Enzyme variants and polynucleotides encoding the same

Abstract

To provide polypeptides having α-amylase activity (α-amylases) which have enhanced wash performance, especially at high temperature (e.g., at or above 40°C).SOLUTION: The present invention relates to polypeptides having α-amylase activity corresponding to variants of an α-amylase derived from Bacillus. In particular, the polypeptides of the invention comprise or consist of an amino acid sequence of SEQ ID NO:1 in which one or more of the amino acids are mutated (for example, by substitution or deletion), such that the polypeptide exhibits enhanced wash performance (especially at temperatures equal to or higher than 40°C).SELECTED DRAWING: None

Description

[Technical field]

[0001] Sequence Listing Reference This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

[0002] The present invention relates to α-amylase variants (ie, polypeptides having α-amylase activity), nucleic acids encoding α-amylases, methods for producing α-amylases, compositions comprising α-amylases, and methods of using α-amylases. [Background technology]

[0003] Alpha-amylases (alpha-1,4-glucan-4-glucanohydrolases, EC 3.2.1.1) constitute a group of enzymes that catalyze the hydrolysis of starch and other linear and branched 1,4-glycosidic oligo- and polysaccharides.

[0004] There is a long history of industrial use of α-amylases in a number of known applications, such as detergents, baking, brewing, starch liquefaction and saccharification, e.g., in the preparation of isomerized sugar or as part of the production of ethanol from starch. These and other uses of α-amylases are known and in particular utilize α-amylases of microbial origin, such as bacterial α-amylases.

[0005] Alpha-amylases from Bacillus such as Termamyl, AA560 (SEQ ID NO: 2; see also WO 2000 / 060060) and SP707 (SEQ ID NO: 8; see also Tsukamoto et al., 1988, Biochem. Biophys. Res. Comm. 151:25-31) form a particular group of alpha-amylases that have found use in detergents. These amylases have been modified to improve their stability in detergents. For example, WO 96 / 23873 discloses deletion mutants of the alpha-amylases SP690, SP722 (SEQ ID NO: 5) and SP707 (SEQ ID NO: 8) (see also SEQ ID NOs: 1, 2 and 7 in WO 96 / 23873) to improve the stability of these amylases. WO 96 / 23873 further discloses substitution mutants to stabilize the amylase against oxidation.Additional α-amylase mutants with improved properties are disclosed in WO 2006 / 002643 and WO 01 / 66712.

[0006] It is therefore known to modify natural amylases to improve certain properties. Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a polypeptide (α-amylase) having α-amylase activity that has high cleaning performance, particularly at high temperatures (eg, 40° C. or higher). [Means for solving the problem]

[0008] The present invention relates to polypeptides having α-amylase activity corresponding to variants of α-amylases derived from the genus Bacillus. In particular, the polypeptides of the invention comprise or consist of the amino acid sequence of SEQ ID NO: 1, in which one or more amino acids have been mutated (e.g. by substitution or deletion) such that the polypeptide exhibits high cleaning performance, especially at temperatures above 40° C.

[0009] The present invention also relates to polynucleotides encoding the variant α-amylase polypeptides; nucleic acid constructs, vectors and host cells comprising the polynucleotides; and methods of producing the variant α-amylase polypeptides.

[0010] The present invention also relates to detergent compositions comprising the variant α-amylase polypeptides and their use in domestic and industrial cleaning processes, such as laundry cleaning and dishwashing.

[0011] definition α-Amylase: The term "α-amylase" (α-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1) constitutes a group of enzymes that catalyze the hydrolysis of starch and other linear and branched 1,4-glucoside oligo- and polysaccharides. For the purposes of the present invention, α-amylase activity is measured according to the procedure described in Example 1. In one embodiment, a variant of the invention has at least 20%, such as at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 100% of the α-amylase activity of the mature polypeptide of SEQ ID NO:1.

[0012] Amino Acid: As used herein, the term "amino acid" includes the standard 20 genetically encoded amino acids and their corresponding stereoisomers in the "D" form (as opposed to the natural "L" form), ω-amino acids, other naturally occurring amino acids, unconventional amino acids (e.g., α,α disubstituted amino acids, N-alkyl amino acids, etc.), and chemically derivatized amino acids. Chemical derivatives of one or more amino acids can be achieved by reaction with a functional side group. Such derivatized molecules include, for example, molecules in which a free amino group has been derivatized to form an amine hydrochloride, a p-toluenesulfonyl group, a carboxybenzoxy group, a t-butyloxycarbonyl group, a chloroacetyl group, or a formyl group. Free carboxyl groups can be derivatized to form salts, methyl and ethyl esters, or other types of esters and hydrazides. Free hydroxyl groups can be derivatized to form O-acyl or O-alkyl derivatives. Chemical derivatives also include peptides containing the naturally occurring amino acid derivatives of the 20 standard amino acids. For example: 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine, and ornithine may be substituted for lysine. Derivatives also include peptides containing one or more additions or deletions, so long as the required activity is maintained. Other modifications included are terminal modifications such as amidation, amino terminal acylation (e.g., acetylation or thioglycolic acid amidation), terminal carboxylamidation (e.g., with ammonia or methylamine), and the like.

[0013] When an amino acid is specifically recited, such as "alanine" or "Ala" or "A", the term refers to both L-alanine and D-alanine unless otherwise specified. Other non-conventional amino acids may also be suitable components of the polypeptides of the invention, so long as the desired functional properties are retained by the polypeptide. For the peptides shown, each of the encoded amino acid residues is represented, where appropriate, by a single letter symbol that corresponds to the trivial name of the conventional amino acid. In one embodiment, the polypeptides of the invention comprise or are composed of L-amino acids.

[0014] cDNA: The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from a mature spliced ​​mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial primary RNA transcript is a precursor of mRNA that is processed through a series of steps, including splicing, before emerging as a mature spliced ​​mRNA.

[0015] Coding sequence: The term "coding sequence" refers to a polynucleotide that directly specifies the amino acid sequence of a variant. The boundaries of a coding sequence are generally determined by an open reading frame, which begins with a start codon, such as ATG, GTG, or TTG, and ends with a stop codon, such as TAA, TAG, or TGA. The coding sequence can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0016] Control sequence: The term "control sequence" refers to a nucleic acid sequence necessary for the expression of a polynucleotide encoding a variant of the invention. Each control sequence may be native (i.e., from the same gene) or foreign (i.e., from different genes) to the polynucleotide encoding the variant, or native or foreign to each other. Such control sequences include, but are not limited to, a leader, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, and a transcription terminator. At a minimum, the control sequence includes a promoter, and a transcriptional and translational stop signal. The control sequence may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequence to the coding region of the polynucleotide encoding the variant.

[0017] High washing performance: The term "high washing performance" or "improved washing performance" refers to the ability of the polypeptides of the invention to provide improved cleaning results (e.g., stain removal) in a washing process, such as laundry or dishwashing, as compared to the parent α-amylase of SEQ ID NO: 1. Washing performance may be determined using methods well known in the art, such as using the Automated Mechanical Stress Assay (AMSA) or Automated Dishwashing (ADW) (see Examples 2 and 3). The skilled artisan will recognize that high washing performance may be achieved under only some, or perhaps all, of the washing conditions, such as, for example, washing temperatures above 40°C (such as 40°C and / or 50°C), and / or the presence or absence of bleach.

[0018] Expression: The term "expression" includes any step involved in the production of a variant, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification and secretion.

[0019] Expression vector: The term "expression vector" refers to a linear or circular DNA molecule that contains a polynucleotide encoding a variant and is operably linked to a control sequence that effects its expression.

[0020] Fragment: The term "fragment" refers to a polypeptide having one or more (e.g., a number of) amino acids not present at the amino and / or carboxyl terminus of the mature polypeptide of SEQ ID NO: 1; wherein the fragment has α-amylase activity. In one embodiment, the fragment contains at least 200 contiguous amino acid residues of SEQ ID NO: 1, such as at least 300 contiguous amino acid residues of SEQ ID NO: 1, or at least 350 contiguous amino acid residues, or at least 400 contiguous amino acid residues, or at least 450 contiguous amino acid residues.

[0021] Host cell: The term "host cell" refers to any cell type that is susceptible to transformation, transfection, transduction, etc., with a nucleic acid construct or expression vector comprising a polynucleotide of the invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0022] Enhancement Factor: The term "enhancement factor" is meant to quantitatively calculate the improvement of a particular property of a variant according to the present invention. The enhancement factor may be calculated according to the following formula:

number

[0023] Other formulas may be used to calculate the enhancement factor, and the formula presented here, as well as alternative methods for calculating the enhancement factor, will be known to those skilled in the art.

[0024] According to the present invention, a value of 1.0 corresponds to the specific activity observed for the parent α-amylase. Values ​​greater than 1.0 indicate an improvement in the specific activity of the tested variant compared to the parent α-amylase. Thus, any value greater than 1.0 is an indication of an improvement in a property, such as the specific activity, of the variant compared to the parent α-amylase. A value of 1.0 is an indication that the property of the variant is at least as good as that of the parent α-amylase.

[0025] Isolated: The term "isolated" refers to a substance in a form or environment that does not occur in nature. Non-limiting examples of isolated substances include: (1) any non-naturally occurring substance; (2) any substance, including but not limited to any enzyme, mutant, nucleic acid, protein, peptide, or cofactor, from which one or more or all of the natural components associated with it in nature have been at least partially removed; (3) any substance that has been artificially modified relative to the substance found in nature; or (4) any substance that has been modified by increasing the amount of the substance relative to other components associated with it in nature (e.g., multiple copies of the gene encoding the substance; use of a stronger promoter than the promoter naturally associated with the gene encoding the substance). An isolated substance may be present in a fermentation broth sample.

[0026] Mature Polypeptide: The term "mature polypeptide" refers to a polypeptide in its final form after translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. It is known in the art that a host cell can produce a mixture of two or more different mature polypeptides (i.e., with different C-terminal and / or N-terminal amino acids) expressed from the same polynucleotide.

[0027] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" refers to a polynucleotide that encodes a mature polypeptide having α-amylase activity.

[0028] Mutation: The term "mutation" in the context of the polypeptides of the invention means that one or more amino acids in a reference amino acid sequence (i.e. SEQ ID NO: 1) have been modified by substitution with a different amino acid or by deletion. Furthermore, a mutation may represent the insertion of one or more extra amino acids into the reference amino acid sequence.

[0029] Nucleic Acid Construct: The term "nucleic acid construct" refers to a nucleic acid molecule having a single or double helix, which is isolated from a naturally occurring gene or which has been modified to contain a segment of nucleic acid in a manner that would not otherwise occur in nature, or which has been synthesized, and which includes one or more regulatory sequences.

[0030] Operably linked: The term "operably linked" refers to a construction in which a control sequence is positioned in an appropriate position relative to a coding sequence of a polynucleotide such that the control sequence effects expression of the coding sequence.

[0031] Parent or Parent α-amylase: The term "parent" or "parent α-amylase" refers to the α-amylase of SEQ ID NO: 1 (commercialized under the trade name "Stainzyme® Plus" by Novozymes A / S; see WO 2006 / 002643).

[0032] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity."

[0033] For the purposes of the present invention, sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), preferably as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), version 5.0.0 or later. The parameters used may be a gap open penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of the Needle labelled "longest identity" (obtained using the -nobrief option) is used as the percentage identity, calculated as follows: (equivalent residues × 100) / (length of alignment-total number of gaps in alignment)

[0034] Alternatively, the parameters used can be a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix. The needle-labeled "longest identity" output (obtained using the -nobrief option) is used as the percent identity, calculated as follows: (equivalent deoxyribonucleotides × 100) / (length of alignment − total number of gaps in the alignment)

[0035] Subsequence: The term "subsequence" means a polynucleotide having one or more (e.g., a number) nucleotides that are absent at the 5' and / or 3' end of a mature polypeptide coding sequence; wherein the subsequence encodes a fragment that has α-amylase activity.

[0036] Variant: The term "variant" refers to a polypeptide having α-amylase activity that contains a mutation (i.e., substitution, insertion and / or deletion) at one or more (e.g., multiple) positions relative to the "parent" α-amylase of SEQ ID NO:1. A substitution refers to the replacement of an amino acid at a position with a different amino acid; a deletion refers to the removal of an amino acid at a position; and an insertion refers to the addition of an amino acid immediately adjacent to the amino acid at a position. Variants of the invention have at least 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the α-amylase activity of the mature polypeptide of SEQ ID NO:1.

[0037] Wild-type α-amylase: The term "wild-type" α-amylase refers to an α-amylase expressed by a naturally occurring microbial organism, such as a bacterium, yeast, or filamentous fungus, as found in nature.

[0038] Conventional mutant determination The polypeptide having α-amylase activity of the present invention corresponds to a variant of the α-amylase from Bacillus shown in SEQ ID NO: 1 (which is commercially available under the name Stainzyme Plus™ by Novozymes A / S). [ka]

[0039] Variant (i.e. mutated) amino acids in the polypeptides of the invention are defined with reference to the amino acid numbering of SEQ ID NO:2 (which corresponds to AA560 of the mature protein of B. subtilis). Differences in amino acid sequence with reference to SEQ ID NO:1 are shown below in bold and underlined type. [ka]

[0040] Thus, amino acid positions 1 to 182 in SEQ ID NO:2 correspond to the same numbers in SEQ ID NO:1, amino acid positions 183 and 184 in SEQ ID NO:2 are absent in SEQ ID NO:1, and amino acid positions 185 to 485 in SEQ ID NO:2 correspond to positions 183 to 483, respectively, of SEQ ID NO:1.

[0041] Therefore, for the purposes of the present invention, the mature polypeptide disclosed in SEQ ID NO:2 is used to determine the corresponding amino acid residues in other α-amylase polypeptides. The amino acid sequence of the other α-amylase is aligned with the mature polypeptide disclosed in SEQ ID NO:2, and based on this alignment, the amino acid position number corresponding to any amino acid residue in the mature polypeptide disclosed in SEQ ID NO:2 is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), preferably version 5.0.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

[0042] Identification of corresponding amino acid residues in other α-amylases can be performed using a number of tools, including but not limited to MUSCLE (multiple sequence comparison by log-expectation; version 3.5 or later; Edgar, 2004, Nucleic Acids Research 32:1792-1797), MAFFT (version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research 30:3059-3066; Katoh et al., 2005, Nucleic Acids Research 33:511-518; Katoh and Toh, 2007, Bioinformatics 23:372-374; Katoh et al., 2009, Methods in Molecular Biology 537:39-64; Katoh and Toh, 2010, Bioinformatics This can be determined by alignment of multiple polypeptide sequences using a number of computer programs, including EMBOSS EMMA employing ClustalW (1.83 or later; Thompson et al., 1994, Nucleic Acids Research 22:4673-4680), all using their default parameters.

[0043] Other pairwise sequence comparison algorithms can be used when other α-amylases diverge from the mature polypeptide of SEQ ID NO:2 such that traditional sequence-based comparisons do not allow for detection of relationships (Lindahl and Elofsson, 2000, J. Mol. Biol. 295:613-615). The sensitivity of sequence-based searches can be increased by using search programs that use probabilistic representations of polypeptide families (profiles) to search databases. For example, the PSI-BLAST program can generate profiles through an iterative database search process to detect distant homologs (Atschul et al., 1997, Nucleic Acids Res. 25:3389-3402). Sensitivity can be further increased when the family or superfamily of polypeptides has more than one representation in the protein structure database. Programs such as GenTHREADER (Jones, 1999, J. Mol. Biol. 287:797-815; McGuffin and Jones, 2003, Bioinformatics 19:874-881) use information from a variety of sources (PSI-BLAST, secondary structure prediction, structural alignment profiles and solvation potentials) as input to neural networks that predict the fold structure of a query sequence. Similarly, the method by Gough et al., 2000, J. Mol. Biol. 313:903-919 can be used to align sequences of unknown structure with superfamily models present in the SCOP database. These alignments can then be used to generate homology models of the polypeptides, which can be accurately evaluated using a variety of tools developed for that purpose.

[0044] For proteins of known structure, numerous tools and resources are available for searching and generating structural alignments. For example, the SCOP superfamily of proteins has been structurally aligned, and these alignments can be accessed and downloaded. Two or more protein structures can be aligned using a variety of algorithms, such as distance alignment matrices (Holm and Sander, 1998, Proteins 33:88-96) or combinatorial extension methods (Shindyalov and Bourne, 1998, Protein Engineering 11:739-747), which can be run incrementally against a query structure database together with the structure of interest to find potential structural homologues (e.g., Holm and Park, 2000, Bioinformatics 16:566-567).

[0045] In describing the α-amylase variants of the present invention, the following nomenclature is adopted for ease of reference: The accepted IUPAC one-letter or three-letter amino acid abbreviations are utilized.

[0046] Substitutions: For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Thus, the substitution of threonine at position 226 with alanine is shown as "Thr226Ala" or "T226A". Multiple mutations are separated by a plus sign ("+"), e.g., "Gly205Arg+Ser411Phe" or "G205R+S411F" represent the substitution of glycine (G) with arginine (R) and serine (S) with phenylalanine (F) at positions 205 and 411, respectively.

[0047] Deletions: For amino acid deletions, the following nomenclature is used: original amino acid, position, *Thus, a deletion of glycine at position 195 is designated as "Gly195*" or "G195*". Multiple deletions are separated by a plus sign ("+"), e.g., "Gly195*+Ser411*" or "G195*+S411*".

[0048] Insertions: For amino acid insertions, the following nomenclature is used: original amino acid, position, original amino acid, inserted amino acid. Thus, an insertion of a lysine after a glycine at position 195 is designated as "Gly195GlyLys" or "G195GK". An insertion of multiple amino acids is designated as [original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2; etc.]. For example, an insertion of a lysine and an alanine after a glycine at position 195 is designated as "Gly195GlyLysAla" or "G195GKA".

[0049] In such cases, the inserted amino acid residue is numbered by adding a subscript to the amino acid residue preceding the inserted amino acid residue. Thus, in the above example, the sequence would be:

[0050] [Table 1]

[0051] Multiple modifications: Variants containing multiple modifications are separated by a plus sign ("+"), e.g., "Arg170Tyr+Gly195Glu" or "R170Y+G195E" represent the replacement of arginine and glycine at positions 170 and 195 with tyrosine and glutamic acid, respectively.

[0052] Different modifications: When different modifications can be introduced at a position, the different modifications are separated by commas, for example, "Arg170Tyr,Glu" represents the substitution of arginine at position 170 with tyrosine or glutamic acid. Thus, "Tyr167Gly,Ala+Arg170Gly,Ala" represents the following variant: "Tyr167Gly+Arg170Gly", "Tyr167Gly+Arg170Ala", "Tyr167Ala+Arg170Gly" and "Tyr167Ala+Arg170Ala". DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] Polypeptides with α-amylase activity The present invention relates to variant α-amylase polypeptides comprising mutations at one or more (e.g., multiple) positions in the amino acid sequence of the mature polypeptide of SEQ ID NO:1, wherein the variants exhibit enhanced cleaning performance relative to the polypeptide of SEQ ID NO:1.

[0054] In one embodiment, the present invention relates to a polypeptide comprising a variant amino acid sequence of SEQ ID NO:1, wherein the polypeptide has α-amylase activity and exhibits enhanced cleaning performance compared to the polypeptide of SEQ ID NO:1.

[0055] In one embodiment, the variant amino acid sequence comprises a mutation at amino acid position 167 of SEQ ID NO:1.

[0056] Thus, in one embodiment, the variant amino acid sequence includes a substitution at amino acid position 167 of SEQ ID NO:1, such as W167Y.

[0057] The amino acid sequence of the parent α-amylase is shown below. [ka]

[0058] The polypeptides of the present invention represent variants of the parent α-amylase of SEQ ID NO: 1, which variants exhibit enhanced cleaning performance in domestic and / or industrial cleaning processes such as laundry cleaning and dishwashing. Cleaning performance can be determined using methods well known in the art such as automatic dishwashing (ADW) (see Example 2) or automated mechanical stress assay (AMSA) (see Example 3).

[0059] In one embodiment, the polypeptide exhibits enhanced washing performance during washing at elevated temperatures, for example temperatures of at least 40°C, such as at least 45°C, such as at least 50°C, such as at least 55°C, and such as at least 60°C.

[0060] In one embodiment, enhanced cleaning performance is assessed in an automatic dishwashing (ADW) assay using melamine tiles soiled with starch. Exemplary washing conditions for determining such enhanced cleaning performance of the polypeptides of the invention include: a. Model ADW detergent with bleach in a 10 minute wash cycle at 40°C; b. Model ADW detergent without bleach in a 10 minute wash cycle at 40°C; c. Model ADW detergent with bleach in a wash cycle of 20 minutes at 50°C; and d. Model ADW detergent without bleach in a 20 minute wash cycle at 50°C; Here, the model ADW detergent contains the trisodium salt of methylglycine diacetate (such as Trilon M granules SG), sodium citrate, sodium carbonate, sodium silicate, sodium sulfate, polyphosphate and a silicate scale inhibitor (such as Acusol 588G) and Surfac 23-6.5.

[0061] One of skill in the art will recognize that in addition to enhanced cleaning performance, the polypeptides of the present invention may also exhibit improvements in one or more of the following properties compared to the parent α-amylase of SEQ ID NO:1: (i) Substrate specificity; (ii) substrate binding; (iii) specific activity; (iv) Thermal stability (v) pH stability profile; (vi) Ca 2+ dependence; (vii) oxidative stability; (viii) high / low pI; and / or (ix) Sensitivity to detergents.

[0062] Assays for measuring the above-mentioned properties of polypeptides are described in WO 2006 / 002643, WO 2001 / 066712 and EP 2 264 460 A.

[0063] A polypeptide of the invention may be longer or shorter than the parent α-amylase of SEQ ID NO: 1. Thus, the polypeptide may be 1000 or less amino acids long, such as 900, 800, 700, 600, 500, 400, 300, 200, 175, 150, 125, 100 or less amino acids long. In one embodiment, the polypeptide is 400-600 amino acids long, such as 450-500, 460-500 or 470-490 amino acids long.

[0064] The variant polypeptides of the present invention contain mutations (ie, substitutions, insertions and / or deletions) at one or more amino acid positions relative to the "parent" α-amylase of SEQ ID NO:1.

[0065] The amino acid alterations may be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as amino-terminal methionine residues; small linker peptides of 20-25 residues or less; or small extensions that facilitate purification by altering the mutation or other function, such as polyhistidine tracts, antigenic epitopes or binding domains.

[0066] Examples of conservative substitutions are within the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, in H. Neurath and RL Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0067] Alternatively, the amino acid mutations are of such a nature that the physicochemical properties of the polypeptide are modified, for example, the amino acid mutations may improve the thermostability, modify the substrate specificity, or change the pH optimum of the polypeptide.

[0068] Essential amino acids in a polypeptide can be identified according to techniques known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resulting mutant molecules are tested for alpha-amylase activity to identify amino acid residues that are important for the activity of the molecule. See also Hilton et al., 1996, J. Biol. Chem. 271:4699-4708. The active site of an enzyme or other biological interactions can also be determined by physical analysis of the structure, as determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, coupled with mutations of putative contact site amino acids. See, e.g., de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identities of essential amino acids can also be inferred from alignments with related polypeptides.

[0069] In an alternative or further embodiment, the polypeptides of the invention comprise a nucleotide sequence at positions 10, 25, 30, 37, 40, 48, 51, 54, 64, 81, 86, 93, 98, 105, 108, 109, 113, 116, 118, 121, 130, 135, 138, 142, 167, 174, 175, 178, 182, 186, 187, 189, 195, 198, 202, 203, 206, 208, 210, 214, 218, 235, 238, 242, 243, 246, 247, 250, 255, 257, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309 59, 260, 261, 265, 267, 269, 270, 274, 275, 276, 281, 295, 298, 299, 311, 319, 320, 334, 339, 360, 365, 366, 383, 384, 385, 394, 398, 402, 404, 416, 434, 460, 469, 474 and 482, where the numbers are according to the amino acid sequence set out in SEQ ID NO:2.

[0070] Thus, the polypeptide may contain one or more of the following substitutions relative to the amino acid sequence of SEQ ID NO:2: M10L, N25K, D30N, K37H, K37L, K37M, K37R, K37V, S40T, W48F, A51Q, A51T, N54S, Y64W, T81S, Q86H, Q86I, Q86L, K93H, K93R, Q98R, M105Y, M105F, M105I, M105L, K108R, G109A, G109M, A113E, M116I, M116L, M116R ... 16A,M116V,M116F,K118Q,K118H,K118N,K118R,E121H,E130H,E130Q,Y135H,E138Q,K142R,K142Q,W167Y,W167H,N174Q,N174*, N175Q,Y178W,T182G,A186D,A186G,W187Y,W189H,F195N,Y198F,L202M,Y203H,Y203N,Y203G,Y203F,I206L,M208F,M208L,M208 V,H210N,V214R,V214T,V214I,R218N,I235V,I235L,I235M,V238T,V238A,K242P,Y243F,Y243M,T246V,T246I,T246L,T246M,R2 47K,I250L,I250V,S255K,I257A,K259N,N260D,M261L,M261A,A265G,F267Y,K269S,K269N,N270G,A274K,I275L,E276Q,K281H, F295Y, F295W, Y298W, Y298F, Y299W, Y299F, Q311T, Q311H, Q311R, Q319H, Q319R, K320H, K320R, S334T, S339A, E360F, S365M, S365C, V366I, K383Q, K383R, S384E, K385H, K385Q, K385R, Q394K, Y398W, N402Y, Y404W, E416L, A434D, G460E, W469F, V474C and W482Y.

[0071] For example, the polypeptide of the present invention may be a polypeptide of the amino acid sequence of SEQ ID NO:1 having the following mutations; E360F+S365C (i.e., a polypeptide having an amino acid sequence based on SEQ ID NO:1 but including the mutations E360F and S365C), K37H+L202M, M261L, H210N, Y243F, K108R, V474C, G460E, T81S, K269S+N270G+A274K, K37V+L202M, L202M+Q311R, N174Q+L202M, K385H, K385Q, K385R, K383Q, K32 0H,K320R,E276Q,K93R,K93H,Q98R,K118Q,K118H,K118N,E130H,E130Q,E1 38Q,K142R,K142Q,E416L,Q394K,S384E,Y64W,K37R,D30N,F295Y,Y243M,Y 178W,K281H,K269N,Y198F,Q311T,F195N,I257A,S255K,R247K,Y404W,Y39 8W,Q319H,Q319R,Q311H,Q311R,Y299W,N260D,K259N,Y299F,A434D,V366I, S365M,S339A,N174*,I235V,I250L,I250V,K37V,K37M,K37L,Y203H,S40T, W187Y,V238T,M105Y,M105F,M105I,I206L,T182G,M116I,Y135H,Y203N,Y2 03G,N25K,M116L,A265G,F295Y,A265G,K242P,V214R,M208F,Y198F,W482Y ,V214T,V214I,M261A,M105F,M208L,M116A,N174Q,N174*+N175Q,I235L,A 265G,M105L,K37H,Q311R,W469F,Y203F,G109A,N175Q,W48F,M116V,M116F ,F295W,Y298W,M208V,M208F,M10L+M261L,W187Y+M208L,Y298F,W167Y,W1 67H,W189H,F295Y,I235M,Y243F+F267Y,K37V+P45R+K383R,D30N+N33D+K3 7V+K383R,W167Y+H210N+S339A+V366I,S339A+V366I,W167Y+H210N+S339A,<h2 style=";text-align:left;direction:ltr">H210N+S339A,W167Y+H210N,W167Y+L202M+H210N+Y299F+S339A+V366I,W167Y+H210N+Y243F+S339A+V366I,W167Y+H210N+S339A+V366I+W482Y,M116F +W167Y+H210N+S339A+V366I,W48F+W167Y+H210N+S339A+V366I,W167Y+H210N+Y299F+S339A+V366I,W167Y+L202M+H210N+S339A+V366I,W167Y+L202M +H210N+Y299F+S339A,W167Y+H210N+S339A+W482Y,M116F+W167Y+H210N+S339A,W48F+W167Y+H210N+S339A,W167Y+H210N+Y299F+S339A,W167Y+L202M +H210N+S339A,W167Y+L202M+Y299F,W167Y+Y243F,M116F+W167Y,W48F+W1 67Y,W167Y+Y299F,W167Y+L202M,W167Y+H210N+V366I,W167Y+V366I,H210N +V366I,W167Y+S339A+V366I,D30N+N33D+K37V+L202M+K383R,D30N+N33D+K37V+W48F+K383R,D30N+N33D+K37V+M116F+K383R,D30N+N33D+K37V+K383R +W482Y,A51T,A51T+N54S,S334T,T246M,T246L,T246V,T246I,A186G,A51Q +G109M+Y203G,V238A+S334T,A51T+L202M,L202M+T246L,A51T+N174Q+L202 M+T246I+S334T,A51T+N174Q+L202M+Q311R+S334T,I235L+T246M+I250L,A 186D+L202M,A186D+L202M+N270G+N402Y,A186D+L202M+S339A,A186D+F195 N+L202M,K37H+A51T+L202M,K37V+,A51T+L202M,A51T+L202M+S365C,A51T+L202M+S339A,A51T+L202M+Q311T,A51T+L202M+M261L,A51T+L202M+H210N,<h2 style=";text-align:left;direction:ltr">A51T+L202M+N270G,A51T+F195N+L202M,A51T+L202M+Q319H,A51T+L202M +Q319R,A51T+L202M+Q311H,A51T+L202M+R247K,A51T+L202M+Q311R,A51 T+L202M+Y398W,A51T+L202M+Y299W,A51T+K108R+L202M,A51T+L202M+Y2 43F,A51T+L202M+V474C,A51T+L202M+G460E,N174Q+L202M+A265G+Q311R +S334T,A51T+L202M+T246V+A265G+Q311R,A51T+L202M+A265G+Q311R,K3 7H+L202M+T246V+S334T,L202M+T246V+S334T+E416L,L202M+T246V+S334T +N402Y,L202M+T246V+S334T+V366I,L202M+T246V+S334T+S365M,L202M+T246V+S334T+S365C,L202M+T246V+M261L+S334T,D30N+L202M+H210N+T246V+S334T,L202M+T246V+N270G+S334T,F195N+L202M+T246V+S334T,L202M+T246V+Q319H+S334T,L202M+T246V+Q319R+S334T,L202M+T246V+Q311H +S334T,L202M+T246V+Q311R+S334T,L202M+T246V+S334T+Y398W,L202M+T246V+K320H+S334T,L202M+T246V+Y299W+S334T,L202M+T246V+K320R+S334T,L202M+Y243F+S334T,L202M+T246V+S334T+V474C,L202M+T246V+S334T+G460E,L202M+I235M+T246V+S334T,K108R+L202M+T246V+S334T,A51T +A186D+L202M+N270G+N402Y,A186D+L202M+N270G+S339A+N402Y,A51T+A 186D+L202M+N270G,A51T+L202M+T246V+N270G,K37H+A51T+L202M+N270G,A51T+L202M+T246L+N270G,A51T+N174Q+L202M+N270G,A51T+L202M+V238A+N270G,A51T+L202M+A265G,A51T+L202M+M261L+N270G,A51T+L202M+F267Y,A51T+L202M+I275L,A51T+L202M+N270G+S365M,A51T+L202M+N270G+S365C,A51T+L202M+N270G+Q311T,A51T+L202M+N270G+E416L,A51T+L202M+N270G+N402Y,A51T+L202M+N270G+S365C,A51T+L202M+N270G+K383R,A51T+L202M+N270G+V474C,A51T+L202M+N270G+G460E,A51T+Q86L+L202M+N270G,A51T+Q86I+L202M+N270G,A51T+A113E+L202M+N270G,A51T+K93H+L202M+N270G,A51T+K108R+L202M+N270G,A51T+L202M+K269N,A51T+L202M+Y243F+N270G,A51T+F195N+L202M+N270G,A51T+L202M+R247K+N270G,A51T+L202M+R218N+N270G,A51T+L202M+S255K+N270G,A51T+L202M+I257A+N270G,A51T+L202M+V214I+R218N+N270G,A51T+L202M+N270G+Q311H,A51T+L202M+N270G+K320H,A51T+L202M+N270G+Y299W,A51T+L202M+N270G+K320R,A51T+L202M+N270G+K383Q,A51T+K142R+L202M+N270G,A51T+E130Q+L202M+N270G,A51T+K118N+L202M+N270G,A51T+E138Q+L202M+N270G,A51T+K118H+L202M+N270G,A51T+K118Q+L202M+N270G,A51T+Q86H+L202M+N270G,A51T+E121H+L202M+N270G,A51T+K118R+L202M+N270G,K37H+A51T+N174Q+L202M+A265G+Q311R+S334T,A51T+N174Q+L202M+T246I+Q311R+S334T,K37V+A51T+L202M+ A265G+F267Y+Q311R+S334T,A51T+N174Q+L202M+A265G+F267Y+Q311R+S334T,A51G+L202M+Q311R+S334T,L202 M+T246I+A265G+F267Y+Q311R+S334T,A51T+L202M+F267Y+Q311R+S334T+S365L,A51T+L202M+S365C,A51T+K10 8R+L202M,A51T+S365C,A51T+K108R,L202M+V238A+S334T,W48F+V238A+S334T,M116F+V238A+S334T,V238A+S3 34T+W482Y,Y243F+S334T,L202M+V238A+Y299F+S334T,L202M+T246V+N270G+S334T,W48F+K118H+V238A+S334T ,W48F+L202M+V238A+S334T,A51T+A186N+L202M+N270G+S365C,W167Y+A186N+H210N+S339A+V366I,W48F+W167 Y+A186N+H210N+S339A, W167Y+A186N+H210N+Y299F+S339A+V366I, L202M+T246V+N270G+S334T+S365C, A186N+L202M+T246V+N270G+S334T, and fragments thereof having alpha amylase activity, wherein the numbering of the amino acid positions is according to the amino acid sequence set forth in SEQ ID NO:2.

[0072] In one embodiment, the polypeptide exhibits enhanced cleaning performance corresponding to an enhancement factor (IF) of at least 1.2 when the polypeptide is evaluated in an ADW assay with detergent containing bleach at 40° C. for 10 minutes (see, e.g., Example 2). A suitable polypeptide may be a variant of SEQ ID NO:1 having a mutation at one or more positions corresponding to positions 37, 51, 93, 98, 108, 118, 167, 186, 202, 210, 235, 243, 246, 247, 250, 255, 259, 260, 261, 270, 299, 311, 319, 334, 339, 365, 385, 398, and 404, where the numbering of the amino acid positions is according to the amino acid sequence set forth in SEQ ID NO:2. For example, the polypeptide may be a polypeptide of the amino acid sequence of SEQ ID NO:1 having the following mutations: K37H+L202M, M261L, Y243F, K385H, K385R, K93H, Q98R, K118Q, K118H, S255K, Y404W, Y398W, Q319R, Y299W, N260D, K259N, Y299F, S339A, S334T, A51T+L202M, I235L+T246M+I250L, K37V+A51T+L202M, A51T+L202M+S365C, A51T+L202M+Q311T, A51T+L202M+M261L, A51T+L202M +R247K, A51T+L202M+Y299W, A51T+K108R+L202M, A51T+L202M+Y243F, A51T+A186D+L202M+N270G, A51T+L202M+N270G+S365C, W167Y+A186N+H210N+S339A+V366I, W48F+W167Y+A186N+H210N+S339A or W167Y+A186N+H210N+Y299F+S339A+V366, and fragments thereof having alpha amylase activity, wherein the numbering of the amino acid positions is according to the amino acid sequence set forth in SEQ ID NO:2.

[0073] In other embodiments, the polypeptides exhibit high cleaning performance, corresponding to an IF of at least 1.2, when the polypeptides are evaluated in an ADW assay at 50° C. for 20 minutes with detergent containing bleach (see, e.g., Example 2). The polypeptide may be a variant of SEQ ID NO: 1 having a mutation at one or more positions corresponding to 265, 266, 267, 269, 270, 299, 311, 319, 320, 334, 339, 360, 365, 366, 383, 385, 402, 416, 474, and 482, where the numbering of the amino acid positions is according to the amino acid sequence set forth in SEQ ID NO: 2. For example, the polypeptide may be a polypeptide of the amino acid sequence of SEQ ID NO: 1 having the following mutations: K37H+L202M, E360F+S365C, M261L, H210N, Y243F, K108R, V474C, T81S, K385H, K385Q, K385R, K93R, K118Q, K118N, E130H, E 130Q,E138Q,K37R,D30N,Y243M,K269N,Y198F,Q311T,F195N,I257A,S255K,R247K,Y4 04W,Y398W,Q319H,Q319R,Q311H,Q311R,Y299W,N260D,K259N,Y299F,V366I,S365M,S3 39A,I250L,I250V,K37M,S40T,V238T,M105F,M105I,I206L,T182G,M116I,M116L,N17 4Q,I235L,A265G,K37H,Q311R,N175Q,W48F,M116F,W167Y+H210N+S339A,H210N+S339A ,W48F+W167Y+H210N+S339A,W48F+W167Y,W167Y+H210N+V366I,D30N+N33D+K37V+K383 R+W482Y,A51T,S334T,T246M,T246L,T246V,T246I,A186G,V238A+S334T,A51T+L202M,A51T+N174Q+L202M+Q311R+S334T,I235L+T246M+I250L,L202M+T246V+S334T+E416L,D30N+L202M+H210N+T246V+S334T,L202M+T246V+N270G+S334T,F195N+L202M+T246V+S334T,L202M+T246V+K320R+S334T,L202M+Y243F+S334T,A51T+A186D+L202M+N270G+N402Y,A186D+L202M+N270G+S339A+N402Y,A51T+A186D+L202M+N270G,A51T+L202M+T246V+N270G,K37H+A51T+L202M+N270G,A51T+L202M+T246L+N270G,A51T+N174Q+L202M+N270G,A51T+L202M+V238A+N270G,A51T+L202M+A265G,A51T+L202M+M261L+N270G,A51T+L202M+F267Y,A51T+L202M+N270G+S365C,A51T+L202M+N270G+S365C,A51T+L202M+N270G+K383R,A51T+Q86I+L202M+N270G,A51T+K93H+L202M+N270G,A51T+K108R+L202M+N270G,A51T+L202M+N270G+K320H,A51T+L202M+N270G+K383Q,A51T+K142R+L202M+N270G,A51T+E130Q+L202M+N270G,A51T+E138Q+L202M+N270G,A51T+L202M+S365C,A51T+S365C,W48F+V238A+S334T,M116F+V238A+S334T,V238A+S334T+W482Y,Y243F+S334T,W48F+K118H+V238A+S334T,A51T+A186N+L202M+N270G+S365C,W167Y+A186N+H210N+S339A+V366I,W48F+W167Y+A186N+H210N+S339A,W167Y+A186N+H210N+Y299F+S339A+V366I,L202M+T246V+N270G+S334T+S365C or A186N+L202M+T246V+N270G+S334T, and fragments thereof having alpha amylase activity, wherein the numbering of the amino acid positions is according to the amino acid sequence set forth in SEQ ID NO:2.

[0074] In further embodiments, the polypeptide exhibits enhanced cleaning performance corresponding to an improvement factor (IF) of at least 1.2 when the polypeptide is evaluated in an ADW assay at 40° C. for 10 minutes with detergent without bleach (see, e.g., Example 2). Suitable polypeptides can be variants of SEQ ID NO:1 having mutations at one or more positions corresponding to positions 37, 48, 51, 64, 81, 108, 116, 167, 174, 186, 187, 189, 195, 198, 202, 208, 210, 235, 238, 243, 246, 250, 261, 265, 267, 269, 270, 275, 311, 319, 334, 339, 365, 366, 385, 460, and 474, where the numbering of the amino acid positions is according to the amino acid sequence set forth in SEQ ID NO:2.For example, the polypeptide may be a polypeptide of the amino acid sequence of SEQ ID NO:1 having the following mutations: K37H+L202M, M261L, H210N, Y243F, K108R, G460E, T81S, N174Q+L202M, K385R, Y64W, Y243M, K269N, Y198F, S365M, S339A, W48F, M116V, M116F, W187Y+M208L, W167Y, W189H, W167Y+L202M+H210N+ Y299F+S339A,W167Y+L202M+H210N+S339A,W167Y+L202M,V238A+S334T,A51T+L202M,L202M+T246L,A51T+N174Q+L202M+T 246I+S334T,I235L+T246M+I250L,A186D+F195N+L202M,K37H+A51T+L202M,K37V+A51T+L202M,A51T+L202M+S365C,A51T+ L202M+Q311T,A51T+L202M+M261L,A51T+L202M+Q319R,A51T+L202M+Q311H,A51T+K108R+L202M,A51T+L202M+V474C,A51 T+A186D+L202M+N270G,K37H+A51T+L202M+N270G,A51T+L202M+A265G,A51T+L202M+M261L+N270G,A51T+L202M+F267Y,A5 The amino acid sequence of SEQ ID NO:2 may be selected from the group consisting of 1T+L202M+I275L, A51T+L202M+N270G+S365C, W167Y+A186N+H210N+S339A+V366I, W48F+W167Y+A186N+H210N+S339A or W167Y+A186N+H210N+Y299F+S339A+V366I, and fragments thereof having alpha amylase activity, wherein the numbering of the amino acid positions is according to the amino acid sequence set forth in SEQ ID NO:2.

[0075] In further embodiments, the polypeptides exhibit high cleaning performance, corresponding to an IF of at least 1.2, when the polypeptides are evaluated in an ADW assay at 50° C. for 20 minutes with detergent without bleach (see, e.g., Example 2). 1 having mutations at one or more positions corresponding to 65, 267, 269, 270, 274, 275, 299, 311, 319, 320, 334, 339, 365, 366, 383, 385, 398, 402, 416, 404, 460, 474, and 482, where the numbering of the amino acid positions is according to the amino acid sequence set forth in SEQ ID NO: 2. For example, the polypeptide may be a polypeptide of the amino acid sequence of SEQ ID NO: 1 having the following mutations; K269S+N270G+A274K, K37V+L202M, N174Q+L202M, K385H, K385R, K93H, K118Q, E130Q, E138Q, K37R, D30N, Y243M, K269N ,Y198F,F195N,I257A,S255K,R247K,Y404W,Y398W,Q319H,Q319R,Q311H,Q311R,Y29 9W,N260D,K259N,Y299F,V366I,S365M,S339A,N174*,K37L,S40T,T182G,A265G,W48 2Y,M116A,N174Q,I235L,A265G,K37H,Q311R,N175Q,W48F,M116V,M116F,W167Y+L20 2M+H210N+S339A+V366I,W167Y+L202M+H210N+Y299F+S339A,W167Y+L202M+H210N+S 339A,W167Y+L202M+Y299F,W48F+W167Y,W167Y+Y299F,W167Y+L202M,D30N+N33D+K3 7V+L202M+K383R,D30N+N33D+K37V+W48F+K383R,A51T,S334T,T246M,T246L,T246I,A186G,A51Q+G109M+Y203G,V238A+S334T,A51T+L202M,L202M+T246L,A51T+N174Q+L202M+T246I+S334T,A51T+N174Q+L202M+Q311R+S334T,I235L+T246M+I250L,A186D+L202M,A186D+L202M+N270G+N402Y,A186D+L202M+S339A,A186D+F195N+L202M,K37H+A51T+L202M,K37V+A51T+L202M,A51T+L202M+S365C,A51T+L202M+S339A,A51T+L202M+Q311T,A51T+L202M+M261L,A51T+L202M+H210N,A51T+L202M+N270G,A51T+F195N+L202M,A51T+L202M+Q319H,A51T+L202M+Q319R,A51T+L202M+Q311H,A51T+L202M+R247K,A51T+L202M+Q311R,A51T+L202M+Y398W,A51T+L202M+Y299W,A51T+K108R+L202M,A51T+L202M+Y243F,A51T+L202M+V474C,A51T+L202M+G460E,N174Q+L202M+A265G+Q311R+S334T,A51T+L202M+T246V+A265G+Q311R,K37H+L202M+T246V+S334T,L202M+T246V+S334T+E416L,L202M+T246V+S334T+N402Y,L202M+T246V+S334T+V366I,L202M+T246V+S334T+S365M,L202M+T246V+S334T+S365C,L202M+T246V+M261L+S334T,D30N+L202M+H210N+T246V+S334T,L202M+T246V+N270G+S334T,F195N+L202M+T246V+S334T,L202M+T246V+Q319H+S334T,L202M+T246V+Q319R+S334T,L202M+T246V+Q311H+S334T,L202M+T246V+Q311R+S334T,L202M+T246V+S334T+Y398W,L202M+T246V+K320H+S334T,L202M+T246V+Y299W+S334T,L202M+T246V+K320R+S334T,L202M+Y243F+S334T,L202M+T246V+S334T+V474C,L202M+T246V+S334T+G460E,L202M+I235M+T246V+S334T,K108R+L202M+T246V+S334T,A51T+A186D+L202M+N270G+N402Y,A186D+L202M+N270G+S339A+N402Y,A51T+A186D+L202M+N270G,A51T+L202M+T246V+N270G,K37H+A51T+L202M+N270G,A51T+L202M+T246L+N270G,A51T+N174Q+L202M+N270G,A51T+L202M+V238A+N270G,A51T+L202M+A265G,A51T+L202M+M261L+N270G,A51T+L202M+F267Y,A51T+L202M+I275L,A51T+L202M+N270G+S365M,A51T+L202M+N270G+S365C,A51T+L202M+N270G+Q311T,A51T+L202M+N270G+E416L,A51T+L202M+N270G+N402Y,A51T+L202M+N270G+S365C,A51T+L202M+N270G+K383R,A51T+L202M+N270G+V474C,A51T+L202M+N270G+G460E,A51T+Q86L+L202M+N270G,A51T+Q86I+L202M+N270G,A51T+A113E+L202M+N270G,A51T+K93H+L202M+N270G,A51T+K108R+L202M+N270G,A51T+L202M+K269N,A51T+L202M+Y243F+N270G,A51T+L202M+R247K+N270G,A51T+L202M+R218N+N270G,A51T+L202M+S255K+N270G,A51T+L202M+I257A+N270G,A51T+L202M+V214I+R218N+N270G,A51T+L202M+N270G+Q311H,A51T+L202M+N270G+K320H,A51T+L202M+N270G+Y299W,A51T+L202M+N270G+K320R,A51T+L202M+N270G+K383Q,A51T+K142R+L202M+N270G,A51T+E130Q+L202M+N270G,A51T+K118N+L202M+ N270G,A51T+E138Q+L202M+N270G,A51T+K118H+L202M+N270G,A51T+E121H+L202M+N270G,K37H+A51T+N174Q+L202M+A265G+Q311R+ S334T,A51T+N174Q+L202M+T246I+Q311R+S334T,K37V+A51T+L202M+A265G+F267Y+Q311R+S334T,A51T+N174Q+L202M+A265G+F267Y +Q311R+S334T,L202M+T246I+A265G+F267Y+Q311R+S334T,A51T+L202M+F267Y+Q311R+S334T+S365L,A51T+L202M+S365C,A51T+K108 R+L202M,L202M+V238A+S334T,W48F+V238A+S334T,M116F+V238A+S334T,V238A+S334T+W482Y,Y243F+S334T,L202M+V238A+Y299F+ S334T,L202M+T246V+N270G+S334T,W48F+K118H+V238A+S334T,W48F+L202M+V238A+S334T,A51T+A186N+L202M+N270G+S365C,W167Y +A186N+H210N+S339A+V366I, W48F+W167Y+A186N+H210N+S339A, W167Y+A186N+H210N+Y299F+S339A+V366I, L202M+T246V+N270G+S334T+S365C or A186N+L202M+T246V+N270G+S334T, and fragments thereof having alpha amylase activity, wherein the numbering of the amino acid positions is according to the amino acid sequence set forth in SEQ ID NO:2.

[0076] In a particular subset of the polypeptides of the invention, the polypeptide is a variant of SEQ ID NO: 1 having a mutation at one or more positions corresponding to positions 48, 118, 167, 186, 210, 235, 238, 243, 246, 250, 299, 334, 339, 366 and 482, where the numbering is according to SEQ ID NO: 2. Thus, the polypeptide may consist of the amino acid sequence of SEQ ID NO: 1 having one or more mutations selected from the group consisting of W48F, K118H, W167Y, H210N, I235L, V238A, Y243F, T246M, I250L, Y299F, S334T, S339A, V366I and W482Y, or a fragment thereof having alpha amylase activity, where the numbering of the amino acid positions is according to the amino acid sequence set forth in SEQ ID NO: 2. For example, the polypeptide may have an amino acid sequence selected from: (a) SEQ ID NO:1 with mutations W167Y, H210N and S339A; (b) SEQ ID NO:1 with mutations W167Y, H210N, S339A and V366I; (c) SEQ ID NO:1 with mutations W167Y, H210N, Y299F, S339A and V366I; (d) SEQ ID NO:1 with mutations W48F and W167Y; (e) SEQ ID NO:1 with mutations W48F, W167Y, H210N and S339A; (f) SEQ ID NO:1 with mutations W48F, W167Y, H210N, Y299F, S339A and V366I; (g) SEQ ID NO:1 with mutations V238A, S334T and W482Y; (h) SEQ ID NO:1 with mutations Y243F and S334T; (i) SEQ ID NO:1 with the mutation W48F; (j) SEQ ID NO:1 with the mutation K118H; (k) SEQ ID NO:1 with the mutation W167Y; (l) SEQ ID NO:1 with mutations W48F, V238A and S334T; (m) SEQ ID NO:1 with mutations I234L, T246M, and I250L; (n) SEQ ID NO: 1 in the mutation S339A; and (o) SEQ ID NO:1 with mutation S334T; and fragments thereof having alpha amylase activity, wherein the numbering of the amino acid positions is according to the amino acid sequence set forth in SEQ ID NO:2.

[0077] The polypeptides of the invention represent variants of the amino acid sequence of the parent α-amylase (SEQ ID NO:1), which exhibit enhanced cleaning performance (e.g., at temperatures of 40° C. or higher). The skilled artisan will recognize that different examples of the polypeptides of the invention will have different degrees of amino acid sequence identity with the sequence of SEQ ID NO:1. Thus, the polypeptides may comprise or consist of an amino acid sequence sharing at least 70% sequence identity with SEQ ID NO:1, such as at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:1. In one embodiment, the number of mutations in the polypeptide relative to the amino acid sequence of SEQ ID NO:1 is 1-20, e.g., 1-10 mutations or 1-5 mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations.

[0078] Exemplary α-amylase polypeptides of the invention are shown in Table 1 (wherein the amino acid sequences of the polypeptides are represented with reference to the parent α-amylase sequence of SEQ ID NO:1).

[0079] [Table 2]

[0080] [Table 3]

[0081] [Table 4]

[0082] [Table 5]

[0083] [Table 6]

[0084] [Table 7]

[0085] [Table 8]

[0086] In certain embodiments, the variant α-amylases of the invention have high cleaning performance at temperatures of both 40° C. and 50° C. Such variant α-amylases have: (a) SEQ ID NO:1 with mutations V238A, S334T and W482Y; (b) SEQ ID NO:1 with mutations Y243F and S334T; (c) SEQ ID NO:1 with mutations W48F, W167Y, H210N, Y299F, S339A, and V366I; (d) SEQ ID NO:1 with mutations W48F, W167F, H210N and S339A; (e) SEQ ID NO:1 with the mutation W48F; and (f) SEQ ID NO:1 with the mutations W167Y, H210N and S339A; and fragments thereof having alpha amylase activity, wherein the numbering of the amino acid positions is according to the amino acid sequence set forth in SEQ ID NO:2.

[0087] Preparation of the Polypeptides of the Invention The variant α-amylases of the invention can be prepared using any mutagenesis method known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, and the like.

[0088] Site-directed mutagenesis is a technique that introduces one or more (eg, several) mutations at one or more defined sites in a parent coding polynucleotide.

[0089] Site-specific mutagenesis can be achieved in vitro by PCR using oligonucleotide primers containing the desired mutation. Site-specific mutagenesis can also be performed in vitro by cassette mutagenesis, which involves cleavage with a restriction enzyme at a site in a plasmid containing a parent coding polynucleotide, followed by ligation of an oligonucleotide containing the mutation in the polynucleotide. Usually, the restriction enzymes that digest the plasmid and the oligonucleotide are the same, allowing the sticky ends of the plasmid and the insert to ligate to each other. See, for example, Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA 76:4949-4955; and Barton et al., 1990, Nucleic Acids Res. 18:7349-4966.

[0090] Site-directed mutagenesis can also be accomplished in vivo by methods known in the art. See, e.g., U.S. Patent Application Publication No. 2004 / 0171154; Storici et al., 2001, Nature Biotechnol. 19:773-776; Kren et al., 1998, Nat. Med. 4:285-290; and Calissano and Macino, 1996, Fungal Genet. Newslett. 43:15-16.

[0091] Any method of site-directed mutagenesis can be used in the present invention. A number of commercial kits are available that can be used to prepare mutants.

[0092] Synthetic gene construction involves the in vitro synthesis of designed polynucleotide molecules to encode a polypeptide of interest. Gene synthesis can be performed using a number of techniques, including the multiplexed microchip-based technology described by Tian et al. (2004, Nature 432:1050-1054), as well as similar technologies in which oligonucleotides are synthesized and assembled into microfluidic chips that can be controlled by light.

[0093] Single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested using known mutagenesis, recombination, and / or shuffling methods, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86:2152-2156; WO 95 / 17413; or WO 95 / 22625, followed by associated screening methods. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:10832-10837; U.S. Pat. No. 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0094] Mutagenesis / shuffling methods can be combined with high-throughput automated screening methods to detect activity of cloned mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17:893-896). Mutagenized DNA molecules that code for active polypeptides can be recovered from the host cells and readily sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.

[0095] Semi-synthetic gene construction is achieved by combining aspects of synthetic gene construction and / or site-directed mutagenesis and / or random mutagenesis and / or shuffling. Semi-synthetic construction is represented by a process that utilizes synthesized polynucleotide fragments combined with PCR technology. Thus, defined regions of a gene can be synthesized de novo, while other regions can be amplified using site-directed mutagenic primers, while still other regions can be subjected to error-prone PCR or non-error-prone PCR amplification. Polynucleotide subsequences can then be shuffled.

[0096] Polynucleotides The present invention also relates to polynucleotides encoding the variants of the invention. Thus, the present invention relates to polynucleotides encoding variant polypeptides having α-amylase activity and exhibiting enhanced cleaning performance compared to the polypeptide of SEQ ID NO: 1. In particular, the present invention relates to polynucleotides encoding variant polypeptides having α-amylase activity and exhibiting enhanced cleaning performance compared to the polypeptide of SEQ ID NO: 1. and 482, wherein the numbers are according to the amino acid sequence set forth in SEQ ID NO:2.

[0097] nucleic acid construct The present invention also relates to a nucleic acid construct comprising a polynucleotide encoding a variant polypeptide of the present invention operably linked to one or more control sequences that effect expression of the coding sequence in a suitable host cell under conditions applicable to the control sequences.Thus, the present invention relates to a nucleic acid construct comprising a polynucleotide encoding a variant polypeptide (having α-amylase activity and exhibiting enhanced cleaning performance compared to the polypeptide of SEQ ID NO:1) operably linked to one or more control sequences that effect expression of the coding sequence in a suitable host cell under conditions applicable to the control sequences. In particular, the present invention provides a method for the expression of a coding sequence in a suitable host cell under conditions applicable to the control sequences, comprising administering to the subject an operably linked sequence comprising one or more of the following: , 247, 250, 255, 257, 259, 260, 261, 265, 267, 269, 270, 274, 275, 276, 281, 295, 298, 299, 311, 319, 320, 334, 339, 360, 365, 366, 383, 384, 385, 394, 398, 402, 404, 416, 434, 460, 469, 474 and 482 (where the numbers are according to the amino acid sequence set forth in SEQ ID NO:2).

[0098] Polynucleotides can be manipulated in a variety of ways to result in expression of the variants. Treatment of the polynucleotide prior to insertion into a vector may be desirable or necessary depending on the expression vector. Techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.

[0099] The control sequence may be a promoter, which is a polynucleotide that is recognized by a host cell for expression of a polynucleotide. The promoter contains a transcriptional control sequence that mediates the expression of a variant. The promoter may be any polynucleotide that exhibits transcriptional activity in a host cell, including mutant, truncated and hybrid promoters, and may be derived from a gene that encodes an extracellular or intracellular polypeptide that is homologous or heterologous to the host cell.

[0100] Examples of suitable promoters for directing transcription of the nucleic acid constructs of the invention in bacterial host cells are the Bacillus amyloliquefaciens α-amylase gene (amyQ), the Bacillus licheniformis α-amylase gene (amyL), the Bacillus licheniformis penicillinase gene (penP), the Bacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillus subtilis levansucrase gene (sacB), the Bacillus subtilis xylA and xylB genes, the Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994, Molecular Microbiology 13:97-107), the E. coli lac operon, the E. coli trc promoter (Egon et al., 1988, Gene 69:301-315), the Streptomyces coelicolor agarase gene (dagA) and the prokaryotic β-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75:3727-3731), and the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in "Useful proteins from recombinant bacteria", Gilbert et al., 1980, Scientific American 242:74-94; and Sambrook et al., supra, 1989. Examples of tandem promoters are disclosed in WO 99 / 43835.

[0101] Examples of suitable promoters for directing transcription of the nucleic acid constructs of the invention in a filamentous fungal host cell include those encoding Aspergillus nidulans acetamidase, Aspergillus niger neutral α-amylase, Aspergillus niger acid-stable α-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum L. oxysporum trypsin-like protease (WO 96 / 00787), Fusarium venenatum amyloglucosidase (WO 00 / 56900), Fusarium venenatum Daria (WO 00 / 56900), Fusarium venenatum Quinn (WO 00 / 56900), Rhizomucor miehei lipase, Rhizomucor miehei aspartic proteinase, Trichoderma reesei β-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, TrichodermaEndoglucanase IV from Trichoderma reesei, Endoglucanase V from Trichoderma reesei, Xylanase I from Trichoderma reesei, Xylanase II from Trichoderma reesei, β-Xylosidase from Trichoderma reesei, and a promoter obtained from a gene of the NA2-tpi promoter (a modified promoter derived from the Aspergillus neutral α-amylase gene, wherein the untranslated leader is replaced by an untranslated leader derived from the Aspergillus triose phosphate isomerase gene; non-limiting examples include a modified promoter derived from the Aspergillus niger neutral α-amylase gene, wherein the untranslated leader is replaced by an untranslated leader derived from the Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerase gene); and mutants, truncated, and hybrid promoters thereof.

[0102] In yeast hosts, useful promoters can be obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2 / GAP), Saccharomyces cerevisiae triosephosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8:423-488.

[0103] The control sequence may also be a transcription terminator recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3'-end of the polynucleotide encoding the variant. Any terminator that functions in the host cell may be used.

[0104] Preferred terminators for bacterial host cells are obtained from the genes for Bacillus clausii alkaline protease (aprH), Bacillus licheniformis alpha-amylase (amyL) and Escherichia coli ribosomal RNA (rrnB).

[0105] Preferred terminators for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger α-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0106] Examples of terminators that may be used include those derived from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1) and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described in Romanos et al., 1992, supra.

[0107] A regulatory sequence can also be an mRNA stabilizer region downstream of the promoter and upstream of the coding sequence of the gene, which enhances expression of the gene.

[0108] Examples of suitable mRNA stabilizer regions are obtained from the Bacillus thuringiensis cryIIIA gene (WO 94 / 25612) and the Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

[0109] The control sequence may also be a leader, a non-translated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5'-end of the polynucleotide encoding the variant. Any leader that is functional in the host cell may be used.

[0110] Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0111] Preferred leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0112] The control sequence may also be a polyadenylation sequence, which is a sequence operably linked to the 3'-end of the variant coding sequence and which, when transcribed, is recognized by a host cell as a signal for the addition of polyadenosine residues to the transcribed mRNA. Any polyadenylation sequence which is functional in the host cell may be used.

[0113] Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger α-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0114] Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15:5983-5990.

[0115] The control sequence may also be a signal peptide coding region that codes for a signal peptide linked to the N-terminus of the variant and directs the variant into the secretory pathway of the cell. The 5'-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence originally linked in translation reading frame with the segment of the coding sequence that codes for the variant. Alternatively, the 5'-end of the coding sequence may contain a signal peptide coding sequence foreign to the coding sequence. A foreign signal peptide coding sequence may be required if the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, the foreign signal peptide coding sequence may simply replace the native signal peptide coding sequence to enhance secretion of the variant. However, any signal peptide coding sequence that directs the expressed variant into the secretory pathway of the host cell may be used.

[0116] Effective signal peptide coding sequences for bacterial host cells are those obtained from the genes of Bacillus NCIB11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis β-lactamase, Bacillus stearothermophilus α-amylase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM) and Bacillus subtilis prsA. Further signal peptides are described in Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

[0117] Effective signal peptide coding sequences for filamentous fungal host cells are those obtained from the genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase.

[0118] Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al., 1992, supra.

[0119] The control sequence may also be a propeptide coding sequence that codes for a propeptide position at the N-terminus of the variant. The resulting polypeptide is known as a zymogen or propolypeptide (or, in some cases, a zymogen). A propolypeptide is generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. Propeptide coding sequences may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95 / 33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae α-factor.

[0120] When both a signal peptide and a propeptide sequence are present, the propeptide sequence is positioned adjacent to the N-terminus of the variant and the signal peptide sequence is positioned adjacent to the N-terminus of the propeptide sequence.

[0121] It may be desirable to add regulatory sequences that regulate the expression of the variant relative to the growth of the host cell. Examples of regulatory systems are those that turn the expression of the gene on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 or GAL1 system may be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter, the Aspergillus oryzae TAKA alpha-amylase promoter, and the Aspergillus oryzae glucoamylase promoter may be used. Other examples of regulatory sequences are those that allow for gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene, which is amplified in the presence of methotrexate, and the metallothionein genes, which are amplified with heavy metals. In these cases, the polynucleotide encoding the variant would be operably linked to the regulatory sequence.

[0122] Expression vector The present invention also relates to recombinant expression vectors comprising a polynucleotide encoding a variant polypeptide of the invention, a promoter, and transcriptional and translational stop signals.Thus, the present invention relates to recombinant expression vectors comprising a polynucleotide encoding a variant polypeptide of the invention, a promoter, and transcriptional and translational stop signals, which have α-amylase activity and exhibit enhanced cleaning performance compared to the polypeptide of SEQ ID NO:1. In particular, the present invention relates to positions 10, 25, 30, 37, 40, 48, 51, 54, 64, 81, 86, 93, 98, 105, 108, 109, 113, 116, 118, 121, 130, 135, 138, 142, 167, 174, 175, 178, 182, 186, 187, 189, 195, 198, 202, 203, 206, 208, 210, 214, 218, 235, 238, 242, 243, 246, 247, 250, 255, 257, 259, 260, 261, 265, 267, 269, 270, 271, 272, 273, 274, 275, 278, 279, 280, 282, 283, 284, 285, 286, 287, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 308, 309, 310, 314, 318, 320, 325, 326, 327, 328, 330, 335, 338, 340, The present invention relates to a recombinant expression vector comprising a polynucleotide encoding a variant polypeptide comprising mutations at one or more positions corresponding to 4, 275, 276, 281, 295, 298, 299, 311, 319, 320, 334, 339, 360, 365, 366, 383, 384, 385, 394, 398, 402, 404, 416, 434, 460, 469, 474 and 482 (where the numbers are according to the amino acid sequence set forth in SEQ ID NO:2), a promoter, and transcriptional and translational stop codons.

[0123] Various nucleotides and control sequences are combined to generate a recombinant expression vector that may contain one or more convenient restriction sites to allow insertion or replacement of a polynucleotide encoding a variant at such site. Alternatively, a polynucleotide may be expressed by inserting a polynucleotide or a nucleic acid construct containing a polynucleotide into a vector suitable for expression. In forming an expression vector, a coding sequence is positioned in a vector such that the coding sequence is operably linked to a control sequence suitable for expression.

[0124] The recombinant expression vector can be any vector (e.g., a plasmid or a virus) that can be easily subjected to recombinant DNA methods and can result in the expression of a polynucleotide. The choice of vector will typically depend on the affinity of the vector for the host cell into which it is introduced. The vector can be a linear or closed circular plasmid.

[0125] The vector may be an autonomously replicating vector (i.e., a vector that exists as an extrachromosomal entity and whose replication is independent of chromosomal replication), such as a plasmid, an extrachromosomal element, a microchromosome, or an artificial chromosome. The vector may contain some means for ensuring autonomous replication. Alternatively, the vector may be one that, when introduced into a host cell, integrates into the genome and replicates together with the chromosome into which it is integrated. Moreover, a single vector or plasmid, or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used.

[0126] Vectors preferably contain one or more selectable markers which facilitate the selection of cells that have been transformed, transfected, transduced, etc. A selectable marker is a gene the product of which provides biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, etc.

[0127] Examples of bacterial selectable markers are the Bacillus licheniformis or Bacillus subtilis dal genes, or markers that confer antibiotic resistance such as ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1 and URA3. Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase) and trpC (anthranilate synthase), and equivalents thereof. Preferred for use in Aspergillus cells are the Aspergillus nidulans or Aspergillus oryzae amdS and pyrG genes, and the Streptomyces hygroscopicus bar gene.

[0128] Preferably, the vector contains elements that allow for integration of the vector into the genome of the host cell, or for autonomous replication of the vector in the cell independent of the genome.

[0129] For integration into the host cell genome, the vector may rely on the sequence of the polynucleotide encoding the variant or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides that result in integration by homologous recombination into the genome of the host cell at a precise location in the chromosome. To increase the likelihood of integration at a precise location, the integration element should contain a sufficient number of nucleic acids, such as 100-10,000 base pairs, 400-10,000 base pairs, and 800-10,000 base pairs, with a high degree of sequence identity to the corresponding target sequence to increase the probability of homologous recombination. The integration element may be any sequence that is homologous to the target sequence in the genome of the host cell. Furthermore, the integration element may be a non-coding or coding polynucleotide. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.

[0130] With regard to autonomous replication, the vector may further comprise an origin of replication that allows the vector to replicate autonomously in the host cell of interest. The origin of replication may be any plasmid replicator that mediates autonomous replication in the cell. The term "origin of replication" or "plasmid replicator" refers to a polynucleotide that allows a plasmid or vector to replicate in vivo.

[0131] Examples of bacterial origins of replication are those of the plasmids pBR322, pUC19, pACYC177 and pACYC184, which permit replication in E. coli, and those of pUB110, pE194, pTA1060 and pAMβ1, which permit replication in Bacillus.

[0132] Examples of origins of replication for use in yeast host cells are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.

[0133] Examples of replication origins useful in filamentous fungal cells are AMA1 and ANS1 (Gems et al., 1991, Gene 98:61-67; Cullen et al., 1987, Nucleic Acids Res. 15:9163-9175; WO 00 / 24883). Isolation of the AMA1 gene and construction of a plasmid or vector containing the AMA1 gene can be accomplished according to the methods disclosed in WO 00 / 24883.

[0134] More than one copy of a polynucleotide of the invention may be inserted into a host cell to enhance the production of variants. The number of copies of the polynucleotide can be increased by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene in the polynucleotide, where cells containing an amplified copy of the selectable marker gene, and thus the additional copies of the polynucleotide, can be selected by culturing the cells in an appropriate selectable agent.

[0135] The techniques used to ligate the above-described elements to construct the recombinant expression vectors of the present invention are well known to those of skill in the art (see, for example, Sambrook et al., 1989, supra).

[0136] host cell The present invention also relates to recombinant host cells comprising a polynucleotide encoding a variant polypeptide of the invention operably linked to one or more control sequences that result in the production of the variant polypeptide of the invention. Thus, the present invention relates to recombinant host cells comprising a polynucleotide encoding a variant polypeptide, which has α-amylase activity and exhibits enhanced cleaning performance compared to the polypeptide of SEQ ID NO:1, operably linked to one or more control sequences that result in the production of the variant polypeptide. In particular, the present invention provides polypeptides having the polypeptide sequences at positions 10, 25, 30, 37, 40, 48, 51, 54, 64, 81, 86, 93, 98, 105, 108, 109, 113, 116, 118, 121, 130, 135, 138, 142, 167, 174, 175, 178, 182, 186, 187, 189, 195, 198, 202, 203, 206, 208, 210, 214, 218, 235, 238, 242, 243, 246, 247, 250, 255, 257, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310 259, 260, 261, 265, 267, 269, 270, 274, 275, 276, 281, 295, 298, 299, 311, 319, 320, 334, 339, 360, 365, 366, 383, 384, 385, 394, 398, 402, 404, 416, 434, 460, 469, 474 and 482 (where the numbers are according to the amino acid sequence set forth in SEQ ID NO:2).

[0137] The construct or vector containing the polynucleotide is introduced into a host cell such that the construct or vector is maintained as a chromosomal integrant or as an autonomously replicating extra-chromosomal vector as previously described. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that arise during replication. The choice of host cell will depend largely on the gene encoding the variant and its source.

[0138] The host cell can be any cell useful in the recombinant production of the variant, e.g., prokaryotic or eukaryotic.

[0139] Prokaryotic host cells can be either gram-positive or gram-negative bacteria, including but not limited to Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0140] Bacterial host cells include, but are not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus The cell may be any Bacillus cell, including Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

[0141] A bacterial host cell may also be any Streptococcus cell, including, but not limited to, any Streptococcus cell, including any of Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus zooepidemicus cells.

[0142] A bacterial host cell can also be any Streptomyces cell, including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

[0143] Introduction of DNA into Bacillus cells can be by protoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen. Genet. 168:111-115), competent cell transformation (see, e.g., Young and Spizizen, 1961, J. Bacteriol. 81:823-829, or Dubnau and Davidoff-Abelson, 1971, J. Mol. Biol. 56:209-221), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6:742-751), or by conjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). Introduction of DNA into E. coli cells can be by protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166:557-580) or electroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res. 16:6127-6145). Introduction of DNA into Streptomyces cells can be by protoplast transformation, electroporation (see, e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49:399-405), conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98:6289-6294). Introduction of DNA into Pseudomonas cells can be by electroporation (see, e.g., Choi et al., 2006, J. Microbiol. Methods 64:391-397) or by conjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71:51-57).Introduction of DNA into Streptococcus cells can be accomplished by natural competence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick, 1991, Microbios 68:189-207), electroporation (see, e.g., Buckley et al., 1999, Appl. Environ. Microbiol. 65:3800-3804), or conjugation (see, e.g., Clewell, 1981, Microbiol. Rev. 45:409-436). However, any method known in the art for introducing DNA into a host cell can be used.

[0144] The host cell can also be a eukaryotic cell, such as a plant or fungal cell.

[0145] The host cell may be a fungal cell. As used herein, "fungi" includes Ascomycota, Basidiomycota, Chytridiomycota and Zygomycota, as well as Oomycota and all vegetative spore-forming fungi (as defined in Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).

[0146] The fungal host cell may be a yeast cell. As used herein, "yeast" includes ascomycete yeasts (Endomycetales), basidiomycete yeasts, and yeasts belong to the fungi imperfecti (Blastomycetes). Because the classification of yeasts may change in the future, for the purposes of the present invention, yeasts shall be defined as described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0147] Yeast host cells may be selected from the group consisting of Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, and Yarrowia aliphatica. The cell may be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces or Yarrowia cell, such as a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces or Yarrowia cell, such as a Candida lipolytica cell.

[0148] The fungal host cell may be a filamentous fungal cell. "Filamentous fungi" includes all filamentous forms of the phylum Eumycota and the subphylum Oomycota (as defined supra by Hawksworth et al., 1995). Filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.

[0149] Filamentous fungal host cells include those of the genera Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neomyces, and others. The cell may be a Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.

[0150] For example, filamentous fungal host cells include those of Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannosinta, and the like. pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bacteridioidesbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolorversicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei or Trichoderma viride cells.

[0151] Fungal cells can be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable techniques for the transformation of Aspergillus and Trichoderma host cells are described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio / Technology 6:1419-1422. Suitable methods for the transformation of Fusarium species are described in Malardier et al., 1989, Gene 78:147-156, and WO 96 / 00787. Yeast can be transformed using the techniques described in Becker and Guarente, In Abelson, JN and Simon, MI, editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75:1920.

[0152] Generation method The present invention also relates to a method for producing a variant α-amylase, comprising: (a) culturing a host cell of the invention under conditions suitable for expression of the variant polypeptide; and (b) recovering the variant polypeptide.

[0153] The host cells are cultured in a nutrient medium suitable for the production of the variant using methods known in the art. For example, the cells can be cultured by shake flask culture or by small- or large-scale fermentation (including continuous, batch, fed-batch or solid-state fermentation) in a suitable medium and under conditions that allow expression and / or isolation of the variant. The culture is carried out in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts, using techniques known in the art. Suitable media are available from commercial suppliers or can be prepared according to published compositions (e.g., catalogs of the American Type Culture Collection). If the variant is secreted into the nutrient medium, it can be recovered directly from the medium. If the variant is not secreted, it can be recovered from cell lysates.

[0154] The variants can be detected using methods known in the art. Suitable detection methods include, but are not limited to, the use of specific antibodies, the formation of an enzyme product, or the disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the alpha amylase activity of the variants (see Examples).

[0155] The variant may be recovered using methods known in the art For example, the variant may be recovered from the nutrient medium by conventional techniques including, but not limited to, harvesting, centrifugation, filtration, extraction, spray drying, evaporation or precipitation.

[0156] The variants may be purified by a variety of techniques known in the art, including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing and size exclusion), electrophoretic techniques (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson and Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure variants.

[0157] In an alternative embodiment, the variants are not recovered and a host cell of the invention expressing the variant may be used as a source of the variant.

[0158] composition The α-amylase polypeptides of the invention may be added and therefore become a component of detergent compositions. Accordingly, the present invention relates to compositions comprising variant polypeptides that have α-amylase activity and exhibit enhanced cleaning performance compared to the polypeptide of SEQ ID NO:1. In particular, the present invention relates to positions 10, 25, 30, 37, 40, 48, 51, 54, 64, 81, 86, 93, 98, 105, 108, 109, 113, 116, 118, 121, 130, 135, 138, 142, 167, 174, 175, 178, 182, 186, 187, 189, 195, 198, 202, 203, 206, 208, 210, 214, 218, 235, 238, 242, 243, 246, 247, 250, 255, 257, 259, 26 and 482, wherein the numbers are in accordance with the amino acid sequence set forth in SEQ ID NO:2.

[0159] Thus, a further aspect of the present invention provides a detergent composition comprising a polypeptide of the present invention and a surfactant, such as an anionic surfactant, a cationic surfactant, a nonionic surfactant and / or an amphoteric surfactant. Concentrates or additives for forming such detergent compositions are also provided, which concentrates or additives comprise a polypeptide of the present invention and, optionally, a surfactant.

[0160] As discussed in detail below, the detergent compositions may further comprise one or more additional components selected from the group consisting of oxidizing agents, bleach activators, fillers, builders, buffers, structuring agents, sequestrants, optical brighteners, defoamers, enzymes, fragrances, anti-redeposition agents, skin conditioning agents, softness enhancing agents, emulsifiers and colorants.

[0161] In one embodiment, the composition is a liquid or powder laundry detergent composition.

[0162] In a further embodiment, the composition is a liquid or powder automatic dishwashing (ADW) detergent composition.

[0163] The detergent compositions of the present invention may be formulated, for example, as hand or machine laundry detergent compositions comprising a laundry additive composition suitable for pre-treating soiled fabrics and a fabric softener composition with added rinse, or as detergent compositions for use in routine household hard surface cleaning operations, or for hand or machine dishwashing operations.

[0164] In certain embodiments, the present invention provides detergent concentrates / additives comprising the α-amylase polypeptides of the present invention. The detergent concentrates / additives, as well as detergent compositions, may contain one or more other enzymes, such as proteases, lipases, peroxidases, other starch degrading enzymes, e.g., other α-amylases, glucoamylases, maltogenic amylases, CGTases and / or cellulases, mannanases (such as MANNAWAY™ from Novozymes, Denmark), pectinases, pectin lyases, cutinases and / or laccases.

[0165] Generally, the properties of the enzyme selected should be compatible with the detergent selected (i.e., pH optimum and compatibility with other enzyme and non-enzyme ingredients, etc.) and the enzyme should be present in an effective amount.

[0166] Proteases: Suitable proteases include those of animal, vegetable or microbial origin. Those of microbial origin are preferred. Chemically modified mutants or protein modified mutants are included. The protease may be a serine protease or a metalloprotease, preferably an alkaline microbial protease or a trypsin-like protease. Examples of alkaline proteases are subtilisins, especially those derived from Bacillus, such as subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89 / 06279). Examples of trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and Fusarium proteases, described in WO 89 / 06270 and WO 94 / 25583.

[0167] Examples of useful proteases are the variants described in WO 92 / 19729, WO 98 / 20115, WO 98 / 20116 and WO 98 / 34946, in particular variants with substitutions at one or more of the following positions: 27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and 274. Preferred commercially available protease enzymes include ALCALASE®, SAVINASE® (SEQ ID NO: 3), PRIMASE®, DURALASE®, ESPERASE® and KANNASE® (from Novozymes A / S), MAXATASE®, MAXACAL, MAXAPEM®, PROPERASE®, PURAFECT®, PURAFECT OXP®, FN2®, FN3®, FN4® (Genencor International Inc.).

[0168] Lipases: Suitable lipases include those of bacterial or fungal origin. Chemically modified mutants or protein modified mutants are included. Examples of useful lipases include lipases from the genus Humicola (syn. Thermomyces), e.g. H. lanuginosa (T. lanuginosus) as described in EP 258 068 and EP 305 216 or H. insolens as described in WO 96 / 13580; lipases from the genus P. alcali or P. pseudoalcali (EP 218 272), P. cepacia (EP 331 281); Pseudomonas lipases, such as those from Pseudomonas sp. SD705 (WO 95 / 06720 and WO 96 / 27002), P. wisconsinensis (WO 96 / 12012); Examples of lipases that may be used include lipases from the genus Bacillus, such as B. stearothermophilus (JP Patent Publication 64 / 744992 A) or B. pumilus (WO 91 / 16422 A).

[0169] Preferred commercially available lipase enzymes include LIPOLASE™ and LIPOLASE ULTRA™ (Novozymes A / S, SEQ ID NO: 4 herein).

[0170] Amylases: Suitable amylases (α and / or β) include those of bacterial or fungal origin, including chemically modified mutants or protein modified mutants. Amylases include α-amylases obtained from Bacillus, for example, special strains of Bacillus licheniformis, which are described in more detail in GB Patent No. 1,296,839. Examples of useful amylases are the variants described in WO 94 / 02597, WO 94 / 18314, WO 96 / 23873 and WO 97 / 43424, in particular variants with substitutions at one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391, 408 and 444. Commercially available α-amylases are DURAMYL™, LIQUEZYME™, TERMAMYL™, NATALASE™, FUNGAMYL™ and BAN™ (Novozymes A / S), Preferenz S100, Preferenz S110, Preferenz S1000, Excellenz S110, Excellenz S1000, Excellenz S2000, RAPIDASE™ and PURASTAR™ (Genencor International Inc.). Thus, suitable amylases may be any one of those listed herein as SEQ ID NOs: 5, 6, 7, 8, 9, 10 and 11, as well as variants thereof.

[0171] Cellulases: Suitable cellulases include those of bacterial or fungal origin, including chemically modified or protein modified mutants. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, and Acremonium, such as fungal cellulases produced from Humicola insolens, Myceliophthora thermophila, and Fusarium oxysporum, as disclosed in U.S. Patent No. 4,435,307, U.S. Patent No. 5,648,263, U.S. Patent No. 5,691,178, U.S. Patent No. 5,776,757, and WO 89 / 09259. Particularly suitable cellulases are alkaline or neutral cellulases with color management benefits. Examples of such cellulases are those described in EP 0 495 257, EP 0 531 372, WO 96 / 11262, WO 96 / 29397, WO 98 / 08940. Other examples are cellulase variants such as those described in WO 94 / 07998, EP 0 531 315, U.S. Pat. No. 5,457,046, U.S. Pat. No. 5,686,593, U.S. Pat. No. 5,763,254, WO 95 / 24471, WO 98 / 12307 and International Patent Application No. PCT / DK98 / 00299.

[0172] Commercially available cellulases include CELLUZYME® and CAREZYME® (Novozymes A / S), CLAZINASE® and PURADAX HA® (Genencor International Inc.), and KAC-500(B)® (Kao Corporation).

[0173] Peroxidase / Oxidase: Suitable peroxidase / oxidases include those of plant, bacterial or fungal origin. Chemically modified mutants or protein modified mutants are included. Examples of useful peroxidases include peroxidases from the genus Coprinus, e.g., C. cinereus, and its variants, such as those described in WO 93 / 24618, WO 95 / 10602 and WO 98 / 15257. Commercially available peroxidases include GUARDZYME® (Novozymes A / S).

[0174] Lechinase: Suitable lekinases include those of bacterial or fungal origin. These may be chemically or proteolytically modified. Examples of useful lekinases include those provided in SEQ ID NOs: 3, 4 and 5 (Novozymes A / S) and those provided in WO 99 / 06516 (Henkel KGAA). Other suitable examples of lechinases, which may be chemically or proteolytically modified, are those provided herein as SEQ ID NOs: 12, 13, 14 and 15.

[0175] The detergent enzymes may be included in the detergent composition by adding individual additives containing one or more enzymes or by adding a composite additive containing all of these enzymes. The detergent additives of the present invention, i.e. individual additives or composite additives, can be formulated, for example, as granules, liquids, slurries, etc. Preferred detergent additive formulations are granules, especially non-dusting granules, liquids, especially stabilized liquids, or slurries.

[0176] Non-dusting granules may be produced, for example, as disclosed in US Pat. Nos. 4,106,991 and 4,661,452, and may optionally be coated by methods known in the art. Examples of waxy coating materials are poly(ethylene oxide) products (polyethylene glycols, PEGs) with an average molecular weight of 1000 to 20000; ethoxylated nonylphenols with 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluidized bed technology are described in GB 1483591. Liquid enzyme preparations may be stabilized, for example, by adding polyols such as propylene glycol, sugars or sugar alcohols, lactic acid or boric acid, according to established methods. The protected enzymes may be prepared according to the method disclosed in EP 238 216.

[0177] The detergent compositions of the present invention may be in any convenient form, for example, as a bar, tablet, powder, granule, paste or liquid. Liquid detergents may be aqueous, typically containing up to 70% water and 0-30% organic solvents, or may be non-aqueous.

[0178] The detergent composition comprises one or more surfactants which may be nonionic, including semi-polar, and / or anionic and / or cationic and / or zwitterionic. The surfactants are typically present at a level of from 0.1% to 60% by weight.

[0179] If included, the detergent will typically contain from about 1% to about 40% of an anionic surfactant such as linear alkylbenzene sulfonates, α-olefin sulfonates, alkyl sulfates (fatty alcohol sulfates), alcohol ethoxy sulfates, secondary alkanesulfonic acids, α-sulfofatty acid methyl esters, alkyl- or alkenyl succinic acids, or soaps.

[0180] If included, the detergent will typically contain from about 0.2% to about 40% of a nonionic surfactant such as an alcohol ethoxylate, nonyl-phenol ethoxylate, alkyl polyglycoside, alkyl dimethylamine-oxide, ethoxylated fatty acid monoethanol-amide, fatty acid mono-ethanolamide, polyhydroxyalkyl fatty acid amide, or N-acyl N-alkyl derivative of glucosamine ("glucamide").

[0181] The detergent may contain 0-65% of detergent builders or complexing agents such as zeolites, diphosphates, triphosphates, phosphonates, carbonates, citric acid, nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinic acids, soluble silicic acid or layered silicates (e.g. SKS-6 from Hoechst). Other complexing agents may be methylglycine diacetate (MGDA) and glutamic acid diacetate (GLDA), which may be used especially in phosphate-free automatic dishwashing detergents.

[0182] The detergent may contain one or more polymers. Examples are sulfonated polymers, carboxymethylcellulose, poly(vinyl-pyrrolidone), poly(ethylene glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole), polycarboxylates such as polyacrylates, maleic acid / acrylic acid copolymers and lauryl methacrylate / acrylic acid copolymers.

[0183] The detergent may contain a bleach system that may include a H2O2 source, such as a perborate or percarbonate, which may be combined with a peracid-forming bleach activator, such as tetraacetylethylenediamine or nonanoyloxybenzenesulfonate. Additionally, the bleach catalyst may be a Mn and / or Co-based component. Alternatively, the bleach system may include peracids of, for example, the amide, imide or sulfone type.

[0184] The enzymes of the detergent compositions of the invention may be stabilized using conventional stabilizers, e.g. polyols such as propylene glycol or glycerol, sugars or sugar alcohols, lactic acid, boric acid or boric acid derivatives, e.g. aromatic boric acid esters, or phenylboronic acid derivatives, e.g. 4-formylphenylboronic acid, and the compositions may be formulated as described, e.g., in WO 92 / 19709 and WO 92 / 19708.

[0185] The detergent may also contain other conventional detergent formulation ingredients such as, for example, fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil suspension agents, anti-redeposition agents, dyes, germicides, optical brighteners, hydrotropes, anti-fading agents or fragrances.

[0186] It is contemplated herein that in the detergent composition any enzyme, particularly the alpha-amylase polypeptide of the invention, may be added in an amount corresponding to 0.01-100 mg enzyme protein / litre of cleaning solution, preferably 0.05-5 mg enzyme protein / litre of cleaning solution, in particular 0.1-1 mg enzyme protein / litre of cleaning solution.

[0187] The alpha amylase polypeptides of the present invention may additionally be incorporated into detergent formulations as disclosed in WO 2006 / 002643, which is incorporated herein by reference.

[0188] Examples of dishwashing detergent compositions of the present invention The α-amylase polypeptides of the invention may also be used in dishwashing detergent compositions, including:

[0189] (a) Powdered automatic dishwashing composition Nonionic surfactant 0.4~2.5% Sodium metasilicate 0~20% Sodium disilicate 3-20% Sodium triphosphate 20-40% Sodium carbonate 0-20% Sodium perborate 2-9% Tetraacetylethylenediamine (TAED) 1-4% Sodium sulfate 5~33% Enzymes (e.g. α-amylase polypeptides) 0.0001-0.1%

[0190] (b) Powdered automatic dishwashing composition Nonionic surfactant 1-2% Sodium disilicate 2~30% Sodium carbonate 10~50% Sodium phosphate 0-5% Trisodium citrate dihydrate 9~30% Sodium nitrilotriacetate (NTA) 0-20% Sodium perborate monohydrate 5-10% Tetraacetylethylenediamine (TAED) 1-2% Polyacrylate polymers (e.g. maleic acid / acrylic acid copolymers) 6-25% Enzymes (e.g. α-amylase polypeptides) 0.0001-0.1% Air freshener 0.1~0.5% Water 5~10%

[0191] (c) Powdered automatic dishwashing composition Nonionic surfactant 0.5~2.0% Sodium disilicate 25~40% Sodium citrate 30~55% Sodium carbonate 0~29% Sodium bicarbonate 0-20% Sodium perborate monohydrate 0~15% Tetraacetylethylenediamine (TAED) 0~6% Maleic acid / acrylic acid copolymer 0~5% Clay 1~3% Polyamino acids 0~20% Sodium polyacrylate 0~8% Enzymes (e.g. α-amylase polypeptides) 0.0001-0.1%

[0192] (d) Powdered automatic dishwashing compositions Nonionic surfactant 1-2% Zeolite MAP 15~42% Sodium disilicate 30-34% Sodium citrate 0~12% Sodium carbonate 0-20% Sodium perborate monohydrate 7~15% Tetraacetylethylenediamine (TAED) 0~3% Polymer 0~4% Maleic acid / acrylic acid copolymer 0~5% Organic phosphonates 0-4% Clay 1~2% Enzymes (e.g. α-amylase polypeptides) 0.0001-0.1% Sodium sulfate remaining

[0193] (e) Powdered automatic dishwashing compositions Nonionic surfactant 1-7% Sodium disilicate 18~30% Trisodium citrate 10~24% Sodium carbonate 12-20% Monopersulfate (2KHSO5.KHSO4.K2SO4) 15~21% Bleach stabilizer 0.1~2% Maleic acid / acrylic acid copolymer 0~6% Diethylenetriaminepentaacetic acid pentasodium 0~2.5% Enzymes (e.g. α-amylase polypeptides) 0.0001-0.1% Sodium sulfate, water remaining

[0194] (f) Powder and liquid dishwashing compositions containing a cleaning surfactant system Nonionic surfactant 0~1.5% Octadecyldimethylamine N-oxide dihydrate 0~5% Octadecyldimethylamine 80:20 wt. C18 / C16 blend N-oxide dihydrate and hexadecyldimethylamine N-Oxide dihydrate 0~4% 70:30 wt. C18 / C16 blend of anhydrous octadecylbis(hydroxyethyl)amine N-oxide and anhydrous hexadecylbis(hydroxyethyl)amine N-oxide 0-5% C with an average ethoxylation degree of 3 13 ~C 15 Alkyl ethoxy sulfate 0~10% C with an average ethoxylation degree of 3 12 ~C 15 Alkyl ethoxy sulfate 0~5% C with an average ethoxylation degree of 12 13 ~C15 Ethoxylated alcohol 0-5% C with an average ethoxylation degree of 9 12 ~C 15 Ethoxylated Alcohol Blend 0-6.5% C with an average degree of ethoxylation of 30 13 ~C 15 Ethoxylated Alcohol Blend 0-4% Sodium disilicate 0~33% Sodium tripolyphosphate 0~46% Sodium citrate 0~28% Citric acid 0~29% Sodium carbonate 0-20% Sodium perborate monohydrate 0~11.5% Tetraacetylethylenediamine (TAED) 0~4% Maleic acid / acrylic acid copolymer 0~7.5% Sodium sulfate 0~12.5% Enzymes (e.g. α-amylase polypeptides) 0.0001-0.1%

[0195] (p) Non-aqueous liquid automatic dishwashing compositions Liquid nonionic surfactants (e.g., alcohol ethoxylates) 2.0-10.0% Alkali metal silicate 3.0~15.0% Alkali metal phosphate 20.0~40.0% Liquid carrier selected from higher glycols, polyglycols, polyoxides, and glycol ethers 25.0-45.0% Stabilizers (e.g., partial esters of phosphoric acid and C 16 ~C 18 Alkanol) 0.5-7.0% Foam suppressors (e.g. silicone) 0-1.5% Enzymes (e.g. α-amylase polypeptides) 0.0001-0.1%

[0196] (q) Non-aqueous liquid dishwashing compositions Liquid nonionic surfactants (e.g. alcohol ethoxylates) 2.0-10.0% Sodium silicate 3.0~15.0% Alkali metal carbonate 7.0~20.0% Sodium citrate 0.0~1.5% Stabilizing system (e.g. mixture of micronized silicone and low molecular weight dialkyl polyglycol ether) 0.5-7.0% Low molecular weight polyacrylate polymer 5.0-15.0% Clay gel thickener (e.g. bentonite) 0.0-10.0% Hydroxypropyl cellulose polymer 0.0~0.6% Enzymes (e.g. α-amylase polypeptides) 0.0001-0.1% Liquid carrier selected from higher glycols, polyglycols, polyoxides and glycol ethers.

[0197] (r) Thixotropic liquid automatic dishwashing compositions C 12 ~C 14 Fatty acids 0~0.5% Block copolymer surfactant 1.5-15.0% Sodium citrate 0~12% Sodium tripolyphosphate 0~15% Sodium carbonate 0~8% Aluminum glycerin tristearate 0~0.1% Sodium cumene sulfonate 0~1.7% Polyacrylate thickener 1.32~2.5% Sodium polyacrylate 2.4~6.0% Boric acid 0~4.0% Sodium Formate 0~0.45% Calcium formate 0~0.2% Sodium n-diphenyl oxide disulfonate 0~4.0% Monoethanolamine (MEA) 0-1.86% Sodium hydroxide (50%) 1.9~9.3% 1,2-Propanediol 0~9.4% Enzymes (e.g. α-amylase polypeptides) 0.0001-0.1% Foam suppressor, dye, fragrance, water remaining

[0198] (s) Liquid automatic dishwashing compositions Alcohol ethoxylate 0~20% Fatty acid ester sulfonate 0~30% Sodium dodecyl sulfate 0~20% Alkyl polyglycoside 0~21% Oleic acid 0~10% Sodium disilicate monohydrate 18~33% Sodium citrate dihydrate 18~33% Sodium stearate 0~2.5% Sodium perborate monohydrate 0~13% Tetraacetylethylenediamine (TAED) 0~8% Maleic acid / acrylic acid copolymer 4~8% Enzymes (e.g. α-amylase polypeptides) 0.0001-0.1%

[0199] (t) Liquid automatic dishwashing compositions containing protected bleach particles Sodium silicate 5-10% Potassium pyrophosphate 15~25% Sodium triphosphate 0~2% Potassium carbonate 4~8% Protected bleach particles, e.g. chlorine 5-10% Polymer thickener 0.7~1.5% Potassium hydroxide 0~2% Enzymes (e.g. α-amylase polypeptides) 0.0001-0.1% Water remaining

[0200] (u) The automatic dishwashing composition of (a), (b), (c), (d), (f) and (j), wherein the perborate is replaced with a percarbonate.

[0201] (v) An automatic dishwashing composition as described in (a)-(f), additionally comprising a manganese catalyst, which may be, for example, one of the compounds described in “Efficient manganese catalysts for low-temperature bleaching”, Nature 369, 1994, pp. 637-639.

[0202] use The present invention also relates to methods of using the α-amylase polypeptides of the invention in detergents, particularly laundry and dishwashing detergent compositions.

[0203] Thus, the present invention provides the use of an α-amylase polypeptide or composition of the invention in domestic or industrial cleaning processes.

[0204] In one embodiment, the use is in cleaning fabrics, for example laundry.

[0205] In another embodiment, the use is cleaning ceramic, plastic or glass materials, for example washing dishes.

[0206] Thus, the α-amylase polypeptides of the invention find application as components in cleaning, dishwashing and hard surface cleaning detergent compositions, either in domestic or industrial settings.

[0207] The α-amylase variants of the invention have valuable properties that enable a variety of other industrial applications. For example, the α-amylase polypeptides of the invention can be used in starch processing, particularly starch conversion, particularly starch liquefaction (see, e.g., U.S. Pat. No. 3,912,590, European Patent Application No. 252 730, European Patent Application No. 63 909, WO 99 / 19467, and WO 96 / 28567, all of which are incorporated herein by reference). Compositions for starch conversion that may include, in addition to the variants of the invention, glucoamylases, pullulanases, and other α-amylases are also contemplated.

[0208] Additionally, the α-amylase variants of the present invention are also particularly useful in producing sweeteners and ethanol, such as fuel, drinking ethanol and industrial ethanol, from starch or whole grains (see, e.g., U.S. Pat. No. 5,231,017, incorporated herein by reference).

[0209] The α-amylase variants of the invention may also be useful in desizing fabrics, cloth and garments (see, e.g., WO 95 / 21247, U.S. Pat. No. 4,643,736, EP 119,920, which are incorporated herein by reference), beer or brewing, and pulp and paper production.

[0210] Starch conversion Conventional starch conversion processes, such as liquefaction and saccharification processes, are described, for example, in U.S. Pat. No. 3,912,590 and European Patent Applications Nos. 252,730 and 63,909, which are hereby incorporated by reference.

[0211] In one embodiment, the starch conversion process that breaks down starch into lower molecular weight carbohydrate components such as sugar or fat substitutes includes a debranching step.

[0212] In the case of starch conversion to sugars, the starch is depolymerized. Such a depolymerization process may consist of a pretreatment step and two or three successive processing steps (i.e., a liquefaction process, a saccharification process, and optionally an isomerization process, depending on the desired end product).

[0213] (i) Pretreatment of native starch Native starch consists of very small granules that are insoluble in water at room temperature. When an aqueous starch slurry is heated, these granules swell and eventually burst, dispersing the starch molecules into solution. During this "gelatinization" process, the viscosity increases significantly. Since the solids level in a typical industrial process is 30-40%, the starch needs to be diluted or "liquefied" so that it can be handled. Today, this reduction in viscosity is most often accomplished by enzymatic degradation.

[0214] (ii) Liquefaction During the liquefaction step, long-chain starch is broken down by α-amylase into branched and linear shorter-chain units (maltodextrins). The liquefaction process is carried out at 105-110°C for 5-10 minutes, followed by 95°C for 1-2 hours. The pH is 5.5-6.2. To ensure optimal enzyme stability under these conditions, 1 mM calcium is added (40 ppm free calcium ions). After this treatment, the liquefied starch has a "glucose equivalent" (DE) of 10-15.

[0215] (iii) Saccharification After the liquefaction process, the maltodextrins are converted to glucose by adding glucoamylase (e.g., AMG) and debranching enzymes such as isoamylase (U.S. Pat. No. 4,335,208) or pullulanase (e.g., Promozyme™) (U.S. Pat. No. 4,560,651). Prior to this step, the pH is lowered to a value below 4.5 and elevated temperatures (above 95° C.) are maintained to inactivate the liquefaction α-amylase and reduce the formation of short-chain oligosaccharides called “panose precursors” that cannot be adequately hydrolyzed by the debranching enzymes.

[0216] The temperature is lowered to 60°C and glucoamylase and debranching enzymes are added. The saccharification process lasts for 24-72 hours.

[0217] Typically, when α-amylase is modified after the liquefaction step, approximately 0.2–0.5% of the saccharification product is a branched trisaccharide. <2> -α-glucosylmaltose (panose), which cannot be degraded by pullulanase. If active amylase from the liquefaction step is present during saccharification (i.e., no denaturation), this level can be as high as 1-2%, which is highly undesirable as it significantly reduces saccharification yields.

[0218] (iv) Isomerization If the desired final sugar product is, for example, isomerized sugar, the glucose syrup may be converted to fructose. After the saccharification process, the pH is increased to a value in the range of 6-8, preferably pH 7.5, and calcium is removed by ion exchange. The glucose syrup is then converted to isomerized sugar, for example, using immobilized glucose isomerase (such as Sweetzyme™ IT).

[0219] Ethanol Production Generally, alcohol production (ethanol) from whole grains can be divided into four main steps. -milling -Liquification -Saccharification -fermentation

[0220] (i) Flour milling The kernels are milled to open up the structure and allow further processing. Two processes are used: wet or dry milling. In dry milling, the whole kernel is milled and used for the rest of the process. Wet milling gives an excellent separation of the germ and the meal (starch granules and proteins) and, with few exceptions, is applied in sites where syrup production is taking place in parallel.

[0221] (ii) Liquefaction In the liquefaction process, starch granules are solubilized by hydrolysis to maltodextrins, mostly with a DP above 4. Hydrolysis can be performed by acid treatment or enzymatically with α-amylase. Acid hydrolysis is used to a limited extent. The raw material can be milled whole grain or by-products from starch processing.

[0222] Enzymatic liquefaction is typically carried out as a three step hot slurry process. The slurry is heated to 60-95°C, preferably 80-85°C, and the enzymes are added. The slurry is then jet cooked at 95-140°C, preferably 105-125°C, cooled to 60-95°C, and a further amount of enzyme is added to achieve final hydrolysis. The liquefaction process is carried out at a pH of 4.5-6.5, typically a pH of 5-6. The milled and liquefied grain is also known as the mash.

[0223] (iii) Saccharification Maltodextrin from the liquefaction must be further hydrolyzed to produce the low molecular weight sugars DP1-3 that are metabolizable by yeast. Hydrolysis is typically performed enzymatically with glucoamylase, but alternatively α-glucosidase or acid α-amylase can be used. The complete saccharification step can last up to 72 hours, however it is common to only perform a pre-saccharification of typically 40-90 minutes and then complete the saccharification during fermentation (SSF). Saccharification is typically carried out at temperatures between 30-65°C, typically around 60°C, and at a pH of 4.5.

[0224] (iv) Fermentation Yeast, typically from Saccharomyces spp., is added to the mash and fermentation is typically carried out for 24 to 96 hours, such as 35 to 60 hours, at a temperature of 26 to 34°C, typically about 32°C, and a pH of pH 3 to 6, preferably about pH 4 to 5.

[0225] It should be noted that the most widely used process is a simultaneous saccharification and fermentation (SSF) process, where there is no holding stage for saccharification, meaning that yeast and enzymes are added together. When performing SSF, it is common to introduce a pre-saccharification step at temperatures above 50° C. just prior to fermentation.

[0226] (v) Distillation Following fermentation, the mash is distilled to extract ethanol.

[0227] The ethanol obtained according to the process of the present invention can be used, for example, as fuel ethanol; potable ethanol, i.e., neutral spirits for drinking; or as industrial ethanol.

[0228] (vi) By-products Fermentation leaves behind grain that is typically used in liquid form or dried for animal feed.

[0229] Further details as to how to carry out the liquefaction, saccharification, fermentation, distillation and recovery of ethanol are known to those skilled in the art.

[0230] According to the process of the present invention, saccharification and fermentation can be carried out simultaneously or separately.

[0231] Pulp and Paper Manufacturing The alkaline α-amylase polypeptides of the invention may also be used in the production of lignocellulosic materials such as pulp, paper and cardboard from waste paper and cardboard fortified with starch, especially where repulping is carried out at a pH above 7 and the amylase promotes disintegration of the waste material via degradation of the fortifying starch. The α-amylases of the invention are particularly useful in a process for producing paper pulp from printed starch-coated paper. This process may be carried out as described in WO 95 / 14807 and comprises the following steps: a) disintegrating paper to produce pulp b) treating with an amylolytic enzyme before, during or after step a); and c) separating the ink particles from the pulp after steps a) and b).

[0232] The α-amylases of the invention may also be extremely useful in starch modification in papermaking, where enzymatically modified starch is used together with alkaline fillers such as calcium carbonate, kaolin and clays. The alkaline α-amylases of the invention allow for starch modification in the presence of fillers, thus allowing for a simpler and more consistent process.

[0233] Desizing fabrics, cloth and clothing The α-amylases of the present invention may also be extremely useful in desizing fabrics, cloths or garments. In the textile processing industry, α-amylases are traditionally used as aids in the desizing process to facilitate the removal of starch-containing textile size, which serves as a protective coating for the weft yarns during weaving. It is important to completely remove the textile size coat after weaving to achieve optimal results in the subsequent processes in which the fabric is subjected to scouring, bleaching and dyeing. Enzymatic starch degradation is preferred to avoid any adverse effects on the textile material. To reduce processing costs and to increase mill throughput, desizing is sometimes combined with scouring and bleaching steps. In such cases, non-enzymatic auxiliaries such as alkalis or oxidizing agents are typically used to degrade starch, since conventional α-amylases are not highly compatible with high pH levels and bleaching agents. Non-enzymatic degradation of starch textile size leads to some fiber damage due to the aggressiveness of the agents. Therefore, it would be desirable to use the α-amylases of the invention for improved performance in alkaline solutions, either used alone or in combination with cellulases when desizing cellulose-containing fabrics or materials.

[0234] Desizing and bleaching processes are well known in the art, for example, such processes are described in WO 95 / 21247, U.S. Pat. No. 4,643,736, and EP 119,920, which are incorporated herein by reference.

[0235] Commercially available desizing products include AQUAZYME® and AQUAZYME® ULTRA, manufactured by Novozymes A / S.

[0236] Beer production The α-amylases of the present invention can also be extremely useful in the beer formation process; the α-amylases will typically be added during the mashing process.

[0237] This invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention.

[0238] The invention described and claimed herein is not limited in scope by the specific embodiments disclosed herein, as these are intended to be illustrative of the various embodiments of the invention. Any equivalent embodiments are intended to be within the scope of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure, including definitions, will control.

[0239] Working Example Example 1: Assay for α-amylase activity 1.Phadebas Assay Alpha-amylase activity can be determined by a method that utilizes Phadebas® tablets as a substrate. Phadebas tablets (Phadebas® Amylase Test, provided by Pharmacia Diagnostic) contain cross-linked insoluble blue starch polymers, which are mixed with bovine serum albumin and buffer substances and tableted.

[0240] For every measurement, one tablet is suspended in a tube containing 5 ml of 50 mM Britton-Robinson buffer (50 mM acetic acid, 50 mM phosphate, 50 mM boric acid, 0.1 mM CaCl2, pH adjusted to target value with NaOH). The test is performed in a water bath at the target temperature. The α-amylase to be tested is diluted in xml of 50 mM Britton-Robinson buffer. 1 ml of this α-amylase solution is added to 5 ml of 50 mM Britton-Robinson buffer. Starch is hydrolyzed by the α-amylase to obtain soluble blue fragments. The absorbance of the resulting blue solution, measured spectrophotometrically at 620 nm, is a function of α-amylase activity.

[0241] It is important that the absorbance at 620 nm measured after 10 or 15 minutes of incubation (test time) is within the range of 0.2-2.0 absorbance units at 620 nm. Within this absorbance range, there is a linear relationship between activity and absorbance (Beer-Lambert law). Therefore, it is necessary to adjust the enzyme dilution to meet this criteria. Under a specific set of conditions (temperature, pH, reaction time, buffer conditions), 1 mg of a given α-amylase will hydrolyze a certain amount of substrate, resulting in the development of a blue color. The color intensity is measured at 620 nm. The measured absorbance is directly proportional to the specific activity (activity / mg of pure α-amylase protein) of the α-amylase under the given set of conditions.

[0242] 2. Alternative method The α-amylase activity is measured by a method that utilizes the PNP-G7 substrate. PNP-G7, short for p-nitrophenyl-α,D-maltoheptaoside, is a blocked oligosaccharide that is cleavable by endo-amylase. Following cleavage, the α-glucosidase included in the kit digests the substrate to liberate free PNP molecules that are yellow in color, allowing measurement by visible spectrophotometry at λ=405 nm (400-420 nm). The kit containing the PNP-G7 substrate and α-glucosidase is from Boehringer-Mannheim (cat. no. 1054635).

[0243] To prepare the reagent solution, add 10 ml of substrate / buffer solution to 50 ml of enzyme / buffer solution as recommended by the manufacturer. The assay is performed by transferring 20 μl of sample to a 96-well microtiter plate and incubating at 25° C. Add 200 μl of reagent solution, previously equilibrated to 25° C. The solution is mixed, preincubated for 1 minute, and absorbance is measured every 30 seconds at OD405 nm in an ELISA reader for 4 minutes.

[0244] The slope of the time-dependent absorption curve is directly proportional to the activity of the α-amylase of interest under a given set of conditions.

[0245] Determination of LAS susceptibility The mutants are incubated with different concentrations of LAS (linear alkylbenzene sulfonate; Nansa 1169 / P) for 10 minutes at 40°C.

[0246] Residual activity is determined using the Phadebas® assay method or an alternative method utilizing the PNP-G7 substrate.

[0247] The LAS is diluted in 0.1 M phosphate buffer pH 7.5.

[0248] Use the following concentrations: 500 ppm, 250 ppm, 100 ppm, 50 ppm, 25 ppm and 10 ppm or no LAS. Dilute the variants in the different LAS buffers to concentrations between 0.01 and 5 mg / l in a total volume of 10 ml and incubate in a temperature-controlled water bath for 10 min. Stop the incubation by transferring a small amount into chilled assay buffer. During the activity measurement, it is important that the LAS concentration is below 1 ppm to not affect the activity measurement.

[0249] Residual activity is then measured in replicates using the Phadebas® assay described above or an alternative method. Activity is measured after blank subtraction. Activity without LAS is 100%.

[0250] Example 2: Assessment of cleaning performance of α-amylase polypeptides of the invention using a full-scale automatic dishwashing (ADW) To evaluate the cleaning performance of the polypeptides of the present invention in detergent-based compositions, cleaning experiments can be carried out using a full-scale automatic dishwashing (ADW) machine. A full-scale ADW machine is used with test conditions that mimic typical consumer settings to test the cleaning performance of the polypeptides.

[0251] In this study, the test conditions were a standard 50°C wash program and a short 40°C program using a Miele Dishwasher Miele G4300 SCU machine.

[0252] Description of general cleaning performance. Starch-soiled melamine tiles (DM-77 / DM-177 / DM-277 / DM-377 from Center For Test materials BV, POBox 120, 3133 KT, Vlaardingen, Netherlands) were used as test materials and washed at 40°C and 50°C with tap water at 19-20°dH as programmed as specified below (see Tables 2, 3 and 4). After opening the detergent dispenser in the dishwasher, detergent and α-amylase were added at a concentration of 1.5 mg polypeptide / wash or 3 mg polypeptide / wash for washing at 40°C, or 0.5 mg polypeptide / wash or 1 mg polypeptide / wash for washing at 50°C. A test at 0 mg polypeptide / L was used as a blank to represent the contribution from the detergent. Full-scale washing performance experiments were carried out under the experimental conditions specified below.

[0253] [Table 9]

[0254] [Table 10]

[0255] [Table 11]

[0256] After cleaning, the melamine tiles were allowed to dry.

[0257] The cleaning performance was measured as the difference in contract reflectance between the cleaned and uncleaned tiles. Contract reflectance was measured by obtaining spectra using a Color-Eye7000 (CE7000) and calculating the contract reflectance. Contract reflectance was measured at 460 nm with a light source that did not contain UV light.

[0258] Wash performance was considered improved if the enhancement factor (IF) was at least 1.0, preferably at least 1.2, under one or more of the conditions listed above (i.e., at either 40°C or 50°C, variant concentrations were 1.5 mg polypeptide / wash or 3 mg polypeptide / wash for the 40°C wash, and 0.5 mg polypeptide / wash or 1 mg polypeptide / wash).

[0259] The washing performance of exemplary polypeptides of the invention obtained by full-scale washing is shown in Table 5a (40°C) and Table 5b (50°C) (wherein the washing performance scores are based on the washing performance of the parent α-amylase of SEQ ID NO:1).

[0260] [Table 12]

[0261] [Table 13]

[0262] [Table 14]

[0263] Example 3: Assessment of the cleaning performance of α-amylase polypeptides of the invention using a full-scale automated mechanical stress assay (AMSA) To evaluate the cleaning performance of the polypeptides of the present invention in detergent-based compositions, cleaning experiments can also be performed using an automated mechanical stress assay (AMSA). The AMSA test allows testing the cleaning performance of large amounts of small-volume enzyme-detergent solutions. The AMSA plate has many slots for test solutions, and the lid tightly holds the swatches to be washed against the openings of all slots. During the washing time, the plate, test solutions, fabrics and lid are vigorously shaken to contact the test solutions with the fabrics and to apply mechanical stress with regular periodic shaking. For further details, see WO 02 / 42740 (especially paragraph "Special Method Embodiments" on pages 23-24, the disclosure of which is incorporated herein by reference).

[0264] General cleaning performance description Test solutions were prepared containing water (21° dH), ADW model detergent with 3.94 g / L bleach or without 3.45 g / L bleach (as specified below (see Tables 6, 7 and 8)), and the polypeptide of the invention at concentrations of 0.03, 0.06, 0.12 and 0.24 mg polypeptide protein / L (40° C.) or 0.01, 0.03, 0.06 and 0.12 mg polypeptide protein / L (50° C.). Starch-stained cloths (CS-28 from Center For Test materials BV, PO Box 120, 3133 KT, Vlaardingen, Netherlands) were added and washed at 40° C. and 50° C. for 10 or 20 minutes as specified below. After thorough rinsing with running tap water and drying in the dark, the light intensity value of the stained cloths was then measured as a measure of the cleaning performance. A test of 0 mg / L enzyme protein was used as a blank to correspond with the contribution from the detergent. It is preferred to apply mechanical action during the wash step, for example in the form of shaking, rolling or agitating the wash solution with the fabrics and tiles. The AMSA wash performance experiments were carried out under the experimental conditions specified below.

[0265] [Table 15]

[0266] [Table 16]

[0267] [Table 17]

[0268] CaCl2, MgCl2 and NaHCO3(Ca 2+ :Mg 2+ :HCO3 - The water hardness was adjusted to 21° dH by adding 100% ethanol (=4:1:10) to the test system. After cleaning, the fabrics were rinsed in tap water and dried.

[0269] The cleaning performance was measured as brightness, which is the intensity of the light reflected from the sample when illuminated with white light. If the sample was dirty, the intensity of the light reflected was lower than that of a clean sample. Therefore, the intensity of the reflected light can be used to measure the cleaning performance.

[0270] Color measurements were taken with a professional flatbed scanner (EPSON Expression 10000XL, EPSON) used to capture images of the washed fabrics.

[0271] To extract light intensity values ​​from the scanned images, the 48 24-bit color pixel values ​​from the image were converted to Red, Green, and Blue (RGB) values. The intensity value (Int) was calculated by adding the RGB values ​​together as a vector and then the length of the resulting vector.

number

[0272] The cleaning performance of the mutants according to the invention obtained with AMSA is as follows:

[0273] [Table 18]

[0274] [Table 19]

[0275] [Table 20]

[0276] [Table 21]

[0277]

Table 22

[0278]

Table 23

[0279]

Table 24

[0280]

Table 25

[0281]

Table 26

[0282]

Table 27

[0283]

Table 28

[0284]

Table 29

Claims

1. A polypeptide comprising a variant amino acid sequence of SEQ ID NO:1, said polypeptide having α-amylase activity and exhibiting enhanced cleaning performance compared to the polypeptide of SEQ ID NO:

1.

2. The polypeptide of claim 1 , wherein the variant amino acid sequence comprises a mutation at amino acid position 167 of SEQ ID NO:

1.

3. 3. The polypeptide of claim 2, wherein the variant amino acid sequence comprises a substitution at amino acid position 167 of SEQ ID NO:1, such as W167Y.

4. The polypeptide of any one of claims 1 to 3, wherein the high cleaning performance is assessed using an Automated Mechanical Stress Assay (AMSA) assay.

5. The polypeptide according to any one of claims 1 to 4, wherein said enhanced washing performance is exhibited during washing at high temperatures.

6. 6. The polypeptide of claim 5, wherein the elevated temperature is at least 40°C, such as at least 45°C, such as at least 50°C, such as at least 55°C, and such as at least 60°C.

7. The polypeptide satisfies one or more of the conditions selected from the group consisting of: (a) Model ADW detergent with bleach in a 10 minute wash cycle at 40°C; (b) Model ADW detergent without bleach in a 10 minute wash cycle at 40°C; (c) Model ADW detergent with bleach in a wash cycle of 20 minutes at 50° C.; and (d) Model ADW detergent without bleach in a 20 minute wash cycle at 50°C; 7. A method for cleaning a detergent comprising the steps of: providing a detergent composition comprising: a) a polysaccharide having a molecular weight of 100 or more; and b) a polysaccharide having a molecular weight of 100 or more; and c) a polysaccharide having a molecular weight of 100 or more;

8. 8. The polypeptide of any one of claims 1 to 7, wherein the polypeptide is 1000 or less amino acids in length, such as 900, 800, 700, 600, 500, 400, 300, 200, 175, 150, 125, 100 or less amino acids.

9. 9. The polypeptide of claim 8, wherein the polypeptide is between 400 and 600 amino acids in length, such as between 450 and 500, 460 and 500, or between 470 and 490 amino acids in length.

10. Positions 10, 25, 30, 37, 40, 48, 51, 54, 64, 81, 86, 93, 98, 105, 108, 109, 113, 116, 118, 121, 130, 135, 138, 142, 167, 174, 175, 178, 182, 186, 187, 189, 195, 198, 202, 203, 206, 208, 210, 214, 218, 235, 238, 242, 243, 246, 247, 250, 255, 257, 259, 260, 261, 2 10. The polypeptide of any one of claims 1 to 9, comprising a mutation at one or more positions corresponding to: 65, 267, 269, 270, 274, 275, 276, 281, 295, 298, 299, 311, 319, 320, 334, 339, 360, 365, 366, 383, 384, 385, 394, 398, 402, 404, 416, 434, 460, 469, 474 and 482, where the numbers are according to the amino acid sequence set forth in SEQ ID NO:

2.

11. The polypeptide of any one of claims 1 to 10, wherein the mutation at one or more positions is a substitution, deletion and / or insertion.

12. The polypeptide has the following substitutions based on the amino acid sequence of SEQ ID NO:2: M10L, N25K, D30N, K37H, K37L, K37M, K37R, K37V, S40T, W48F, A51Q, A51T, N54S, Y64W, T81S, Q86H, Q86I, Q86L, K93H, K93R, Q98R, M105Y, M105F, M105I, M105L, K108R, G109A, G109M, A113E, M116I, M116L, M116A, M116V, M116F, K118Q, K118H, K118N, K118R, E121H, E130H, E130Q, Y135H, E138Q, K142R, K142Q, W167Y, W167H, N174Q, N174*, N175Q, Y178W, T182G, A186D, A186 G, W187Y, W189H, F195N, Y198F, L202M, Y203H, Y203N, Y203G, Y203F, I206L, M208F, M208L, M208V, H210N, V214R, V214T, V214I, R218N, I23 5V, I235L, I235M, V238T, V238A, K242P, Y243F, Y243M, T246V, T246I, T246L, T246M, R247K, I250L, I250V, S255K, I257A, K259N, N260D, M 261L, M261A, A265G, F267Y, K269S, K269N, N270G, A274K, I275L, E276Q, K281H, F295Y, F295W, Y298W, Y298F, Y299W, Y299F, Q311T, Q311H, 12. The polypeptide of any one of claims 1 to 11, comprising one or more of Q311R, Q319H, Q319R, K320H, K320R, S334T, S339A, E360F, S365M, S365C, V366I, K383Q, K383R, S384E, K385H, K385Q, K385R, Q394K, Y398W, N402Y, Y404W, E416L, A434D, G460E, W469F, V474C and W482Y, wherein the numbers are in accordance with the amino acid sequence set forth in SEQ ID NO:

2.

13. The polypeptide is; K37H+L202M, E360F+S365C, M261L, H210N, Y243F, K108R, V474C, G460E, T81S, K269S+N270G+A274K, K37V+L202M, L202M+Q311R, N174Q+L202M, K385H, K385Q, K385R, K383Q, K320H, K320R, E276Q, K93R, K93H, Q98R, K118Q, K118H, K118N, E130H, E130Q, E138Q, K142R, K142Q, E416L, Q394K, S38 4E, Y64W, K37R, D30N, F295Y, Y243M, Y178W, K281H, K269N, Y198F, Q311T, F1 95N, I257A, S255K, R247K, Y404W, Y398W, Q319H, Q319R, Q311H, Q311R, Y299 W, N260D, K259N, Y299F, A434D, V366I, S365M, S339A, N174*, I235V, I250L, I250V, K37V, K37M, K37L, Y203H, S40T, W187Y, V238T, M105Y, M105F, M105I, I206L, T182G, M116I, Y135H, Y203N, Y203G, N25K, M116L, A265G, F295Y, A2 65G, K242P, V214R, M208F, Y198F, W482Y, V214T, V214I, M261A, M105F, M208 L, M116A, N174Q, N174*+N175Q, I235L, A265G, M105L, K37H, Q311R, W469F, Y 203F, G109A, N175Q, W48F, M116V, M116F, F295W, Y298W, M208V, M208F, M10L +M261L, W187Y+M208L, Y298F, W167Y, W167H, W189H, F295Y, I235M, Y243F+F 267Y, K37V+P45R+K383R, D30N+N33D+K37V+K383R, W167Y+H210N+S339A+V3 66I, S339A+V366I, W167Y+H210N+S339A, H210N+S339A, W167Y+H210N, W167 Y+L202M+H210N+Y299F+S339A+V366I, W167Y+H210N+Y243F+S339A+V366I,<h2 style=";text-align:left;direction:ltr">W167Y+H210N+S339A+V366I+W482Y,M 116F+W167Y+H210N+S339A+V366I,W4 8F+W167Y+H210N+S339A+V366I,W167 Y+H210N+Y299F+S339A+V366I,W167Y +L202M+H210N+S339A+V366I,W167Y+ L202M+H210N+Y299F+S339A,W167Y+H 210N+S339A+W482Y,M116F+W167Y+H2 10N+S339A,W48F+W167Y+H210N+S339A ,W167Y+H210N+Y299F+S339A,W167Y+L202M+H210N+S339A,W167Y+L202M+Y 299F,W167Y+Y243F,M116F+W167Y,W4 8F+W167Y,W167Y+Y299F,W167Y+L202 M,W167Y+H210N+V366I,W167Y+V366I ,H210N+V366I,W167Y+S339A+V366I, D30N+N33D+K37V+L202M+K383R,D30N+N33D+K37V+W48F+K383R,D30N+N33D+ K37V+M116F+K383R,D30N+N33D+K37V+K383R+W482Y,A51T,A51T+N54S,S33 4T,T246M,T246L,T246V,T246I,A186 G,A51Q+G109M+Y203G,V238A+S334T, A51T+L202M,L202M+T246L,A51T+N17 4Q+L202M+T246I+S334T,A51T+N174Q +L202M+Q311R+S334T,I235L+T246M+ I250L,A186D+L202M,A186D+L202M+N2 70G+N402Y,A186D+L202M+S339A,A186D+F195N+L202M,K37H+A51T+L202M, K37V+,A51T+L202M,A51T+L202M+S365C,A51T+L202M+S339A,A51T+L202M+ Q311T,A51T+L202M+M261L,A51T+L20 2M+H210N,A51T+L202M+N270G,A51T+ F195N+L202M,A51T+L202M+Q319H,A5 1T+L202M+Q319R,A51T+L202M+Q311H,<h2 style=";text-align:left;direction:ltr">A51T+L202M+R247K,A51T+L202M+Q3 11R,A51T+L202M+Y398W,A51T+L202M 10 ... T+L202M+G460E,N174Q+L202M+A265 G+Q311R+S334T,A51T+L202M+T246V+ 22562 ... M+T246V+S334T+E416L,L202M+T246 V+S334T+N402Y,L202M+T246V+S334T +V366I,L202M+T246V+S334T+S365M,L202M+T246V+S334T+S365C,L202M+ T246V+M261L+S334T,D30N+L202M+H 210N+T246V+S334T,L202M+T246V+N2 70G+S334T,F195N+L202M+T246V+S334T,L202M+T246V+Q319H+S334T,L202 M+T246V+Q319R+S334T,L202M+T246 V+Q311H+S334T,L202M+T246V+Q311R +S334T,L202M+T246V+S334T+Y398W,L202M+T246V+K320H+S334T,L202M+ 24 hours ago 202M+T246V+S334T+V474C,L202M+T2 46V+S334T+G460E,L202M+I235M+T24 6V+S334T,K108R+L202M+T246V+S33 4T,A51T+A186D+L202M+N270G+N402Y ,A186D+L202M+N270G+S339A+N402Y, A51T+A186D+L202M+N270G,A51T+L20 25 days 51T+N174Q+L202M+N270G,A51T+L202 M+V238A+N270G,A51T+L202M+A265G,A51T+L202M+M261L+N270G,A51T+L202M+F267Y,A51T+L202M+I275L,A51T+L202M+N270G+S365M,A51T+L202M+N270G+S365C,A51T+L202M+N270G+Q311T,A51T+L202M+N270G+E416L,A51T+L202M+N270G+N402Y,A51T+L202M+N270G+S365C,A51T+L202M+N270G+K383R,A51T+L202M+N270G+V474C,A51T+L202M+N270G+G460E,A51T+Q86L+L202M+N270G,A51T+Q86I+L202M+N270G,A51T+A113E+L202M+N270G,A51T+K93H+L202M+N270G,A51T+K108R+L202M+N270G,A51T+L202M+K269N,A51T+L202M+Y243F+N270G,A51T+F195N+L202M+N270G,A51T+L202M+R247K+N270G,A51T+L202M+R218N+N270G,A51T+L202M+S255K+N270G,A51T+L202M+I257A+N270G,A51T+L202M+V214I+R218N+N270G,A51T+L202M+N270G+Q311H,A51T+L202M+N270G+K320H,A51T+L202M+N270G+Y299W,A51T+L202M+N270G+K320R,A51T+L202M+N270G+K383Q,A51T+K142R+L202M+N270G,A51T+E130Q+L202M+N270G,A51T+K118N+L202M+N270G,A51T+E138Q+L202M+N270G,A51T+K118H+L202M+N270G,A51T+K118Q+L202M+N270G,A51T+Q86H+L202M+N270G,A51T+E121H+L202M+N270G,A51T+K118R+L202M+N270G,K37H+A51T+N174Q+L202M+A265G+Q311R+S334T,A51T+N174Q+L202M+T246I+Q311R+S334T,K37V+A51T+L202M+A265G+F267Y+Q311R+S334T,A51T+N174Q+L202M+A265G+F267Y+Q311R+S334T, A51G+L202M+Q311R+S334T, L202M+T246I+A265 G+F267Y+Q311R+S334T, A51T+L202M+F267Y+Q311R+S334T+S365L, A51T+L202M+S365C, A51T+K10 8R+L202M, A51T+S365C, A51T+K108R, L202M+V238A+S334T, W48F+V238A+S334T, M116F+V238A+S3 34T, V238A+S334T+W482Y, Y243F+S334T, L202M+V238A+Y299F+S334T, L202M+T246V+N270G+S334T , W48F+K118H+V238A+S334T, W48F+L202M+V238A+S334T, A51T+A186N+L202M+N270G+S365C, W167 Y+A186N+H210N+S339A+V366I, W48F+W167Y+A186N+H210N+S339A, W167Y+A186N+H210N+Y299F+S The polypeptide according to any one of claims 1 to 12, which consists of the amino acid sequence of SEQ ID NO: 2 having mutations selected from the group consisting of 339A + V366I, L202M + T246V + N270G + S334T + S365C or A186N + L202M + T246V + N270G + S334T, or a fragment thereof having alpha amylase activity.

14. 14. The polypeptide of any one of claims 1 to 13, wherein said enhanced cleaning performance corresponds to an enhancement factor (IF) of at least 1.2 when said polypeptide is evaluated in an ADW assay at 40°C for 10 minutes with a detergent containing bleach.

15. 15. The polypeptide of claim 14, wherein the polypeptide comprises mutations at one or more positions corresponding to positions 37, 51, 93, 98, 108, 118, 167, 186, 202, 210, 235, 243, 246, 247, 250, 255, 259, 260, 261, 270, 299, 311, 319, 334, 339, 365, 385, 398 and 404, where the numbers are according to SEQ ID NO:

2.

16. The polypeptide is; M261L, Y243F, K385H, K385R, K93H, Q98R, K118Q, K118H, S255K, Y404W, Y398W, Q319R, Y299W, N260D, K259N, Y299F, S339A, S334T, A51T+L202M , I235L+T246M+I250L, K37V+A51T+L202M, A51T+L202M+S365C, A51T+L202M+ Q311T, A51T+L202M+M261L, A51T+L202M+R247K, A51T+L202M+Y299W, A51T+K1 2 having mutations selected from the group consisting of: 08R+L202M, A51T+L202M+Y243F, A51T+A186D+L202M+N270G, A51T+L202M+N270G+S365C, W167Y+A186N+H210N+S339A+V366I, W48F+W167Y+A186N+H210N+S339A and W167Y+A186N+H210N+Y299F+S339A+V366I, or a fragment thereof having alpha amylase activity.

17. 14. The polypeptide of any one of claims 1 to 13, wherein said enhanced cleaning performance corresponds to an IF of at least 1.2 when said polypeptide is evaluated in an ADW assay at 50°C for 20 minutes with a detergent containing bleach.

18. 18. The polypeptide of claim 17, wherein the polypeptide comprises mutations at one or more positions corresponding to positions 30, 33, 37, 40, 48, 51, 81, 86, 93, 105, 108, 116, 118, 130, 138, 142, 167, 174, 175, 182, 186, 195, 198, 202, 206, 210, 235, 238, 243, 246, 247, 250, 255, 257, 259, 260, 261, 265, 266, 267, 269, 270, 299, 311, 319, 320, 334, 339, 360, 365, 366, 383, 385, 402, 416, 474 and 482, where the numbers are according to SEQ ID NO:

2.

19. The polypeptide is; K37H+L202M, E360F+S365C, M261L, H210N, Y243F, K108R, V474C, T81S, K385H, K385Q, K385R, K93R, K118Q, K118N, E130H, E130Q, E138Q, K37R, D30N, Y243M, K269N, Y198F, Q311T, F195N, I257A, S255K, R247K, Y404W, Y398W, Q319H, Q319R, Q311H, Q311R, Y299W, N260D, K259N, Y299F, V366I, S365M, S339A, I250L, I250V, K37M, S40T, V238T, M105F, M105I, I206L, T18 2G, M116I, M116L, N174Q, I235L, A265G, K37H, Q311R, N175Q, W48F, M116F, W 167Y+H210N+S339A, H210N+S339A, W48F+W167Y+H210N+S339A, W48F+W167 Y, W167Y+H210N+V366I, D30N+N33D+K37V+K383R+W482Y, A51T, S334T, T246 M, T246L, T246V, T246I, A186G, V238A+S334T, A51T+L202M, A51T+N174Q+L 202M+Q311R+S334T, I235L+T246M+I250L, L202M+T246V+S334T+E416L, D30 N+L202M+H210N+T246V+S334T, L202M+T246V+N270G+S334T, F195N+L202M +T246V+S334T, L202M+T246V+K320R+S334T, L202M+Y243F+S334T, A51T+A1 86D+L202M+N270G+N402Y, A186D+L202M+N270G+S339A+N402Y, A51T+A186 D+L202M+N270G, A51T+L202M+T246V+N270G, K37H+A51T+L202M+N270G, A51 T+L202M+T246L+N270G, A51T+N174Q+L202M+N270G, A51T+L202M+V238A+N 270G, A51T+L202M+A265G, A51T+L202M+M261L+N270G, A51T+L202M+F267Y,A51T+L202M+N270G+S365C, A51T+L202M+N270G+S365C, A51T+L202M+N270G+K383R, A51T+Q86I+ L202M+N270G, A51T+K93H+L202M+N270G, A51T+K108R+L202M+N270G, A51T+L202M+N270G+K320H, A51T+L202M+N270G+K383Q, A51T+K142R+L202M+N270G, A51T+E130Q+L202M+N270G, A51T+E138Q +L202M+N270G, A51T+L202M+S365C, A51T+S365C, W48F+V238A+S334T, M116F+V238A+S334T, V238 A+S334T+W482Y, Y243F+S334T, W48F+K118H+V238A+S334T, A51T+A186N+L202M+N270G+S365C, W 167Y+A186N+H210N+S339A+V366I, W48F+W167Y+A186N+H210N+S339A, W167Y+A186N+H210N+Y299 19. The polypeptide of claim 17 or 18, which consists of the amino acid sequence of SEQ ID NO: 2 having mutations selected from the group consisting of F+S339A+V366I, L202M+T246V+N270G+S334T+S365C and A186N+L202M+T246V+N270G+S334T, or a fragment thereof having alpha amylase activity.

20. 14. The polypeptide of any one of claims 1 to 13, wherein said increased cleaning performance corresponds to an enhancement factor (IF) of at least 1.2 when said polypeptide is evaluated in an ADW assay at 40°C for 10 minutes with detergent without bleach.

21. 21. The polypeptide of claim 20, wherein the polypeptide comprises mutations at one or more positions corresponding to positions 37, 48, 51, 64, 81, 108, 116, 167, 174, 186, 187, 189, 195, 198, 202, 208, 210, 235, 238, 243, 246, 250, 261, 265, 267, 269, 270, 275, 311, 319, 334, 339, 365, 366, 385, 460 and 474, where the numbers are according to SEQ ID NO:

2.

22. The polypeptide is; K37H+L202M, M261L, H210N, Y243F, K108R, G460E, T81S, N174Q+L202M, K385R, Y64W, Y243M, K269N, Y198F, S365M, S339A, W48F, M116V, M116F, W187Y+M208L, W167Y, W189H, W167Y+L202M+H210N+Y299F+S339A, W167Y+L202M+H210N +S339A, W167Y+L202M, V238A+S334T, A51T+L202M, L202M+T246L, A51T+N174Q+L202M+T246I+S334T, I235L+T246M+I25 0L, A186D+F195N+L202M, K37H+A51T+L202M, K37V+A51T+L202M, A51T+L202M+S365C, A51T+L202M+Q311T, A51T+L202M+M 261L, A51T+L202M+Q319R, A51T+L202M+Q311H, A51T+K108R+L202M, A51T+L202M+V474C, A51T+A186D+L202M+N270G, K3 7H+A51T+L202M+N270G, A51T+L202M+A265G, A51T+L202M+M261L+N270G, A51T+L202M+F267Y, A51T+L202M+I275L, A51T+ 22. The polypeptide of claim 20 or 21, consisting of the amino acid sequence of SEQ ID NO: having mutations selected from the group consisting of: L202M+N270G+S365C, W167Y+A186N+H210N+S339A+V366I, W48F+W167Y+A186N+H210N+S339A and W167Y+A186N+H210N+Y299F+S339A+V366I, or a fragment thereof having alpha amylase activity.

23. 14. The polypeptide of any one of claims 1 to 13, wherein said high cleaning performance corresponds to an IF of at least 1.2 when said polypeptide is evaluated in an ADW assay at 50°C for 20 minutes with detergent without bleach.

24. 202, 203, 210, 218, 235, 238, 243, 246, 247, 250, 255, 257, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 340, 341, 342, 343, 346, 347, 347, 348, 349, 350, , 261, 265, 267, 269, 270, 274, 275, 299, 311, 319, 320, 334, 339, 365, 366, 383, 385, 398, 402, 416, 404, 460, 474 and 482, where the numbers are according to SEQ ID NO:

2.

25. The polypeptide is: K269S + N270G + A274K, K37V + L202M, N174Q + L202M, K385H, K385R, K93H, K118Q, E130Q, E138Q, K37R, D30N, Y243M, K269N, Y198F, F195N, I257A, S255K, R247K, Y404W, Y398W, Q319H, Q319R, Q311H, Q311R, Y299W, N260D, K259N, Y299F, V366I, S365M, S339A, N174*, K37L, S40T, T182G, A265G, W4 82Y, M116A, N174Q, I235L, A265G, K37H, Q311R, N175Q, W48F, M116V, M116F , W167Y+L202M+H210N+S339A+V366I, W167Y+L202M+H210N+Y299F+S339A, W 167Y+L202M+H210N+S339A, W167Y+L202M+Y299F, W48F+W167Y, W167Y+Y299 F, W167Y+L202M, D30N+N33D+K37V+L202M+K383R, D30N+N33D+K37V+W48F+K 383R, A51T, S334T, T246M, T246L, T246I, A186G, A51Q+G109M+Y203G, V238 A+S334T, A51T+L202M, L202M+T246L, A51T+N174Q+L202M+T246I+S334T, A5 1T+N174Q+L202M+Q311R+S334T, I235L+T246M+I250L, A186D+L202M, A186 D+L202M+N270G+N402Y, A186D+L202M+S339A, A186D+F195N+L202M, K37H+A 51T+L202M, K37V+A51T+L202M, A51T+L202M+S365C, A51T+L202M+S339A, A 51T+L202M+Q311T, A51T+L202M+M261L, A51T+L202M+H210N, A51T+L202M+N 270G, A51T+F195N+L202M, A51T+L202M+Q319H, A51T+L202M+Q319R, A51T+L 202M+Q311H, A51T+L202M+R247K, A51T+L202M+Q311R, A51T+L202M+Y398W,<h2 style=";text-align:left;direction:ltr">1 ... +V474C,A51T+L202M+G460E,N174Q+L 202M+A265G+Q311R+S334T,A51T+L20 250 ... +E416L,L202M+T246V+S334T+N402Y, L202M+T246V+S334T+V366I,L202M+T 24 hours ago 34, 2019 +L202M+T246V+S334T,L202M+T246V+ Q319H+S334T,L202M+T246V+Q319R+S 33442 6V+S334T+Y398W,L202M+T246V+K32 0H+S334T,L202M+T246V+Y299W+S334 T,L202M+T246V+K320R+S334T,L202M +Y243F+S334T,L202M+T246V+S334T+ 44556 ... 02M+T246V+S334T,A51T+A186D+L202 M+N270G+N402Y,A186D+L202M+N270G +S339A+N402Y,A51T+A186D+L202M+ N270G,A51T+L202M+T246V+N270G,K3 7H+A51T+L202M+N270G,A51T+L202M+ T246L+N270G,A51T+N174Q+L202M+N2 70G,A51T+L202M+V238A+N270G,A51T +N 270G,A51T+L202M+F267Y,A51T+L202 M+I275L,A51T+L202M+N270G+S365M,A51T+L202M+N270G+S365C,A51T+L202M+N270G+Q311T,A51T+L202M+N270G+E416L,A51T+L202M+N270G+N402Y,A51T+L202M+N270G+S365C,A51T+L202M+N270G+K383R,A51T+L202M+N270G+V474C,A51T+L202M+N270G+G460E,A51T+Q86L+L202M+N270G,A51T+Q86I+L202M+N270G,A51T+A113E+L202M+N270G,A51T+K93H+L202M+N270G,A51T+K108R+L202M+N270G,A51T+L202M+K269N,A51T+L202M+Y243F+N270G,A51T+L202M+R247K+N270G,A51T+L202M+R218N+N270G,A51T+L202M+S255K+N270G,A51T+L202M+I257A+N270G,A51T+L202M+V214I+R218N+N270G,A51T+L202M+N270G+Q311H,A51T+L202M+N270G+K320H,A51T+L202M+N270G+Y299W,A51T+L202M+N270G+K320R,A51T+L202M+N270G+K383Q,A51T+K142R+L202M+N270G,A51T+E130Q+L202M+N270G,A51T+K118N+L202M+N270G,A51T+E138Q+L202M+N270G,A51T+K118H+L202M+N270G,A51T+E121H+L202M+N270G,K37H+A51T+N174Q+L202M+A265G+Q311R+S334T,A51T+N174Q+L202M+T246I+Q311R+S334T,K37V+A51T+L202M+A265G+F267Y+Q311R+S334T,A51T+N174Q+L202M+A265G+F267Y+Q311R+S334T,L202M+T246I+A265G+F267Y+Q311R+S334T,A51T+L202M+F267Y+Q311R+S334T+S365L,A51T+L202M+S365C,A51T+K108R+L202M,L202M+V238A+S334T,W48F+V238A+S334T, M116F+V238A+S334T, V238A+S334T+W482Y, Y243F+S334T, L202M+V238A+Y299F+S334T, L202M+T246V+N270G+ S334T, W48F+K118H+V238A+S334T, W48F+L202M+V238A+S334T, A51T+A186N+L202M+N270G+S365C, W167Y+A186N+H210N+S339A+V3 25. The polypeptide of claim 23 or 24, which comprises the amino acid sequence of SEQ ID NO: 2 having mutations selected from the group consisting of: 66I, W48F + W167Y + A186N + H210N + S339A, W167Y + A186N + H210N + Y299F + S339A + V366I, L202M + T246V + N270G + S334T + S365C and A186N + L202M + T246V + N270G + S334T, or a fragment thereof having alpha amylase activity.

26. 26. The polypeptide of any one of claims 1 to 25, wherein the polypeptide comprises mutations at one or more positions corresponding to positions 48, 167, 210, 299, 339, 366, where the numbering is according to SEQ ID NO:

2.

27. 27. The polypeptide of claim 26, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO:2 having one or more mutations selected from the group consisting of W48F, W167Y, H210N, Y299F, S339A and V366I, or a fragment thereof having alpha amylase activity.

28. The polypeptide is (a) SEQ ID NO:1 with mutations W167Y, H210N and S339A; (b) SEQ ID NO:1 with the mutations W167Y, H210N, S339A and V366I; (c) SEQ ID NO:1 with the mutations W167Y, H210N, Y299F, S339A and V366I; (d) SEQ ID NO:1 with the mutations W48F and W167Y; (e) SEQ ID NO:1 with the mutations W48F, W167Y, H210N and S339A; (f) SEQ ID NO:1 with the mutations W48F, W167Y, H210N, Y299F, S339A and V366I; (g) SEQ ID NO:1 with the mutations V238A, S334T and W482Y; (h) SEQ ID NO:1 with the mutations Y243F and S334T; (i) SEQ ID NO:1 with the mutation W48F; (j) SEQ ID NO:1 with the mutation K118H; (k) SEQ ID NO:1 with the mutation W167Y; (l) SEQ ID NO:1 with the mutations W48F, V238A and S334T; (m) SEQ ID NO:1 with mutations I234L, T246M, and I250L; (n) SEQ ID NO: 1 with the mutation S339A; and (o) SEQ ID NO:1 with the mutation S334T; 28. The polypeptide of claim 26 or 27, comprising or consisting of an amino acid sequence selected from the group consisting of:

29. 29. The polypeptide of any one of claims 1 to 28, wherein the polypeptide comprises or consists of an amino acid sequence sharing at least 70% sequence identity with SEQ ID NO:1, such as at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:

1.

30. 30. The polypeptide of any one of claims 1 to 29, wherein the number of mutations relative to SEQ ID NO:1 is between 1 and 20, such as between 1 and 10 and between 1 and 5 mutations, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

31. The polypeptide of any one of claims 1 to 30, wherein the polypeptide is selected from the group of polypeptides in Table 1.

32. A polynucleotide encoding the polypeptide of any one of claims 1 to 31.

33. A nucleic acid construct comprising the polynucleotide of claim 32.

34. An expression vector comprising the polynucleotide of claim 32.

35. A host cell comprising the polynucleotide of claim 32.

36. (a) culturing the host cell of claim 35 under conditions suitable for expression of the polypeptide; (b) recovering the polypeptide; The present invention relates to a method for producing an α-amylase polypeptide comprising the steps of:

37. 202, 203, 206, 208, 210, 214, 218, 235, 238, 242, 243, 246, 247, 250, 255, 257, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 289, 290, 295, 298, 299, 300, 301, 302, 303, 304, 305, 306, 308, 309, 310, 314, 318, 320, 325, 326, 327, 328, 330, 335, 338, 342, 343, 346, 347, 348, 349, 350, 355, 357, 359, 360, 361, 362, 363, 364, 365, 366, 367, 370, 375, 378, 379, 380, 382, ​​383, 384, 385, 386, 387, 389, 390, 395, 396, 397, 37. The method of claim 36, comprising mutations at one or more positions corresponding to: 260, 261, 265, 267, 269, 270, 274, 275, 276, 281, 295, 298, 299, 311, 319, 320, 334, 339, 360, 365, 366, 383, 384, 385, 394, 398, 402, 404, 416, 434, 460, 469, 474 and 482, where the numbers are according to the amino acid sequence set forth in SEQ ID NO:

2.

38. A detergent composition, or a concentrate or additive for forming the same, comprising a polypeptide according to any one of claims 1 to 31 and a surfactant.

39. 39. The composition of claim 38, wherein the surfactant is selected from the group consisting of anionic surfactants, cationic surfactants, nonionic surfactants and amphoteric surfactants.

40. 40. The composition of claim 38 or 39, further comprising one or more additional components selected from the group consisting of oxidizing agents, bleach activators, chelating agents, fillers, builders, buffers, structuring agents, sequestrants, optical brighteners, defoamers, enzymes, fragrances, anti-redeposition agents, skin conditioning agents, softening agents, emulsifiers and colorants.

41. A composition according to any one of claims 38 to 40, wherein the composition is a liquid or powder laundry detergent composition.

42. A composition according to any one of claims 38 to 40, wherein the composition is a liquid or powder automatic dishwashing (ADW) detergent composition.

43. The composition of any one of claims 38 to 40, wherein the composition is a liquid hand dishwashing detergent composition.

44. Use of a variant according to any one of claims 1 to 31 or a composition according to any one of claims 38 to 43 in domestic or industrial cleaning processes.

45. 45. Use according to claim 44 in the cleaning of fabrics, for example in laundry.

46. 45. Use according to claim 44 for cleaning ceramic, plastic or glass materials, for example washing dishes.