitutions N172Q, N227R, or F231L relative to SEQ ID NO:

5.

5. A recombinant, naturally occurring variant of a parent alpha-amylase having 80% identity to SEQ ID NO:5 and having the following amino acid substitutions relative to SEQ ID NO:5: (a) N29Q+T51V+S125R+N227R+S253L+G272E+K319R+S418A (b) T51V+S125R+F231L; (c) T51V+S125R+N172Q+N227R; (d) N029Q + T051V + T244I + S253L + K268R + K319R + S418A; or (e) E415G The alpha-amylase variant has the formula:

6. A detergent composition comprising the α-amylase variant according to any one of claims 1 to 5.

7. 7. The detergent composition of claim 6, further comprising a subtilisin protease variant from Bacillus gibsonii having amino acid substitutions X39E, X99R, X126A, X127E, and X128G.

8. A method for converting starch into oligosaccharides, comprising contacting the starch with an effective amount of an α-amylase variant according to any one of claims 1 to 5.

9. 10. A method for removing starchy stains or soils from a surface, the method comprising contacting the surface with an effective amount of an α-amylase variant according to any one of claims 1 to 5 and allowing the polypeptide to hydrolyse starch components present in the starchy stains to produce smaller starch derived molecules which are soluble in an aqueous composition, thereby removing the starchy stains from the surface.

10. A nucleic acid encoding an α-amylase variant according to any one of claims 1 to 5.

11. A host cell comprising the nucleic acid of claim 10.

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