Alpha-amylase variants and polynucleotides encoding them

Abstract

This invention relates to α-amylase variants. It also relates to polynucleotides encoding these variants; nucleic acid constructs, vectors, and host cells comprising these polynucleotides; and methods of using these variants.

Description

[0001] References to sequence lists

[0002] This application contains a sequence list in computer-readable form, which is incorporated herein by reference. Background of the Invention Technical Field

[0004] This invention relates to α-amylase variants, polynucleotides encoding these variants, methods for producing these variants, and methods for using these variants. Background Technology

[0006] Alpha-amylase (α-1,4-glucan-4-glucan hydrolase, EC 3.2.1) is a group of enzymes that hydrolyze starch, glycogen, and other related polysaccharides by cleaving internal α-1,4-glycosidic bonds. For many years, it has been used, for example, in laundry, where its beneficial effects in removing starch-containing or starch-based stains are well-known. However, this enzyme has become important in other commercial applications, such as in the initial stages of starch processing (liquefaction), in textile desizing, in alcohol production, and as a cleaning agent in detergent compositions.

[0007] In recent years, there has been a desire to improve the properties of various amylases. Specifically, when it comes to the home care industry, the main focus is on lowering the washing temperature of clothes to reduce energy consumption. Therefore, much effort has been made to find improved α-amylase variants.

[0008] Nevertheless, there remains a need for α-amylase variants with altered properties to provide improved performance in various industrial applications. Therefore, this invention provides such a further improved α-amylase variant with improved properties compared to its parent. Summary of the Invention

[0009] This invention relates to α-amylase variants of parental polypeptides having α-amylase activity, wherein the variant is a mature form of an α-amylase having amylase activity and includes substitutions at one or more positions selected from the group consisting of: H1, H2, D3, G4, T5, G7, T8, I9, M10, Q11, Y12, F13, E14, W15, N16, V17, P18, N19, D20, Q22, H23, W24, N25, R26, L27, H28, N29, N30, A31, Q32, N33, L34, K35, N36, A37, G38, I39, T40, A41, I42, W43, I44, P45. P46, A47, W48, K49, G50, T51, S52, Q53, N54, D55, V56, G59, A60, Y61, L63, Y6 4. L66, E68, F69, N70, Q71, K72, G73, T74, V75, R76, T77, K78, T81, K82, A83, E 84, L85, E86, R87, A88, I89, R90, S91, L92, K93, N95, G96, I97, Q98, V99, Y10 0. V103, H107, K108, G109, G110, A111, D112, F113, T114, E115, R116, V117, Q 118, A119, V120, E121, V122, N123, P124, Q125, N126, R127, N128, Q129, E13 0. V131, S132, G133, T134, Y135, Q136, I137, E138, A139, W140, T141, G142, F143, N144, F145, P146, G147, G149, N150, Q151, S153, S154, F155, K156, W1 57. R158, W159, Y160, H161, F162, D163, G164, T165, D166, W167, D168, Q169, S170, R171, Q172, L173, A174, N175, R176, I177, Y178, K179, F180, R181, G1 84. K185, A186, W187, D188, W189, E190, V191, D192, T193, E194, N195, G196, N197, Y198, D199, Y200, M202, Y203, A204, D205, V206, M208, D209, H210, P2 11. E212, V213, I214, N215, L217, N218, R219, W220, G221, V222, W223, Y224,A225, N226, T227, L228, N229, L230, D231, F233, V238, H240, I241, K242, S244, F245, M246, R247, D248, W249, L250, G251, H252, V253, R254, G255, Q256 T257, G258, K259, N260, L261, F262, A263, V264, Y267, W268, K269, N270, D271, L272, G273, A274, L275, E276, N277, Y278, L279, S280, K281, T282, N283 W284, T285, M286, S287, A288, V291, P292, L293, H294, Y295, N296, L297, Y298, Q299, A300, S301, N302, S303, S304, G305, N306, Y307, M309, R310, N311 L312, L313, N314, G315, T316, V318, Q319, R320, H321, P322, S323, A325, V326, T327, F328, V329, N331, T334, P336, G337, E338, E341, S342, V344, Q345 G346, W347, F348, K349, P350, L351, A352, A354, T355, L357, T358, E360, Q361, G362, Y363, Q365, V366, F367, Y368, G369, D370, Y372, G373, I374, P375 S376, D377, V379, P380, S381, Y382, R383, Q384, I386, D387, P388, L389, L390, K391, A392, Q394, Q395, Y396, A397, Y398, R400, H402, D403, Y404, F405 D406、H407、W408、D409、V410、I411、T414、R415、E416、N418、A419、S420、H4 21、P422、S424、G425、L426、A427、T428、I429、M430、S431、D432、G435、G436、 S437、K438、W439、M440、Y441、V442、R444、Q445、K446、A447、G448、V450、W4 51、H452、D453、M454、T455、G456、N457、S459、G460、T463、I464、N465、Q466、D467, W469, H471, F473, N475, G476, G477, V481, and W482; wherein these positions correspond to amino acid positions in the amino acid sequence as defined in SEQ ID NO:3; and wherein the substitutions at one or more of these positions provide α-amylase variants having an improvement factor of ≥1.0 for measures of stability, and / or an improvement factor of ≥1.0 for measures of specific activity.

[0010] The present invention also relates to polynucleotides encoding these variants; nucleic acid constructs, vectors, and host cells including these polynucleotides; and methods for generating these variants.

[0011] The present invention also relates to methods for improving the washing performance and / or stability of parental α-amylase. Attached Figure Description

[0012] Figure 1 The improvement factor (IF) for the measure of specific activity of the variants according to the invention, tested in standard detergent A, is shown. The individual positions of the modifications are given on the X-axis, and the specific IF for each variant is given on the Y-axis.

[0013] Figure 2 The improvement factor (IF) for the measure of specific activity of the variants according to the invention, tested in standard X detergent, is shown. The individual positions of the modifications are given on the X-axis, and the specific IF for each variant is given on the Y-axis.

[0014] Figure 3 The improvement factor (IF) for measures of stability of the variants according to the invention, tested in standard detergent A, is shown. The individual positions of the modifications are given on the X-axis, and the specific IF for each variant is given on the Y-axis.

[0015] Variant Naming Rules

[0016] For the purposes of this invention, the mature polypeptide described in SEQ ID NO:3 is used to determine the corresponding amino acid residues in another α-amylase. The amino acid sequence of another α-amylase was compared with the mature polypeptide elucidated in SEQ ID NO:3, and based on this comparison, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453) implemented in the Needle program of the EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (preferably version 5.0.0 or later) was used to determine the amino acid position number corresponding to any amino acid residue in the mature polypeptide elucidated in SEQ ID NO:3. The parameters used are a starting penalty of 10 for empty spaces, a penalty of 0.5 for extending empty spaces, and an EBLOSUM62 (the EMBOSS version of BLOSUM62) replacement matrix.

[0017] The identification of the corresponding amino acid residues in another α-amylase can be determined by comparing multiple polypeptide sequences using several computer programs (with their respective default parameters). These computer programs include, but are not limited to, MUSCLE (multiple sequence comparison by log-expectation value; 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); and Katoh et al., 2005, Nucleic Acids Research. 33:511-518 [Katoh et al., 2005, Nucleic Acid Research, 33:511-518]; Katoh and Toh, 2007, Bioinformatics 23:372-374 [Katoh and Toh, 2007, Bioinformatics, 23:372-374]; Katoh et al., 2009, Methods in Molecular Biology 537:39-64 [Katoh et al., 2009, Methods in Molecular Biology, 537:39-64]; Katoh and Toh, 2010, Bioinformatics 26:1899-1900 [Katoh and Toh, 2010, Bioinformatics, 26:1899-1900]; and using ClustalW's EMBOSS EMMA (version 1.83 or later; Thompson et al.) et al., 1994, Nucleic Acids Research 22:4673-4680 [Thompson et al., 1994, Nucleic Acids Research, 22:4673-4680]).

[0018] When other enzymes deviate from the mature polypeptide described in SEQ ID NO:3, thus preventing conventional sequence-based comparison methods from detecting their relationships (Lindahl and Elofsson, 2000, J.Mol.Biol.295:613-615), alternative pairwise sequence comparison algorithms can be used. Higher sensitivity in sequence-based searches can be achieved using search programs that employ probabilistic representations (spectrals) of polypeptide families to search databases. For example, the PSI-BLAST program generates multiple spectra through an iterative database search process and can detect distant homologs (Atschul et al., 1997, Nucleic Acids Res.25:3389-3402). Even higher sensitivity can be achieved if the polypeptide family or superfamily has one or more representatives in a protein structure database. Programs such as GenTHREADER (Jones, 1999, J.Mol.Biol.287:797-815; McGuffin and Jones, 2003, Bioinformatics 19:874-881) utilize information from multiple sources (PSI-BLAST, secondary structure prediction, structure alignment spectra, and solvation potential) as input to a neural network that predicts the structural folding of a query sequence. Similarly, the method described by Gough et al., 2000, J.Mol.Biol.313:903-919 can be used to align sequences of unknown structures with superfamily models existing in the SCOP database. These alignments can then be used to generate homology models of peptides, and the accuracy of such models can be assessed using a variety of tools developed for this purpose.

[0019] For proteins with known structures, several tools and resources are available for retrieving and generating structure alignments. For example, the SCOP superfamily of proteins has already been structurally aligned, and those alignments are accessible and downloadable. Various algorithms, such as distance alignment matrix (Holm and Sander, 1998, Proteins 33:88-96) or combined extension (Shindyalov and Bourne, 1998, Protein Engineering 11:739-747), can be used to align two or more protein structures. Furthermore, implementations of these algorithms can be used to query structure databases containing structures of interest to discover potential structural homologs (e.g., Holm and Park, 2000, Bioinformatics 16:566-567).

[0020] In the description of variations of the invention, the following nomenclature is provided for ease of reference. The accepted IUPAC single-letter and three-letter amino acid abbreviations are used.

[0021] replace For amino acid substitutions, the following nomenclature is used: initial amino acid, position, substituted amino acid. Therefore, the substitution of threonine at position 226 with alanine is represented as "Thr226Ala" or "T226A". Multiple mutations are separated by plus signs ("+"), for example, "Gly205Arg+Ser411Phe" or "G205R+S411F" represent the substitution of glycine (G) with arginine (R) at positions 205 and 411, respectively, and the substitution of serine (S) with phenylalanine (F).

[0022] Missing For amino acid deletions, use the following nomenclature: initial amino acid, position, * Therefore, a glycine deletion at position 195 is represented as "Gly195*" or "G195*". Multiple deletions are separated by a plus sign ("+"), for example, "Gly195". * +Ser411 * "or "G195 * +S411 * ".

[0023] insert.For amino acid insertions, the following nomenclature is used: initial amino acid, position, initial amino acid, inserted amino acid. Therefore, inserting lysine after glycine at position 195 is represented as "Gly195GlyLys" or "G195GK". Insertions of multiple amino acids are represented as [initial amino acid, position, initial amino acid, inserted amino acid #1, inserted amino acid #2; etc.]. For example, inserting lysine and alanine after glycine at position 195 is represented as "Glyl95GlyLysAla" or "G195GKA".

[0024] In such cases, the inserted amino acid residues are numbered by adding lowercase letters to the position numbers of the amino acid residues preceding them. In the example above, the sequence would therefore be:

[0025] <![CDATA[ Parents: ]]> <![CDATA[ Variants: ]]> 195 195 195a 195b G GKA

[0026] Multiple modifications Variants with multiple modifications are separated by a plus sign ("+"), such as "Arg170Tyr+Gly195Glu" or "R170Y+G195E", which represent that arginine and glycine at positions 170 and 195 are replaced by tyrosine and glutamic acid, respectively.

[0027] Different modifiers When different modifications can be introduced at a position, these different changes are separated by commas. For example, "Arg170Tyr,Glu" means that arginine at position 170 is replaced by tyrosine or glutamic acid. Therefore, "Tyr167Gly,Ala+Arg170Gly,Ala" represents the following variants: "Tyr167Gly+Arg170Gly", "Tyr167Gly+Arg170Ala", "Tyr167Ala+Arg170Gly", and "Tyr167Ala+Arg170Ala". Detailed Implementation

[0028] This invention relates to a set of sequences. For convenience, these sequences are listed below;

[0029] SEQ ID NO:1 is the nucleotide sequence of mature α-amylase (AAI10).

[0030] SEQ ID NO:2 is the amino acid sequence of AAI10, including the signal peptide (i.e., the full length).

[0031] SEQ ID NO:3 is the amino acid sequence of mature AAI10.

[0032] SEQ ID NO:4 is the amino acid sequence of mature AAI10, which includes a double deletion at positions G182 and D183 corresponding to SEQ ID NO:3.

[0033] SEQ ID NO:5 is the forward primer of PnMi4490.

[0034] SEQ ID NO:6 is the forward primer of PnMi4491.

[0035] Variations of the present invention

[0036] In one aspect, the present invention relates to variants of parental polypeptides having α-amylase activity. Therefore, in a specific aspect, the present invention relates to α-amylase variants of parental α-amylases having α-amylase activity, wherein said variants are mature forms of α-amylases having amylase activity and include substitutions at one or more positions selected from the group consisting of: H1, H2, D3, G4, T5, G7, T8, I9, M10, Q11, Y12, F13, E14, W15, N16, V17, P18, N19, D20, Q22, H23, W24, N25, R26, L27, H28, N29, N30, A31, Q32, N33, L34, K35, N36, A37, G38, I39, T40. A41, I42, W43, I44, P45, P46, A47, W48, K49, G50, T51, S52, Q53, N54, D55, V56, G59, A60, Y61, L63, Y64, L66, E68, F69, N70, Q71, K72, G73, T74, V75, R 76. T77, K78, T81, K82, A83, E84, L85, E86, R87, A88, I89, R90, S91, L92, K9 3. N95, G96, I97, Q98, V99, Y100, V103, H107, K108, G109, G110, A111, D112 , F113, T114, E115, R116, V117, Q118, A119, V120, E121, V122, N123, P124 , Q125, N126, R127, N128, Q129, E130, V131, S132, G133, T134, Y135, Q136, I137, E138, A139, W140, T141, G142, F143, N144, F145, P146, G147, G149, N 150, Q151, S153, S154, F155, K156, W157, R158, W159, Y160, H161, F162, D1 63. G164, T165, D166, W167, D168, Q169, S170, R171, Q172, L173, A174, N1 75, R176, I177, Y178, K179, F180, R181, G184, K185, A186, W187, D188, W18 9. E190, V191, D192, T193, E194, N195, G196, N197, Y198, D199, Y200, M202 , Y203, A204, D205, V206, M208, D209, H210, P211, E212, V213, I214, N215,L217, N218, R219, W220, G221, V222, W223, Y224, A225, N226, T227, L228, N229, L230, D231, F233, V238, H240, I241, K242, S244, F245, M246, R247, D248 W249, L250, G251, H252, V253, R254, G255, Q256, T257, G258, K259, N260, L261, F262, A263, V264, Y267, W268, K269, N270, D271, L272, G273, A274, L275 E276, N277, Y278, L279, S280, K281, T282, N283, W284, T285, M286, S287, A288, V291, P292, L293, H294, Y295, N296, L297, Y298, Q299, A300, S301, N302 S303, S304, G305, N306, Y307, M309, R310, N311, L312, L313, N314, G315, T316, V318, Q319, R320, H321, P322, S323, A325, V326, T327, F328, V329, N331 T334, P336, G337, E338, E341, S342, V344, Q345, G346, W347, F348, K349, P350, L351, A352, A354, T355, L357, T358, E360, Q361, G362, Y363, Q365, V366 F367, Y368, G369, D370, Y372, G373, I374, P375, S376, D377, V379, P380, S381, Y382, R383, Q384, I386, D387, P388, L389, L390, K391, A392, Q394, Q395 Y396、A397、Y398、R400、H402、D403、Y404、F405、D406、H407、W408、D409、V4 10、I411、T414、R415、E416、N418、A419、S420、H421、P422、S424、G425、L426、 A427、T428、I429、M430、S431、D432、G435、G436、S437、K438、W439、M440、Y4 41、V442、R444、Q445、K446、A447、G448、V450、W451、H452、D453、M454、T455、G456, N457, S459, G460, T463, I464, N465, Q466, D467, W469, H471, F473, N475, G476, G477, V481, and W482; wherein these positions correspond to amino acid positions in the amino acid sequence as defined in SEQ ID NO:3; and wherein the substitutions at one or more of these positions provide α-amylase variants having an improvement factor of ≥1.0 for measures of stability, and / or an improvement factor of ≥1.0 for measures of specific activity.

[0037] It has been shown that, compared with the parental α-amylase, the variants according to the invention, including monosubstituted variants, have improved specific activity and / or stability.

[0038] As used herein, the term "amylase" or "α-amylase" refers to an enzyme capable of catalyzing the degradation of starch. Typically, α-amylase (EC3.2.1.1, α-D-(1->4)-glucan-glucan hydrolase) is an intracellular enzyme that cleaves the α-D(1->4)O-glycosidic bonds within starch molecules in a random sequence.

[0039] As used herein, the term "starch" refers to a complex polysaccharide carbohydrate composed of amylose and amylopectin, with the molecular formula (C6H12H2O). 10 O5) x Any material (where X can be any number). Specifically, the term refers to plant-based materials such as rice, barley, wheat, corn, rye, potatoes, etc.

[0040] As used herein, the term "α-amylase variant" refers to a polypeptide having α-amylase activity comprising modifications (i.e., substitution, insertion, and / or deletion) at one or more (e.g., several) positions. Substitution means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; and insertion means adding an amino acid adjacent to and immediately following an amino acid occupying a position, all as defined above. Variants of the invention have at least 20%, for example 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 amino acid sequence as illustrated in SEQ ID NO:3, of α-amylase activity. The α-amylase variants of the present invention comprise an amino acid sequence having at least 60%, for example at least 65%, for example at least 70%, for example at least 75%, for example at least 80%, for example at least 85%, for example at least 90%, for example at least 95%, for example at least 97%, for example at least 99%, but less than 100%, sequence consistency with any one of SEQ ID NO:2, 3 or 4.

[0041] As used herein, the term "α-amylase activity" or "amylase activity" refers to the activity of α-1,4-glucan-4-glucan hydrolase (EC 3.2.1.1), which constitutes a group of enzymes that catalyze the hydrolysis of starch and other linear and branched 1,4-glycosidic oligosaccharides and polysaccharides. Therefore, as used herein, the term "α-amylase" refers to an enzyme (enzyme class: EC 3.2.1.1) having α-amylase activity, which hydrolyzes the α-bonds of large, α-linked polysaccharides (such as starch and glycogen) to produce glucose and maltose. The terms "α-amylase" and "amylase" are used interchangeably and constitute the same meaning and purpose within the scope of this invention. For the purposes of this invention, α-amylase activity is determined according to the procedure described in the examples. 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% α-amylase activity of the polypeptide of SEQ ID NO:3 or 4 or the mature polypeptide of SEQ ID NO:2.

[0042] As used herein, the term "parental α-amylase" refers to an α-amylase that has been modified to produce an enzyme variant. For example, an α-amylase having the amino acid sequence as set forth in SEQ ID NO:2, 3, or 4 can be a parent for these variants of the invention. Any polypeptide having at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 97%, such as at least 99%, sequence identical to any of SEQ ID NO:2, 3, or 4 can also be a parent polypeptide for variants of the invention, such as a parental α-amylase.

[0043] The parental α-amylase can be a fusion polypeptide or a cleavable fusion polypeptide. Such a fusion polypeptide can consist of a subsequence of one parental polypeptide and a subsequence of a second parental polypeptide. Specifically, the fusion polypeptide can consist of the A and B domains of one α-amylase and the C domain of another α-amylase, thereby providing the parental polypeptide to produce the variants of the invention. Techniques for generating fusion polypeptides are known in the art and include linking coding sequences of the polypeptides such that they are within a frame and that the expression of the fusion polypeptide is under the control of the same one or more promoters and terminators. Inteinization techniques can also be used to construct fusion peptides, where fusion peptides are generated post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583 [Cooper et al., 1993, Journal of the European Society for Molecular Biology, 12: 2575-2583]; Dawson et al., 1994, Science 266: 776-779 [Dawson et al., 1994, Science, 266: 776-779]).

[0044] As used herein, the terms “domain A,” “domain B,” and “domain C” refer to three distinct domains A, B, and C, all of which are part of the α-amylase structure, see, for example, Machius et al., 1995, J. Mol. Biol. 246: 545-559 [Machius et al., 1995, Journal of Molecular Biology, 246: 545-559]. Therefore, α-amylases (e.g., parental polypeptides and variants according to the invention) may all include domains A, B, and C. The term “domain” refers to a distinct and independent substructure of a polypeptide that forms itself as a complete molecule. α-Amylases consist of a β / α-8 barrel structure carrying the active site residues (denoted as domain A), a fairly long loop structure between β-sheet 3 and α-helix 3 (denoted as domain B) (together; “domains A and B”), and a domain C, and in some cases also include a carbohydrate-binding domain (e.g., WO 2005 / 001064; Machius et al., see above).

[0045] The domains of α-amylases can be determined through structural analysis, such as using crystallography. An alternative method for determining the domains of α-amylases is by sequence alignment of the amino acid sequence of the α-amylase with that of another α-amylase whose domain has been determined. A sequence aligned with, for example, the C-domain sequence of an α-amylase whose C-domain has been determined can be considered the C-domain of the given α-amylase.

[0046] As used herein, the term "A and B domains" refers to the two domains of α-amylase considered as one unit, while the C domain is another unit of α-amylase. Therefore, the amino acid sequence of the "A and B domains" is understood to be a sequence or part of a sequence of an α-amylase that includes the "A and B domains" and other domains (such as the C domain). In one embodiment, the A and B domains of the parental α-amylase correspond to amino acids 1 to 397 as illustrated in SEQ ID NO:3.

[0047] As used herein, the term "C-domain" refers to a domain of α-amylase as a unit. The "C-domain" of α-amylase corresponds to amino acids 400 to 485 of SEQ ID NO:3.

[0048] Fusion peptides may also include a cleavage site between the two peptides. This site is cleaved during the secretion of the fusion protein, thereby releasing both peptides. Examples of cleavage sites include, but are not limited to, those disclosed in the following literature: Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3:568-576 [Martin et al., 2003, Journal of Industrial Microbiology and Biotechnology, 3:568-576]; Svetina et al., 2000, J. Biotechnol. 76:245-251 [Svetina et al., 2000, Journal of Biotechnology, 76:245-251]; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63:3488-3493 [Rasmussen-Wilson et al., 1997, Applied Environmental Microbiology, 63:3488-3493]; Ward et al., 1995, Biotechnology 13:498-503 [Ward et al., 1995, Biotechnology, 13:498-503]; and Contreras et al., 1991, Biotechnology 9:378-381 [Contreras et al., 1991, Biotechnology, 9:378-381]; Eaton et al., 1986, Biochemistry 25:505-512 [Eaton et al., 1986, Biochemistry, 25:505-512]; Collins-Racie et al., 1995, Biotechnology 13:982-987 [Collins-Racie et al., 1995, Biotechnology, 13:982-987]; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6:240-248 [Carter et al., 1989, Proteins: Structure, Function and Genetics, 6:240-248]; and Stevens, 2003, Drug Discovery World 4:35-48 [Stevens, 2003, Drug Discovery World, 4:35-48].

[0049] The parent polypeptide can be obtained from any genus of microorganisms. For the purposes of this invention, the term "obtained from" as used herein in conjunction with a given source shall mean that the parent encoded by the polynucleotide is produced by that source or by a strain in which a polynucleotide from that source has been inserted.

[0050] It will be understood that, for the species mentioned above, this invention covers both perfect and imperfect states, as well as other taxonomic equivalents, such as asexual forms, regardless of their known species names. Those skilled in the art will readily identify the appropriate equivalents.

[0051] Strains of these species are readily available to the public at many culture collections, such as the American Type Culture Collection (ATCC), the German Microbial Culture Collection (DSMZ), the Netherlands Culture Collection (CentraalbureauVoor Schimmelcultures, CBS), and the Northern Research Center (NRRL) of the Patent Culture Collection of the U.S. Agricultural Research Service.

[0052] The parent polypeptide can be identified and obtained from other sources, including microorganisms isolated from nature (e.g., soil, compost, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, compost, water, etc.), using the probes mentioned above. Techniques for directly isolating microorganisms and DNA from their natural environment are well known in the art. The polynucleotide encoding the parent polypeptide can then be obtained by similarly screening a genomic DNA or cDNA library of another microorganism or a mixed DNA sample. Once the polynucleotide encoding the parent polypeptide is detected with one or more probes, it can be isolated or cloned using techniques known to those skilled in the art (see, for example, Sambrook et al., 1989, above).

[0053] In one embodiment, the parental α-amylase comprises or is composed of the amino acid sequence set forth in SEQ ID NO:3 or 4. In another embodiment, the parental α-amylase comprises or is composed of the mature polypeptide of SEQ ID NO:2. Specifically, the parental α-amylase may comprise or is composed of amino acids 1 to 485 of SEQ ID NO:2.

[0054] In another embodiment, the parental α-amylase is a fragment as illustrated in SEQ ID NO:3 containing at least 350, such as at least 390, amino acid residues (e.g., at least 395 and at least 397 amino acid residues) of SEQ ID NO:3.

[0055] In another embodiment, the parental α-amylase is an allelic variant of the mature polypeptide of SEQ ID NO:3.

[0056] As used herein, the term "sequence identity" refers to the correlation between two amino acid sequences or two nucleotide sequences. For the purposes of this invention, the Niedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) implemented in the Niedleman program of the EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (preferably version 5.0.0 or later) is used to determine the sequence identity between two amino acid sequences. The parameters used are a vacancy start penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The Niedel output marked as "Longest Consistency" (obtained using the -nobrief option) is used as the percentage consistency and calculated as follows:

[0057] (Consistent residues x 100) / (Alignment length - Total number of vacancies in the alignment)

[0058] For the purposes of this invention, the Niedleman-Wunsch algorithm (Needleman and Wunsch, 1970, see above) implemented in the Niedle program, such as in the EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, see above) (preferably version 5.0.0 or later), is used to determine sequence consistency between two deoxyribonucleotide sequences. The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The Niedle output labeled "Longest Consistency" (obtained using the -nobrief option) is used as the percentage consistency and calculated as follows:

[0059] (Consistent deoxyribonucleotides x 100) / (Alignment length - total number of vacancies in the alignment)

[0060] As used herein, the term "fusion polypeptide" refers to a polypeptide comprising an amino acid sequence derived from more than one (e.g., two, three, or four) species. Such fusion polypeptides can be produced using molecular techniques well known to those skilled in the art. The fusion polypeptides of the present invention possess α-amylase activity. Specifically, the fusion polypeptides of the present invention comprise, for example, A and B domains of an α-amylase, and a C domain from another α-amylase.

[0061] As used herein, the terms "mature polypeptide" and "mature amino acid sequence" refer 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. Also considered within the term "mature polypeptide" is a polypeptide whose signal peptide has been cleaved, for example, during the natural maturation process within the cell expressing the polypeptide. In one aspect, the mature polypeptide is the amino acid sequence of SEQ ID NO:3. It is known in the art that host cells can produce mixtures of two or more different mature polypeptides (i.e., with different C-terminal and / or N-terminal amino acids) expressed from the same polynucleotide. It is also known in the art that different host cells process polypeptides differently, and therefore a host cell expressing a polynucleotide can produce different mature polypeptides (e.g., with different C-terminal and / or N-terminal amino acids) compared to another host cell expressing the same polynucleotide. In one embodiment, the mature polypeptide comprises up to 485 amino acid residues (e.g., amino acids 1 to 485 of SEQ ID NO:2). Thus, in one embodiment, the mature polypeptide comprises up to 486 amino acid residues corresponding to the amino acid sequence set forth in SEQ ID NO:3.

[0062] As used herein, the term "improvement factor" refers to a quantitative method for calculating the improvement of a specific characteristic of a variant according to the invention. The improvement factor can be determined according to the following formula:

[0063]

[0064] Other formulas can be used to determine the improvement factor. Those skilled in the art are aware of the currently proposed formula and alternative methods for calculating the improvement factor.

[0065] According to the present invention, a value of 1.0 corresponds to the specific activity observed against the parental α-amylase. Values ​​higher than 1.0 indicate an improvement in the specific activity of the tested variant compared to the parental α-amylase. Therefore, any value ≥1.0 indicates an improvement in the properties (e.g., specific activity) of the variant compared to the parental α-amylase.

[0066] Similarly, the term "improvement factor" can be used when measuring the stability (e.g., thermal stability) of a variant in a detergent composition. Therefore, the term "improvement factor" can also be used as an indicator of the stability of a variant. According to the invention, a value of 1.0 corresponds to the stability observed against the parental α-amylase. Values ​​higher than 1.0 indicate an improvement in the stability of the tested variant compared to the parental α-amylase. Therefore, any value ≥1.0 indicates an improvement in the stability of the variant compared to the parental α-amylase.

[0067] As used herein, the term "measure of stability" refers to a measure of enzyme stability. These measures of stability include stability in detergents, stability with chelating agents, pH stability, stability against proteases, chlorides and / or bleach, and thermal stability.

[0068] As used herein, the term "stability" refers to the residual activity of a given variant, which can be determined by incubating the variant in a standard detergent composition preferably including chelating agents such as EDTA, EGTA, DTPA, DTMPA, MGDA, or HEDP. Therefore, residual activity specifically relates to "chelating agent stability," meaning that the variant has shown a given residual activity after incubation in a standard detergent containing a chelating agent. For example, as shown in the Examples section, a variant can be tested in a standard detergent composition A comprising 0.1% EDTA at a final concentration. Residual activity can then be determined after a given incubation period at a given temperature. Residual activity can be determined, for example, by using the EnzCheck or Phadebas assays described in the Examples section. The activity of the variant tested after a given time and temperature can be compared to the activity of a reference incubated for the same period in the same detergent composition at low temperatures (e.g., 4°C and 0°C). The less difference between the two treatments, the higher the detergent stability. Similar tests can be performed using other detergents containing chelating agents such as DTMPA, sodium citrate, and HEDP. Similarly, surfactant stability can be determined in a similar manner. Therefore, variants of the present invention may preferably have improved stability against surfactants such as anionic surfactants and LAS, wherein surfactant stability can be determined by the same determinations as described above.

[0069] As used herein, “improved storage stability” refers to the stability of a given variant compared to the parent peptide, where residual activity is measured when stored at a given time and temperature. Therefore, the stability of the variant is improved under storage conditions compared to a reference (such as the parent peptide).

[0070] In one embodiment, this variant exhibits improved thermal stability.

[0071] As used herein, "improved thermal stability" refers to an improvement in the stability of a given variant compared to the parent peptide, wherein stability is determined after incubation at an elevated temperature for a given period of time. For example, the elevated temperature could be 50°C or higher.

[0072] In one embodiment, improved stability is determined by the following method, which includes the following steps;

[0073] a) Incubate α-amylase variant samples and parental α-amylase samples for a period of time in standard detergent compositions (such as standard A, standard J, standard T, or standard X);

[0074] b) Measure the activities of the variant α-amylase and the parent peptide, respectively; and

[0075] c) Calculate the residual activity of the sample.

[0076] If stability is determined in a standard powder detergent (such as standard T or standard X), stability can preferably be measured by incubation in a washing solution of the standard powder detergent, for example, by dissolving the powder in water in which the variant is incubated.

[0077] In another embodiment, improved stability is determined by the following method, which includes the following steps;

[0078] a) Incubate α-amylase variant samples and parental peptide samples in standard detergent compositions (such as Standard A, Standard J, Standard T, or Standard X) at 40°C to 60°C for 2 to 168 hours respectively;

[0079] b) Measure the activities of the variant α-amylase and the parent peptide, respectively; and

[0080] c) The residual activity of the sample is calculated as the average activity in the sample relative to the average activity of the frozen control sample.

[0081] As used herein, the term "measure of specific activity" refers to a measure of enzyme activity. These measures of activity include washability at pH 8.0 and 25°C and activity using synthetic substrates such as Phadebas tablets (Magle Life Sciences).

[0082] As used herein, the term "specific activity" refers to the number of moles of substrate converted into a product per unit time by an enzyme preparation under specific conditions. Specific activity is expressed as units (U) / mg of protein. Specific activity can be determined as described in Example 2.

[0083] As used herein, the term "washing performance" refers to the ability of an enzyme to remove starch or starch-containing stains present on an object to be cleaned during, for example, laundry washing or hard surface cleaning (such as dishwashing). The term "washing performance" includes general cleaning such as hard surface cleaning, as in dishwashing, but also includes washing performance on textiles such as clothing, and also includes industrial and institutional cleaning. Washing performance can be quantified by calculating a so-called intensity value, and the result can be displayed as an "improvement factor" (IF). Washing performance can be determined as described in the examples herein.

[0084] As used herein, the term "intensity value" refers to a washing performance that can be measured as luminance, expressed as the intensity of light reflected from a sample when illuminated with white light. When a sample is stained, the intensity of the reflected light is lower than that of a clean sample. Therefore, the intensity of the reflected light can be used to measure washing performance, with higher intensity values ​​associated with higher washing performance.

[0085] Color measurements were performed using a professional flatbed scanner (Kodak iQsmart, Kodak), which captures images of the washed textiles.

[0086] To extract light intensity values ​​from a scanned image, the 24-bit pixel values ​​from the image are converted into red, green, and blue (RGB) values. The intensity value (Int) can be calculated by summing the RGB values ​​as vectors and then taking the length of the resulting vector.

[0087]

[0088] As used herein, "improved washing performance" refers to the improvement in washing performance of the α-amylase of the present invention relative to the washing performance of the parent polypeptide. Improved washing performance can be measured by comparing so-called strength values ​​and calculating the improvement factor (IF). The improved washing performance was determined according to the "Washing Performance of α-Amylase as Determined Using Automatic Mechanical Stress" section and using standard detergent J at 15°C. Other standard detergents, such as standard detergent A, standard detergent X, or standard detergent T, can be used.

[0089] Therefore, in one embodiment, improved washing performance is determined by the following method, which includes the following steps;

[0090] a) Add α-amylase variants and parental polypeptide samples to standard detergent compositions, such as Standard A, Standard J, Standard T, or Standard X, respectively, and wash starch-dyed fabrics with the above compositions;

[0091] b) Measure the intensity of light reflected from the sample when illuminated with white light; and

[0092] c) Optionally calculate the improvement factor (IF), which is the ratio of the δ intensity of the α-amylase sample to the δ intensity of the parental peptide sample.

[0093] In one embodiment, improved washing performance is determined by the following method, which includes the following steps:

[0094] a) Add α-amylase variant and parental polypeptide sample to standard detergent composition, such as standard A, standard J, standard T, or standard X, respectively, and wash starch-dyed fabrics with the above composition for 20 minutes at 15°C and 30°C.

[0095] b) Measure the intensity of light reflected from the sample when illuminated with white light; and

[0096] c) Optionally calculate the improvement factor (IF), which is the ratio of the δ intensity of the α-amylase sample to the δ intensity of the parental peptide sample.

[0097] From Example 4 and Figure 1-3 The data obtained shows that the hit average at each test location indicates that many variants with modifications at one of the locations have an IF of at least 1.0 for at least one test condition, i.e., specific activity or stability in standard A or standard X.

[0098] As used herein, the term "hit mean" refers to the mean of the improvement factors measured for all generated variants with an IF greater than 1.0 for each test condition, i.e., the specific activity or stability of these variants tested in detergent compositions of standard A or standard X. Therefore, the improvement factors for each individual variant at a particular location are summed and divided by the number of variants for that particular location, thus providing the hit mean.

[0099] In one embodiment, the variant includes one or more substitutions selected from the group consisting of: H1A, H1G, H1V, H1C, H1S, H1L, H2T, H2G, H2L, H2M, H2E, H2P, D3A, D3C, D3R, D3Y, D3W, D3S, D3F, G4D, G4Q, G4R, T5L, T5Q, T5E, T5V, T5G, T5F, T5W, G7F, G7E, G7V, G7D, G7R, G7A, G7L, G7S, G7N, T8A, T8S, I9C, I9M, I9L, M10 L, Q11C, Q11L, Q11V, Q11M, Y12V, Y12A, Y12F, F13I, F13Y, E14M, E14L, E14S, E14Q, E14F W15G, W15F, W15S, W15I, W15H, N16G, N16Q, N16R, V17Q, V17A, V17T, V17L, V17D, V17G, V17E, P18L, N19G, N19S , N19Y, N19T, N19V, D20R, Q22C, Q22A, Q22N, Q22S, Q22M, Q22T, Q22E, Q22Y, H23W, W24H, W24F, N25V, N25G, N25 E, N25E, N25S, N25R, N25L, N25T, R26L, R26G, R26D, R26I, R26S, R26W, R26A, R26F, R26M, R26E, L27A, L27M, H2 8E, H28A, H28W, H28K, H28I, H28R, H28M, N29G, N29C, N29L, N29K, N29V, N29W, N29A, N30S, N30H, N30L, N30T, N3 0E, N30R, A31S, A31W, A31V, A31G, A31R, Q32G, Q32L, Q32A, Q32D, N33F, N33A, N33R, N33D, L34Y, L34F, K35L, K 35E, K35W, K35Q, N36R, N36V, N36Q, N36A, N36P, N36W, N36E, N36T, A37R, A37T, A37S, A37G, A37E, G38N, G38M, G 38Y, G38K, I39L, I39M, I39V, T40D, T40A, T40Q, T40E, T40S, A41Q, A41E, A41N, A41M, A41D, I42A, I42V, I42L, W43R, I44T, I44L, I44V, P45A, P46G, A47S, A47G, A47T, W48C, W48I, W48L, W48G, W48M, W48P, W48Q, W48H, W48A,W48Y、W48F、K49Q、G50S、G50N、G50P、T51S、T51G、T51C、T51P、T51L、T51A、S52V、S52M、S52I、S52L、S52A、S52D、S52K、S52N、S52E、S52T、S52C、S52F、Q53L、Q53V、Q53G、Q53M、N54G、N54K、N54V、N54T、N54L、N54I、N54S、D55G、D55V、D55I、V56L、V56M、V56T、G59E、G59A、G59S、A60S、A60W、A60L、A60G、A60V、A60F、A60T、Y61F、L63P、L63I、L63V、L63H、L63K、L63C、L63G、L63A、Y64W、Y64S、Y64V、Y64I、Y64H、Y64M、Y64C、Y64R、Y64G、Y64L、Y64A、L66M、E68D、E68S、E68M、E68C、E68T、E68V、E68Q、E68Y、F69H、F69W、N70G、N70A、N70T、N70S、N70E、Q71E、Q71N、Q71D、Q71I、Q71V、K72Y、K72R、G73V、G73L、G73E、G73C、T74L、T74V、T74N、V75T、V75G、V75E、R76S、R76G、R76A、R76V、T77A、T77S、T77V、K78L、K78T、K78A、T81W、T81L、T81G、K82A、K82G、A83G、A83E、A83Q、A83R、A83S、A83F、A83V、E84F、E84D、E84M、E84W、L85V、E86L、E86D、E86W、E86R、R87M、R87A、R87E、A88S、A88G、A88V、I89A、I89P、I89H、I89L、R90L、R90G、S91A、S91G、S91E、S91V、S91T、L92C、L92F、K93W、K93G、K93L、N95G、N95E、N95L、N95S、N95W、N95C、N95D、N95R、G96Q、G96R、I97M、I97V、I97L、Q98V、Q98L、Q98C、Q98M、Q98T、Q98F、Q98W、Q98P、Q98H、V99I、V99L、V99S、V99T、V99G、V99E、Y100W、Y100V、Y100F、V103G、V103A、V103S、V103T、H107Q、H107M、K108V、G109A、G109S、G109F、G109D、G110C、A111S、D112E、D112K、D112V、D112F、D112C、F113M、F113V、F113E、F113L、F113C、F113D、F113K、F113Q、F133Y、T114S、T114V、T114L、E115C、E115A、R116Q、R116N、R116L、R116I、R116D、R116T、R116G、R116E、R116S、R116C、R116V、R116F、R116K、R116H、R116W、V117G、V117C、V117S、V117W、V117A、V117E、V117L、V117I、V117M、Q118E、118F、Q118R、Q118V、Q118G、A119Y、A119W、V120I、V120D、V120L、V120C、V120A、V120N、E121V、E121M、E121D、E121P、E121G、V122E、V122P、V122L、V122I、V122F、N123G、N123A、N123E、N123W、N123T、N123P、N123K、N123V、P124R、P124G、Q125E、Q125T、N126G、N126A、N126E、N126T、N126R、R127G、R127S、R127C、R127W、R127L、R127V、R127F、R127Y、N128S、N128E、N128C、N128A、N128P、N128G、Q129L、Q129E、Q129C、E130C、V131L、V131E、V131I、S132A、S132H、S132P、S132C、S132E、S132D、S132V、S132Q、S132N、G133T、G133L、G133A、G133D、G133S、G133V、G133I、G133E、G133R、T134V、T134D、T134E、Y135L、Y135V、Y135S、Y135R、Y135T、Y135W、Y135K、Y135G、Y135P、Y135M、Y135D、Y135H、Y135F、Y135E、Q136E、Q136W、Q136G、I137A、I137S、I137G、I137V、E138A、E138L、E138Y、E138T、A139S、A139M、A139G、A139V、A139T、A139P、W140H、W140Y、W140S、W140A、T141C、T141G、T141S、T141M、G142D、G142E、G142A、G142R、F143M、F143L、F143I、N144C、N144G、N144R、N144M、N144K、F145A、F145C、F145S、F145N F145Q、F145T、F145H、P146L、P146S、P146V、P146C、P146M、P146T、G147V、G147P、G147D、G147S、G147I、G147L、G147C、G149C、G149Y、G149L、G149A、N150C、N150D、N150E、Q151L、Q151T、Q151C、Q151I、S153C、S153G、S153T、S154M、S154E、S154A、S154V、F155T、F155H、F155L、F155W、K156Q、K156F、K156L、K156S、K156G、K156Y、K156V、K156A、K156D、K156W、K156P、K156E、K156C、K156N、K156T、W157L、R158S、R158M、R158A、R158G、R158Q、R158W、R158L、R158T、R158V、R158E、R158Y、W159N、W159G、W159T、W159C、W159E、W159D、W159S、W159H、Y160M、Y160A、Y160Q、Y160T、Y160S、Y160V、Y160G、Y160I、Y160L、Y160E、Y160W、Y160R、Y160H、H161N、H161C、F162M、F162L、F162Y、F162I、D163A、D163R、D163S、D163K、D163L、G164S、G164T、G164C、T165L、T165S、T165I、T165M、T165G、T165V、T165E、D166S、D166T、D166N、W167G、W167Y、W167D、W167F、D168M、D168S、D168G、Q169L、Q169A、Q169S、Q169V、Q169F、Q169E、S170A、S170V、S170Q、S170W、S170G、S170F、S170T、S170L、R171A、R171F、R171S、R171K、R171V、R171M、Q172E、Q172L、Q172S、Q172R、Q172G、Q172W、Q172C、L172K、L173I、L173G、173N、L173M、L173S、L173Y、L173V、L173A、L174F、A174V、A174G、A174K、A174L、A174M、A174S、N175G、N175L、N175V、N175I、N175S、R176W、R176E、R176G、R176M、R176M、R176T、R176K、R176Y、I177L、I177S、I177G、Y178V、Y178R、Y178K、Y178W、K179L、K179C、K179E、K179S、K179W、K179A、K179G、K179T、K179F、F180A、F180L、R181A、R181G、R181K、R181P、R181S、R181V、R181Q、R181E、R181D、G184L、G184R、G184V、G184T、G184D、K185G、K185A、K185D、K185N、K185V、A186S、A186D、A186C、A186T、A186Q、A186V、A186N、A186E、A186H、W187G、D188R、D188Q、D188G、D188M、D188L、D188V、W189R、W189A、W189G、W189C、E190D、E190N、E190A、V191M、V191A、D192E、D192Q、D192S、T193K、T193V、T193C、T193P、T193S、T193G、T193D、T193R、E194S、E194D、E194L、E194Q、N195R、N195A、N195F、N195T、G196A、G196V、G196P、N197G、N197V、N197Y、N197C、N197D、Y198W、Y198A、Y198G、D199G、D199S、D199A、Y200W、Y200F、Y200C、Y200V、Y200T、Y200L、Y200A、Y200G、Y200S、M202T、M202L、Y203L、Y203W、Y203F、Y203G、Y203C、Y203I、Y203Q、Y203H、Y203T、Y203M、Y203V、Y203S、A204P、A204C、A204R、A204H、A204W、A204F、A204L、A204Y、A204D、A204G、D205C、D205A、D205L、D205R、V206H、V206Q、V206P、V206A、V206S、V206K、V206L、V206I、V206M、V206F、V206Y、M208R、M208P、M208L、M208C、D209C、D209E、D209H、D209F、D209Y、D209S、D209M、D209V、D209L、D209I、D209Q、H210V、H210F、H210A、H210W、H210L、H210G、H210D、H210E、H210Q、H210N、P211G、P211W、P211C、E212G、E212I、E212F、E212M、E212D、E212K、E212Y、E212R、E212C、V213M、V213T、V213G、V213C、V213W、V213R、V213F、I214P、I214V、N215C、N215D、N215V、N215T、L217V、L217S、L217T、L217G、L217C、L217P、L217A、L217W、N218S、N218D、N218R、N218L、N218M、R219L、R219W、R219S、R219A、R219H、R219I、R219M、R219T、R219E、R219Y、R219D、R219C、W220I、W220P、W220E、W220R、W220A、W220S、W220L、G221S、G221A、V222F、V222C、V222E、V222L、W223Y、W223F、W223L、Y224L、A225K、N226C、N226Q、N226M、N226E、N226K、N226R、T227S、T227M、T227C、T227L、T227Y、T227R、L228A、L228F、L228M、N229T、N229S、L230S、L230A、L230T、L230V、D231E、D231C、D231G、D231P、F233A、F233L、F233T、F233P、F233M、V238C、V238W、V238A、H240W、I241L、K242A、K242C、K242Y、K242P、K242W、K242G、K242L、K242R、K242S、K242E、K242Q、K242H、S244Q、F245G、F245C、F245V、F245D、F245A、M246L、M246F、M246V、M246I、R247Y、R247G、R247M、D248G、D248R、W249L、W249F、L250W、L250T、L250A、L250C、L250Y、L250F、G251K、G251M、G251Q、H252E, H252W, H252C, H252V, H252T, H252F, V253S, V253W, V253A, V253L, R254Q, R254S, G255C, G255E, G255L, Q256P, T257V, G258V, G258C, G258I, G258 P、G258L、G258D、G258F、K259V、K259Q、K259I、K259L、K259A、K259S、K259D、 K259C、N260E、N260C、N260W、N260D、N260S、N260R、N260G、L261G、L261D、L26 1T, F262Q, F262L, F262E, F262R, F262P, F262V, A263L, A263C, V264T, V264L, Y267V, Y267L, W268V, W268Y, K269W, K269L, K269Q, K269R, N270D, N270A, N 270M, N270G, D271E, D271V, D271C, L272M, G273W, G273R, G273V, G273E, A274S, A274D, A274E, L275G, L275E, L275A, L275R, L275K, E276R, E276L, N277D Y278G, Y278S, L279V, L279Q, L279G, L279M, S280C, S280R, S280E, S280D, K281L, K281A, K281W, K281G, T282G, T282A, T282C, T282R, T282I, N283G, N283 E、N283Q、N283V、N283A、W284T、W284I、W284S、W284E、W284C、W284V、W284A、 T285E、T285F、T285S、T285P、T285W、T285M、M286D、M286L、M286C、S287T、S28 7E、S287V、S287Q、S287P、S287L、S287W、S287C、A288L、A288Y、A288V、V291T 、P292A、P292Q、P292L、P292R、P292G、L293A、L293E、L293T、L293G、L293Q、L 293Y, H294G, H294A, H294W, H294Q, H294S, Y295H, Y295S, Y295I, Y295A, N296A, N296Y, N296M, N296F, N296H, N296S, N296K, L297S, L297W, L297H, Y298VY298P, Q299S, Q299L, Q299R, Q299I, Q299P, Q299A, Q299H, A300V, A300C, S301A, S301R, S301Q, N302I, N302T, N302M, N302S, S3 03I、S303E、S303A、S303M、S304I、S304P、S304G、S304L、S304F、 S304A、S304M、G305S、G305V、G305L、G305F、G305I、G305Q、N306V N306L, N306D, N306S, N306T, Y307F, Y307W, M309L, R310A, R310T, N311C, N311Y, N311L, N311R, L312F, L312V, L312I, L313S, L3 13M, L313I, N314Y, N314D, N314F, G315V, G315I, G315T, G315R, G315F, T316K, T316S, T316C, T316G, V318C, V318E, V318L, V318A 、Q319L、Q319I、Q319A、Q319H、Q319D、Q319F、Q319P、R320C、R32 0V、R320L、R320A、R320S、H321T、H321E、H321V、H321C、H321S、H 321G, H321R, P322E, P322C, S323N, A325V, A325S, V326I, V326G, V326A, V326C, V326T, T327S, T327L, T327V, T327E, T327Q, F328 M, F328C, V329A, 329S, V329C, V329I, N331C, N331V, N331I, T334S, T334A, P336M, P336E, P336D, P336R, P336L, P336S, P336K, P3 36W, G337T, G337V, G337C, G337M, G337F, E338A, E338H, E338T, E341C, E341D, E341I, E341M, E341F, E341K, E341S, E341P, E341T S342G、S342T、S342A、V344S、V344L、Q345K、Q345V、Q345I、Q345L、Q345C、Q345W、Q345N、G346H、G34 6A、G346L、G346N、G346R、G346Q、G346V、G346S、W347L、W347R、W347V、W347C、F348Q、F348S、F348C、F348P, F348M, K349G, K349A, K349Y, K349L, K349S, P350S, P350Q, P350L, P350G, P350D, P350E, L351W, L351M, L351S, L351Q, L351H, A3 52S, A352C, A354C, T355V, T355A, T355L, T355Y, T355F, L357C, L357S, L357T, L357H, T358V, T358F, T358S, T358L, E360S, E360D, E360C E360G, E360A, E360M, Q361P, Q361R, Q361V, Q361A, Q361E, Q361G, Q361D, Q361S, G362Y, G362R, G362C, G362N, G362M, G362V, Y363Q, Y3 63L, Y363I, Y363A, Y363V, Y363R, Q365D, Q365M, Q365L, Q365T, Q365C, Q365S, V366L, V366A, V366I, V366C, F367L, F367S, F367A, Y368M Y368T, Y368H, Y368A, Y368S, Y368Q, Y368I, Y368L, Y368V, G369K, G369S, G369E, G369A, G369R, G369I, G369L, D370A, D370T, Y372A, Y3 72S, Y372I, Y372L, Y372G, G373C, G373S, G373R, G373T, G373Q, G373W, I374Y, I374L, I374S, I374M, P375L, P375A, P375T, P375V, P375R P375E, P375S, S376A, S376N, S376P, S376W, S376R, S376L, S376E, S376T, S376V, S376I, S376Q, S376F, D377C, D377L, D377V, D377T, D3 77S, D377G, V379L, V379F, V379P, V379S, P380L, P380E, P380A, P380V, P380G, P380I, P380Y, S381N, S381L, S381A, S381I, S381V, Y382I Y382A、Y382E、Y382L、Y382V、Y382R、Y382K、R383G、R383S、R383P、 R383V、Q384A、Q384P、Q384L、Q384H、Q384R、Q384Y、Q384S、Q384V、Q384T、Q384G、Q384E、I386T、I386L、I386M、I386W、I386V、D387H、D387V、D387Q、D387L、P388G、P388T、P388M、P388V、P388W、P388K、P388L、P388S、P388R、L389S、L389H、L389A、L389V、L389F、L389M、L390S、L390F、L390I、L390G、L390V、L390T、L390N、L390H、L390C、K391L、K391Y、K391A、K391Q、A392I、A392M、A392T、Q394S、Q394R、Q394L、Q394T、Q394N、Q394M、Q395W、Q395N、Q395L、Q395E、Q395K、Q395R、Q395V、Q395S、Q395M、Y396T、Y396L、Y396R、Y396W、A397C、A397G、A397S、Y398K、Y398S、Y398C、Y398N、R400L、R400C、R400K、R400S、R400M、R400E、R400I、H402V、H402E、H402M、H402A、H402Y、H402S、H402P、H402L、H402D、H402R、H402T、D403E、D403P、D403Q、D403I、Y404F、Y404W、Y404K、F405C、F405V、F405I、F405P、F405W、F405G、F405A、D406E、D406V、D406T、D406M、D406S、H407C、H407D、W408R、W408G、W408D、W408P、W408C、W408Y、D409G、V410I、V410Q、V410E、V410L、I411A、I411L、I411V、T414A、T414V、R415F、R415L、R415A、R415Q、E416C、E416V、E416L、E416A、E416Q、N418L、N418A、N418C、N418M、N418G、N418R、N418F、A419E、A419C、A419M、A419Q、A419G、A419V、A419L、A419R、S420E、S420T、S420V、S420M、H421V、H421C、H421A、H421R、H421T、P422Y、P422H、P422D、P422R、S424C、S424A、S424L、G425A G425V、L426S、L426A、A427V、A427S、T428L、T428N、T428A、T428G、I429V、I429A、M430R、M430L、M430A、M430T、S431A、S431T、S431F、D432Q、D432P、G435E、G435N、G435M、G435I、G435S、G436L、G436M、G436E、G436T、S437G、S437Q、S437L、K438T、W439C、W439H、W439E、W439A、W439R、W439T、W439N、W439T、W439R、W439N、M440L、Y441N、Y441H、Y441Q、Y441K、V442I、444A、R444S、R444L、R444P、R444V、Q445L、Q445S、Q445C、Q445E、Q445R、K446Q、K446G、K446T、K446P、A447G、A447Q、A447E、A447T、A447R、A447S、G448S、G448R、V450A、V450T、V450Q、V450R、W451L、H452A、H452T、H452C、H452G、H452Y、H452R、H452V、D453E、M454R、M454L、M454V、M454Q、M454S、T455L、T455S、G456V、G456R、G456Y、G456K、G456L、N457V、N457I、N457G、N457R、S459E、S459V、S459C、S459P、S459M、S459T、S459Q、S459R、G460S、G460D、G460E、T463C、T463V、T463L、T463D、T463H、T463M、T463A、T463Q、I464V、N465G、N465Q、N465L、N465R、Q466L、Q466C、Q466E、D467V、D467C、W469T、W469M、W469E、W469N、H471Q、F473Y、F473C、F473R、F473E、F473G、F473T、F473V、N475T、N475H、N475G、G476F、G476Y、G476K、G477Q、G477F、G477K、G477L、V481Q、V481S、And W482F, wherein the number is based on the amino acid sequence as illustrated in SEQ ID NO:3, and wherein the substitutions at one or more of these positions provide variants having an IF of ≥1.0 for a measure of stability, and / or an IF of ≥1.0 for a measure of specific activity.

[0100] As used herein, the term "corresponds to" refers to the manner in which a specific amino acid in a sequence is identified (where a specific amino acid sequence is referenced). For example, for the purposes of this invention, when referring to a specific amino acid position, those skilled in the art can align another amino acid sequence with the already referenced amino acid sequence to determine which specific amino acid in the other amino acid sequence may be of interest. Alignments of another amino acid sequence with, for example, the sequence illustrated in SEQ ID NO:3 or any other sequence listed herein have been described elsewhere herein. Alternative alignment methods may be used, and these methods are well known to those skilled in the art.

[0101] In one embodiment, the parental α-amylase has at least 85%, for example, at least 90%, for example, at least 95%, for example, 97%, for example, at least 98% sequence identity with the amino acid sequence described in SEQ ID NO:3. In another embodiment, the parental α-amylase has at least 85%, for example, at least 90%, for example, at least 95%, for example, 97%, for example, at least 98% sequence identity with the amino acid sequence described in SEQ ID NO:4.

[0102] In one embodiment, the parental α-amylase consists of or includes the amino acid sequence described in SEQ ID NO:3 or 4.

[0103] This invention provides variants of such parental α-amylases.

[0104] Therefore, variations of the invention include amino acid sequences having at least 60%, for example at least 65%, for example at least 70%, for example at least 75%, for example at least 80%, for example at least 85%, for example at least 90%, for example at least 95%, for example at least 97%, for example at least 98%, for example at least 99%, but less than 100%, sequence consistency with the amino acid sequences illustrated in SEQ ID NO:3 and 4.

[0105] Variations according to the invention may include modifications other than substitution, such as insertion or deletion. Such modifications may also be referred to as “alterations.” Thus, as used herein, the term “alteration” refers to the substitution and deletion of amino acids within the amino acid sequence of the polypeptide. The terms “alteration” and “modification” may be used interchangeably herein. This should not be construed as any limitation, and therefore, unless explicitly stated otherwise, these terms have the same meaning and purpose.

[0106] In one embodiment, the variant includes substitutions at one or more locations selected from the group consisting of: H1, H2, D3, G4, T5, G7, T8, I9, Q11, Y12, F13, E14, W15, N16, P18, N19, D20, Q22, W24, N25, R26, L27, H28, N29, N30, A31, Q32, L34, K35, N36, A37, I39, T40, A41, W43, I44, P45, P46, A47, W48, G50, T51, S52, Q53, N54, D55, V56, G59, A60, Y61, L63, Y64, L66, E68. F69, N70, Q71, K72, G73, T74, V75, R76, T77, K78, K82, A83, E84, L85, R87, A8 8. I89, R90, S91, L92, K93, N95, G96, I97, Q98, V99, Y100, V103, H107, K108, G 109, D112, F113, E115, R116, V117, A119, V120, E121, V122, N123, Q125, N12 6. R127, N128, Q129, E130, V131, S132, G133, T134, Y135, I137, A139, W140, T 141, G142, F143, N144, F145, P146, G147, G149, N150, Q151, S153, F155, K15 6. W157, R158, W159, Y160, F162, D163, G164, T165, D166, W167, D168, Q169, S170, R171, Q172, L173, A174, N175, R176, I177, Y178, K179, F180, R181, G1 84. K185, A186, W187, D188, W189, E190, V191, D192, T193, E194, N195, G196, N197, Y198, D199, Y200, M202, Y203, A204, D205, V206, M208, D209, H210, P2 11. E212, V213, I214, N215, L217, R219, V222, W223, A225, N226, T227, L228, N229, L230, D231, F233, V238, I241, K242, F245, M246, W249, L250, H252, R2 54. G255, T257, G258, K259, N260, L261, F262, A263, Y267, W268, K269, N270,D271, G273, A274, Y278, L279, S280, K281, T282, N283, W284, T285, M286, S287, A288, V291, P292, L2 93. H294, N296, L297, Q299, A300, S301, S303, S304, G305, N306, L312, L313, N314, G315, T316, V318 , Q319, R320, H321, P322, A325, P336, G337, E341, S342, V344, Q345, G346, W347, F348, K349, P350, L 351, A352, L357, T358, Q361, G362, Y363, Q365, V366, Y368, G369, D370, Y372, G373, I374, P375, S376 , D377, V379, P380, S381, Y382, R383, Q384, I386, D387, P388, L389, L390, K391, A392, Q394, Q395, Y 396, A397, R400, H402, F405, D406, H407, W408, V410, T414, R415, E416, N418, A419, H421, G425, A427 The amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 1, 1, 1, 2, 3 ...

[0107] In a specific embodiment, the variant includes one or more substitutions selected from the group consisting of: H1V, H1G, H1S, H1C, H2T, H2L, H2M, H2E, H2P, H2G, D3A, G4Q, G4D, G4R, T5Q, T5G, T5E, T5V, G7F, G7V, G7E, G7E, G7D, G7R, T8S, I9M, I9L, Q11L, Q11V, Y12V, Y12A, Y12F, F13Y, F13I, E14L, E14M, E14S, W15G, W 15S, W15F, N16G, N16Q, P18L, N19G, N19S, N19Y, N19T, N19V, D20R, Q22C, Q22A, Q22N, Q22S, Q22M, Q22T, Q22E, W24F, N25V, N2 5S, N25G, N25E, N25E, R26L, R26G, R26D, R26M, R26I, R26S, R26W, R26A, R26F, L27A, L27M, H28A, H28E, H28I, N29G, N29C, N29L N30S, N30H, N30L, N30T, N30E, A31S, A31V, Q32D, Q32G, Q32L, Q32A, L34F, L34Y, K35L, K35E , K35W, K35Q, N36R, N36Q, N36A, N36E, A37T, A37S, I39M, I39L, T40E, T40A, A41E, A41N, A41 M, A41Q, A41D, W43R, I44T, P45A, P46G, W48G, W48L, W48C, W48I, W48M, W48P, W48Q, W48Y, W4 8F, G50P, G50S, G50N, T51S, T51G, T51P, T51L, T51C, S52M, S52V, S52I, S52L, Q53V, Q53L, Q5 3G, N54G, N54K, N54V, D55V, D55I, V56L, V56M, G59E, G59A, A60S, A60W, A60L, A60G, A60T, Y 61F, L63V, L63P, L63I, L63H, L63K, Y64W, Y64S, Y64V, Y64I, Y64H, Y64M, Y64C, Y64R, Y64G, Y 64L, Y64A, L66M, E68D, F69H, F69W, N70G, N70A, N70T, N70S, N70E, Q71N, Q71E, Q71D, Q71I, Q71V, K72Y, K72R, G73V, G73L, G73E, T74V, T74L, V75T, V75G, R76S, R76G, R76A, T77S, K78L,K78T、K78A、K82A、A83G、A83E、E84D、L85V、R87M、R87A、R87E、A88V、I89H、I89P、I89A、R90L、R90G、S91E、S91A、S91G、L92C、K93W、K93G、K93L、N95L N95W、N95C、N95E、N95S、N95G、G96Q、I97V、Q98C、Q98M Q98T、Q98V、Q98L、V99I、V99L、V99S、V99T、Y100V、Y100F、Y100W、V103G、H107Q、K108V、G109A、G109S、D112E、F113L、F113M、F113V、F113E、F113C、F113D、F113Y、E115A、R116N、R116L、R116I、R116D、R116T、R116G、R116E、R116S、R116C、R116V、R116Q、R116F、V117C、V117L、V117S、V117W、V117A、V117G、V117E、A119Y、V120D、V120L、E121P、E121G、E121V、E121M、E121D、V122E、V122P、V122I、N123G、N123A、N123E、N123W、N123T、N123P、Q125E、Q125T、N126E、N126G、N126A、R127G、R127S、R127C、R127W、R127L、R127V、R127F、R127Y、N128C、N128S、N128E、Q129L、Q129C、Q129E、E130C、V131L、V131E、S132A、S132H、S132P、S132C、S132E、S132D、S132N、G133S、G133T、G133L、G133V、G133I、G133E、G133A、G133DT134V、T134D、T134E、Y135L、Y135V、Y135S、Y135R、Y135T、Y135W、Y135K、Y135G、Y135P、Y135M、Y135D、Y135H、Y135F、Y135E、I137A、I137S、I137G、A139M、A139S、W140Y、W140S、W140AW140H、T141M、T141S、T141C、T141G、G142D、G142E、F143M、F143L、N144C、F145C、F145S、F145A、P146S、P146V、P146C、P146L、G147V、G147P、G147D、G147S、G147I、G147L、G147C、G149C、N150C、N150D、N150E、Q151T、Q151C、Q151L、S153C、F155H、F155T、F155L、K156Y、K156V、K156A、K156D、K156W、K156P、K156E、K156C、K156F、K156Q、K156L、K156S、K156G、W157L、R158M、R158A、R158G、R158Q、R158W、R158L、R158T、R158V、R158E、R158Y、R158S、W159N、W159G、W159T、W159C、W159E、W159D、W159S、W159H、Y160Q、Y160M、Y160T、Y160A、Y160S、Y160V、Y160G、Y160I、Y160L、Y160E、F162M、F162L、F162Y、F162I、D163K、D163A、D163R、D163L、G164S、G164T、G164C、T165L、T165M、T165S、T165G、T165I、D166T、D166N、D166S、W167Y、W167G、W167D、D168M、D168S、D168G、Q169A、Q169L、Q169E、S170A、S170V、R171F、R171S、R171K、R171V、R171A、Q172S、Q172L、Q172E、L173G、L173N、L173M、L173S、L173Y、L173V、L173I、A174G、A174V、N175L、N175V、N175I、N175G、R176E、R176W、R176G、R176M、I177L、I177S、I177G、Y178V、Y178R、Y178K、K179C、K179E、K179S、K179W、K179A、K179G、K179T、K179F、K179L、F180A、F180L、R181K、R181P、R181S、R181V、R181Q、R181E、R181A、R181G、G184L、G184R、G184V、G184D、K185A、K185D、K185N、K185V、K185G、A186C、A186S、A186D、W187G、D188Q、D188G、D188R、D188M、D188L、D188V、W189R、W189A、W189G、W189C、E190D、E190N、E190A、V191M、V191A、D192Q、D192S、T193K、T193V、T193C、T193P、T193S、E194D、E194L、E194Q、N195R、N195A、G196A、G196V、N197G、N197V、N197Y、N197C、N197D、Y198W、D199G、D199S、D199A、Y200W、Y200F、Y200C、Y200V、Y200T、Y200L、Y200A、Y200G、Y200S、M202T、Y203F、Y203G、Y203C、Y203I、Y203Q、Y203H、Y203T、Y203M、Y203S、Y203V、Y203W、Y203L、A204P、A204R、A204C、A204H、A204W、A204F、A204L、A204Y、A204D、A204G、D205C、V206H、V206Q、V206P、M208C、D209E、D209H、D209F、D209Y、D209S、D209M、D209V、H210V、H210A、H210W、H210L、H210G、H210D、H210E、H210N、P211G、P211W、P211C、E212F、E212M、E212D、E212G、E212I、V213W、I214P、N215D、N215V、N215C、L217C、L217P、L217T、L217G、L217S、R219H、R219W、R219S、R219I、R219A、R219M、R219T、R219E、R219Y、R219D、R219C、R219L、V222F、V222C、V222E、W223Y、A225K、N226Q、N226C、T227S、T227M、T227C、T227L、L228A、L228F、N229S、L230S、L230A、L230T、L230V、D231G、F233A、F233L、V238C、V238W、I241L、K242C、K242Y、K242P、K242W、K242G、K242L、K242R、K242S、K242E、K242A、K242Q、F245C、F245V、F245D、F245G、M246L、M246F、W249L、L250T、L250A、H252E、H252W、H252C、R254Q、R254S、G255E、G255L、T257V、G258V、G258C、G258I、G258L、G258P、K259V、K259Q、K259I、K259L、N260E、N260D、N260C、N260W、L261G、L261D、F262Q、F262L、F2 62E、F262R、F262P、F262V、A263L、A263C、Y267V、Y267L、W268V、K269L、K269W N270D, N270A, N270M, D271V, D271C, G273W, G273R, G273V, A274S, A274D, A274E, Y278G, Y278S, L279V, L279G, L279Q, S280C, K281L, K281W, K281G, K281A 、T282R、T282I、N283V、N283A、W284E、W284C、W284V、W284A、T285E、T285F、T2 85S、M286D、M286L、S287V、S287Q、S287T、S287E、S287P、S287L、S287W、S287C A288L, A288Y, V291T, P292A, P292Q, P292R, P292L, L293E, L293T, L293G, H294G, H294A, Y295F, Y295N, N296A, N296Y, N296M, N296F, L297S, L297W, Q299I Q299S, Q299L, Q299R, A300V, S301R, S301Q, S303I, S303E, S304P, G305S, N306V, L312F, L312V, L313S, N314F, N314D, G315I, G315V, T316S, V318C, V318 E, V318A, V318L, Q319L, Q319I, Q319A, R320L, R320V, R320C, H321E, H321V, H321C, H321T, P322E, P322C, A325V, A325S, P336M, P336E, P336D, P336R, P3 36L, G337T, G337C, G337M, G337V, E341C, E341D, E341I, S342G, S342T, S342A, V344S, V344L, Q345K, Q345C, Q345V, Q345I, Q345L, G346H, G346A, G346L G346N, G346Q, G346V, G346R, W347L, F348Q, F348S, F348C, K349G, K349A, K349Y, K349L, K349S, P350L, 350G, P350S, P350Q, L351M, L351S, A352S, A352CL357C, L357T, L357S, T358V, T358F, T358S, Q361P, Q361R, Q361A, Q361E, Q361V, G362Y, G362R, G362C, G362N, G362M, G362V, Y363Q, Y363I, Y363A, Y363 L, Q365D, V366A, Y368M, Y368T, Y368I, Y368L, Y368A, Y368S, Y368Q, Y368V, Y368H, G369K, G369S, G369A, G369E, G369R, G369I, D370A, D370T, Y372S, Y37 2I, Y372L, Y372G, G373C, G373S, I374Y, P375L, P375A, P375T, S376P, S376W, S376R, S376L, D377C, D377L, D377V, V379F, V379P, V379L, P380L, P380G, P 380E、P380A、P380I、P380V、S381N、S381L、S381A、S381I、S381V、Y382I、Y38 2A、Y382E、Y382V、Y382L、R383G、R383S、R383P、Q384L、Q384H、Q384R、Q384Y、 I386T, D387H, D387V, D387Q, D387L, P388G, P388T, P388M, P388W, P388V, L389S, L389H, L390I, L390G, K391Y, K391A, A392I, Q394S, Q394R, Q395N, Q395 L、Q395E、Q395K、Q395R、Y396L、A397C、A397G、R400L、R400C、R400K、R400S、 R400M、H402V、H402E、H402M、H402A、H402S、H402P、H402Y、H402L、F405C、F40 5V、F405I、F405P、F405W、F405G、D406E、H407C、H407D、W408R、W408P、W408C 、W408Y、W408G、W408D、V410I、V410Q、T414V、T414A、R415F、R415L、E416C、E 416V、N418L、N418A、N418C、N418M、N418G、A419E、A419C、A419M、H421V、H42 1C、H421A、H421R、G425A、A427V、T428G、I429V、I429A、M430R、S431A、G435E、G435N, G436M, G436E, G436L, G436T, S437Q, S437L, W439C, W439H, W439E, W439R, W439N, M440L, V442I, R444A, R444S, R444L, R444V, R444P, Q445L, Q445S, Q445C, Q445E, A447G, A447Q, A447E, A447T, G448S, H452A, H452T, M454R, M454L, M454V, T455S, G456V, G456R, G456Y, N457R, S459V, S459C, S459P, S459M, S459T, S459E, S459Q, G460S, G460D, G460E, T463D, T463H, N465Q, N465L, N465R, D467V, D467C, W469E, W469T, H471Q, F473Y, F473C, F473R, N475T, N475H, G476K, G477Q, G477F, G477K, G477L, V481Q, and V481S, where the numbers are based on SEQ. The amino acid sequence is defined in ID NO:3, and wherein the one or more substitutions provide an IF ≥ 1.2 for a measure of specific activity in the standard A detergent composition and optionally an IF ≥ 1.0 for a measure of stability.

[0108] In one embodiment, the variant includes one or more substitutions selected from the group consisting of: H1V, H2T, G7F, G7V, G7E, G7E, G7D, I9M, I9L, Q11L, Q11V, W15G, W15S, N19G, N19S, Q22C, Q22A, W24F, N25V, R26L, H28E, N29G, N30S, A31S, L34Y, K35L, I39L, T40A, A41Q, I44T, W48C, W48I, W48Y, W48F, S52M, S52V, Q53V, Q53L, V56L, G59E, T74V, T74L, V75T, I89A, R9 0L, S91A, S91G, L92C, N95E, N95S, N95G, I97V, Q98V, Q98L, Y100W, K108V, F1 13M, F113V, F113E, F113Y, R116Q, V117G, N126G, N126A, N128S, N128E, Q129E , G133A, G133D, A139S, W140H, T141S, T141C, T141G, F143M, F143L, F145A, P 146L, Q151L, K156F, K156Q, K156L, K156S, K156G, R158S, T165I, D166S, Q169 L, S170A, S170V, R171A, Q172L, Q172E, L173I, A174V, N175G, K179L, R181A, R181G, K185G, A186S, A186D, T193S, Y203W, Y203L, D209S, D209M, D209V, H21 0N, E212G, E212I, V213W, N215C, L217S, R219L, V222E, N226C, T227L, L228F , K242A, F245G, H252C, T257V, G258P, K259L, N260W, K269W, L279Q, K281A, M2 86L, P292L, L293T, L293G, Y295F, L297W, Q299S, Q299L, Q299R, N306V, L312 F, L312V, G315V, T316S, V318L, R320V, R320C, H321T, P336L, G337V, S342A, V 344L, Q345L, G346R, W347L, P350S, P350Q, Q361V, Y363L, Y368H, I374Y, S37 6R, S376L, V379L, P380V, S381V, S381N, D387Q, D387L, A392I, Q395E, Q395K,Q395R, H402L, F405G, T414A, E416V, N418M, N418G, A419M, S431A, G435N, W439E, W439R, W439N, G456Y, S459E, S459Q, N465L, N465R, and D467C, wherein the numbering is based on the amino acid sequence as defined in SEQ ID NO:3, and wherein one or more substitutions provide an IF ≥ 1.2 for a measure of specific activity in a standard A detergent composition and an IF ≥ 1.0 for a measure of stability.

[0109] In one embodiment, the variant includes substitutions at one or more locations selected from the group consisting of: H1, H2, G4, T5, G7, Q11, F13, E14, W15, N16, P18, N19, D20, Q22, N25, R26, N29, N30, A31, Q32, L34, A41, P46, W48, G50, T51, S52, Q53, D55, V56, G59, A60, Y61, Y64, F69, N70, Q71, K72, G73, T74, V75, R76, T77, K78, K82, A83, R87, I89, R90, S91, L92, K93, N95, Q9 8. V99, Y100, H107, K108, G109, D112, F113, E115, R116, V117, A119, V120, E 121, V122, N123, N126, R127, N128, Q129, E130, V131, S132, G133, T134, Y135 , I137, A139, W140, T141, G142, F143, N144, F145, P146, G147, G149, N150, Q 151, S153, F155, K156, W157, R158, W159, Y160, F162, D163, G164, T165, D166 , W167, D168, Q169, S170, R171, Q172, L173, A174, N175, R176, I177, Y178, K 179, F180, R181, G184, K185, A186, W187, D188, W189, V191, T193, E194, N197 , D199, Y200, M202, Y203, A204, D205, V206, D209, H210, P211, E212, N215, L 217, R219, W223, N226, V238, I241, K242, F245, G258, K259, N260, F262, A263 , K269, N270, D271, G273, A274, Y278, L279, K281, W284, T285, S287, P292, N 296, L297, Q299, S301, N306, L312, G315, V318, Q319, R320, H321, P322, A325 , P336, G337, E341, S342, V344, G346, F348, K349, P350, L351, Q361, G362, Y3 63. Y368, G369, D370, Y372, G373, S376, D377, V379, P380, S381, Y382, Q384,D387, Q394, Q395, A397, R400, H402, H407, W408, V410, R415, N418, H421, S431, G435, G436, W439, M440, Q445, A447, M454, G456, N465, D467, N475, G477, and V481, wherein these positions correspond to amino acid positions in the amino acid sequence as defined in SEQ ID NO:3, and the one or more substitutions provide an improvement factor of ≥1.4 for a measure of specific activity in the standard A detergent composition and optionally an improvement factor of ≥1.0 for a measure of stability.

[0110] In a specific embodiment, the variant includes one or more substitutions selected from the group consisting of: H1V, H1G, H1S, H1C, H2E, H2P, H2G, G4D, G4R, T5E, T5V, G7F, G7V, G7E, G7E, G7D, G7R, Q11V, F13Y, F13I, E14L, E14M, E14S, W15G, W15S, W15F, N16G, N16Q, P18L, N19G, N19S, N19Y, N19T, N19V, D20R, Q22C, Q22A, Q22S, Q22M, Q22T, Q22E, N25V, N25S, N25G, N25E, N25E, R26L, R26G, R2 6D, R26M, R26I, R26S, R26W, R26A, R26F, N29G, N29L, N30L, N30T, N30E, A31S, A3 1V, Q32D, Q32G, Q32L, Q32A, L34Y, A41Q, A41D, P46G, W48C, W48I, W48M, W48P, W48 Q, W48Y, G50P, G50S, G50N, T51G, T51P, T51L, T51C, S52M, S52V, S52I, S52L, Q53 V, Q53L, Q53G, D55V, D55I, V56L, V56M, G59E, G59A, A60S, A60W, A60L, A60G, A60T Y61F, Y64W, Y64S, Y64V, Y64I, Y64H, Y64M, Y64C, Y64R, Y64G, Y64L, Y64A , F69W, N70G, N70A, N70T, N70S, N70E, Q71N, Q71E, Q71D, Q71I, Q71V, K72Y , K72R, G73V, G73L, G73E, T74V, T74L, V75G, R76G, R76A, T77S, K78L, K78 T, K78A, K82A, A83E, R87A, R87E, I89H, I89P, I89A, R90L, R90G, S91E, S91 A. S91G, L92C, K93W, K93G, K93L, N95W, N95C, N95E, N95S, N95G, Q98C, Q9 8M, Q98T, Q98L, V99S, V99T, Y100V, Y100F, Y100W, H107Q, K108V, G109A, G 109S, D112E, F113L, F113M, F113V, F113E, F113C, F113D, E115A, R116N, R116L, R116I, R116D, R116T, R116G, R116E, R116S, R116C, R116V, R116Q,R116F、V117C、V117L、V117S、V117W、V117A、V117G、V117E、A119Y、V120L、E121P、E121G、E121V、E121M、E121D、V122E、V122P、V122I、N123A、N123E、N123W、N123T、N123P、N126E、N126G、N126A、R127G、R127S、R127C、R127W、R127L、R127V、R127F、R127Y、N128C、N128S、N128E、Q129C、Q129E、E130C、V131L、V131E、S132A、S132H、S132P、S132C、S132E、S132D、G133V、G133I、G133E、G133A、G133D、T134V、T134D、T134E、Y135L、Y135V、Y135S、Y135R、Y135T、Y135W、Y135K、Y135G、Y135P、Y135M、Y135D、Y135E、I137A、I137S、I137G、A139M、A139S、W140Y、W140S、W140A、W140H、T141M、T141S、T141C、T141G、G142D、G142E、F143M、F143L、N144C、F145C、F145S、F145A、P146S、P146V、P146C、P146L、G147V、G147P、G147D、G147S、G147I、G147L、G147C、G149C、N150C、N150D、N150E、Q151T、Q151C、Q151L、S153C、F155H、F155T、F155L、K156Y、K156V、K156A、K156D、K156W、K156P、K156E、K156C、K156F、K156Q、K156L、K156S、K156G、W157L、R158M、R158A、R158G、R158Q、R158W、R158L、R158T、R158V、R158E、R158Y、R158S、W159N、W159G、W159T、W159C、W159E、W159D、Y160Q、Y160M、Y160T、Y160A、Y160S、Y160V、Y160G、Y160I、Y160L、Y160E、F162I、D163K、D163A、D163R、D163L、G164S、G164T、G164C、T165L、T165M、T165S、T165G、T165I、D166T、D166N、D166S、W167Y、W167G、W167D、D168G、Q169A、Q169L、S170A、S170V、R171F、R171S、R171K、R171V、R171A、Q172S、Q172E、L173G、L173N、L173M、L173S、L173Y、L173V、L173I、A174G、A174V、N175V、N175I、N175G、R176E、R176W、R176G、R176M、I177L、I177S、I177G、Y178V、Y178R、Y178K、K179C、K179E、K179S、K179W、K179A、K179G、K179T、K179F、K179L、F180L、R181K、R181P、R181S、R181V、R181Q、R181E、R181A、R181G、G184R、G184V、K185A、K185D、K185N、K185V、K185G、A186C、A186S、A186D、W187G、D188Q、D188G、D188R、D188M、D188L、D188V、W189R、W189A、W189G、W189C、V191M、V191A、T193C、T193P、E194Q、N197C、N197D、D199G、D199S、D199A、Y200W、Y200F、Y200C、Y200V、Y200T、Y200L、Y200A、Y200G、Y200S、M202T、Y203F、Y203G、Y203C、Y203I、Y203Q、Y203H、Y203T、Y203M、Y203S、Y203V、Y203W、Y203L、A204P、A204R、A204C、A204H、A204W、A204F、A204L、A204Y、A204D、A204G、D205C、V206P、D209V、H210V、H210A、H210W、H210L、H210G、H210D、H210E、P211C、E212M、E212D、E212I、N215D、N215V、N215C、L217P、L217T、L217G、L217S、R219H、R219W、R219S、R219I、R219A、R219M、R219T、R219E、R219Y、R219D、R219C、R219L、W223Y、N226Q、L230V、V238C、V238W、I241L、K242C、K242Y、K242P、K242W、K242G、K242L、K242R、K242S、K242E、K242A、K242Q、F245C、F245V、F245D、F245G、G258L、K259I、N2 60D、N260C、F262Q、F262L、F262E、F262R、F262P、F262V、A263L、A263C、K269 L, K269W, N270D, N270A, N270M, D271C, G273V, A274S, A274D, A274E, Y278S, L279G, K281G, W284V, W284A, T285F, T285S, S287V, S287Q, S287T, S287E, S28 7P、S287L、S287W、S287C、P292R、N296Y、N296M、N296F、L297W、Q299I、S301Q 、N306V、L312F、L312V、G315V、V318E、V318A、Q319A、R320C、H321E、H321V、H 321C, P322E, P322C, A325V, A325S, P336E, P336D, P336R, P336L, G337M, G337V, E341D, E341I, S342A, V344L, G346V, F348Q, F348S, F348C, K349G, K349A K349Y, K349L, K349S, P350L, P350G, P350S, P350Q, L351S, Q361A, Q361E, G362Y, G362R, G362C, G362N, G362M, G362V, Y363Q, Y363I, Y363A, Y368I, Y368 L, Y368A, Y368S, Y368Q, Y368V, Y368H, G369K, G369S, G369A, G369E, G369R, G369I, D370T, Y372L, Y372G, G373S, S376L, D377L, D377V, V379P, P380I, S38 1L、S381A、S381I、S381V、Y382A、Y382E、Y382V、Y382L、Q384R、Q384Y、D387V 、D387Q、D387L、Q394S、Q394R、Q395R、A397G、R400L、R400C、R400K、R400S、R 400M、H402S、H402P、H402Y、H407D、W408R、W408P、W408C、W408Y、W408G、W40 8D、V410Q、R415F、R415L、N418A、N418C、N418M、N418G、H421C、H421A、H421R、S431A, G435E, G435N, G436T, W439R, W439N, M440L, R444V, R444P, Q445C, Q445E, A447E, A447T, M454V, G456R, S459T, N465Q, N465L, N465R, D467V, N475H, G477Q, G477F, G477K, G477L, and V481S, wherein the numbering is based on the amino acid sequence as defined in SEQ ID NO:3, and wherein one or more substitutions provide an IF ≥ 1.4 for a measure of specific activity in a standard A detergent composition and optionally an IF ≥ 1.0 for a measure of stability.

[0111] In one embodiment, the variant includes one or more substitutions selected from the group consisting of: H1V, G7F, G7V, G7E, G7E, G7D, Q11V, W15G, W15S, N19G, N19S, Q22C, Q22A, N25V, R26L, N29G, A31S, L34Y, A41Q, W48C, W48I, W48Y, S52M, S52V, Q53V, Q53L, V56L, G59E, T74V, T74L, I8 9A, R90L, S91A, S91G, L92C, N95E, N95S, N95G, Q98L, Y100W, K108V, F113M, F113V, F113E, R116Q, V117G, N126G, N126A, N128S, N128E, Q129E, G133A, G133D, A139S, W140H, T141S, T141C, T141G, F143M, F143L, F145A, P146L, Q 151L, K156F, K156Q, K156L, K156S, K156G, R158S, T165I, D166S, Q169L, S170A, S170V, R171A, Q172E, L173I, A1 74V, N175G, K179L, R181A, R181G, K185G, A186S, A186D, Y203W, Y203L, D209V, E212I, N215C, L217S, R219L, K24 2A, F245G, K269W, L297W, N306V, L312F, L312V, G315V, R320C, P336L, G337V, W439R, W439N, S342A, V344L, P350S, P350Q, Y368H, S376L, S381V, D387Q, D387L, Q395R, N418M, N418G, S431A, G435N, N465L, and N465R, wherein the numbering is based on the amino acid sequence as defined in SEQ ID NO:3, and wherein one or more substitutions provide an IF ≥ 1.4 for a measure of specific activity in a standard A detergent composition and an IF ≥ 1.0 for a measure of stability.

[0112] In one embodiment, the variant includes substitutions at one or more locations selected from the group consisting of: H1, G7, F13, E14, W15, N16, N19, D20, Q22, R26, N29, N30, A31, Q32, P46, W48, G50, T51, S52, Q53, D55, G59, A60, Y64, N70, Q71, K72, G73, T74, V75, K78, R90, Y100, H107. K108, G109, D112, F113, R116, V117, A119, E121, N123, N126, R127, N128, E130, V131, S132, G133, T134, Y135 , I137, A139, W140, T141, G142, N144, F145, P146, G147, G149, N150, Q151, F155, K156, R158, W159, Y160, D16 3. G164, T165, D166, W167, S170, R171, Q172, L173, R176, Y178, K179, R181, K185, A186, W187, D188, W189, V1 91. E194, D199, Y200, M202, Y203, A204, D205, H210, N215, L217, R219, W223, V238, K242, F245, G258, F262, K 269, N270, A274, T285, S287, N296, L312, V318, H321, P322, A325, G337, E341, S342, K349, P350, Q361, G362, Y368, G369, D377, S381, Q384, D387, R400, W408, R415, N418, G435, Q445, M454, G477, and V481, wherein these positions correspond to amino acid positions in the amino acid sequence as defined in SEQ ID NO:3, and the one or more substitutions provide an improvement factor of ≥1.6 for a measure of specific activity in the standard A detergent composition and optionally an improvement factor of ≥1.0 for a measure of stability.

[0113] In a specific embodiment, the variant includes one or more substitutions selected from the group consisting of: H1C, G7V, G7E, G7E, G7D, F13I, E14L, E14M, E14S, W15S, W15F, N16Q, N19G, N19S, N19V, D20R, Q22A, R26L, R26W, R26A, R26F, N29G, N29L, N30E, A31S, Q32L, Q32A, P46G, W48Y, G50N, T51G, T51P, T51L, T51C, S52V, S52I, S52L, Q53V, Q53L, D55V, D55I, G59A, A60L, A60G , Y64V, Y64I, Y64H, Y64M, Y64C, Y64R, Y64G, Y64L, Y64A, N70S, N70E, Q71I, Q 71V, K72R, G73L, G73E, T74V, T74L, V75G, K78T, K78A, R90L, R90G, Y100F, H10 7Q, K108V, G109A, G109S, D112E, F113E, F113C, F113D, R116I, R116D, R116T , R116G, R116E, R116S, R116C, R116V, R116Q, R116F, V117S, V117W, V117A, V1 17G, V117E, A119Y, E121G, E121V, E121M, E121D, N123P, N126G, N126A, R127 C, R127W, R127L, R127V, R127F, R127Y, N128C, N128E, E130C, V131E, S132H, S 132P, S132C, S132E, S132D, G133E, G133D, T134D, T134E, Y135T, Y135W, Y13 5K, Y135G, Y135P, Y135M, Y135D, I137S, I137G, A139S, W140Y, W140S, W140A, W140H, T141C, T141G, G142E, N144C, F145S, F145A, P146C, P146L, G147I, G1 47L, G147C, G149C, N150D, N150EQ151T, Q151C, Q151L, F155L, K156V, K156A, K156D, K156W, K156P, K156E, K156C, K156F, K156Q, K156L, K156S, K156G, R15 8A, R158G, R158Q, R158W, R158L, R158T, R158V, R158E, R158Y, R158S, W159G,W159T、W159C、W159E、W159D、Y160S、Y160V、Y160G、Y160I、Y160L、Y160E、D163L、G164T、G164C、T165G、T165I、D166T、D166N、D166S、W167D、Q169A、Q169L、S170V、R171V、R171A、Q172S、L173V、R176M、Y178R、Y178K、K179C、K179E、K179S、K179W、K179A、K179G、K179T、K179F、R181V、R181Q、R181E、R181G、K185A、K185D、K185N、K185V、K185G、A186D、W187G、D188M、D188L、D188V、W189R、W189A、W189G、W189C、V191M、V191A、E194Q、D199S、D199A、Y200W、Y200F、Y200C、Y200V、Y200T、Y200L、Y200A、Y200G、Y200S、M202T、Y203I、Y203Q、Y203H、Y203T、Y203M、Y203S、Y203V、Y203W、Y203L、A204H、A204W、A204F、A204L、A204Y、A204D、A204G、D205C、H210W、H210L、H210G、H210D、H210E、N215D、N215V、N215C、L217S、R219Y、R219D、R219C、W223Y、V238C、V238W、K242W、K242G、K242L、K242R、K242S、K242E、K242Q、F245C、F245V、F245D、F245G、G258L、F262E、F262R、F262P、F262V、K269L、N270D、N270A、N270M、A274S、A274D、A274E、T285F、T285S、S287W、S287C、N296M、N296F、L312F、L312V、V318A、H321V、H321C、P322C、A325S、G337M、G337V、E341I、S342A、K349A、K349Y、K349L、K349S、P350G、P350Q、Q361E、G362R、G362C、G362N、G362M、G362V、Y368L、Y368A、Y368S、Y368Q、Y368V、G369K、G369S、G369A、G369E、G369R、G369I、D377V、S381L, S381A, S381I, Q384R, Q384Y, D387L, R400M, W408Y, W408G, W408D, R415L, N418G, G435E, G435N, Q445E, M454V, G477Q, G477F, G477K, G477L, and V481S, wherein these positions correspond to amino acid positions in the amino acid sequence as defined in SEQ ID NO:3, and wherein the one or more substitutions provide an IF ≥ 1.6 for a measure of specific activity in the standard A detergent composition and optionally an IF ≥ 1.0 for a measure of stability.

[0114] In one embodiment, the variant includes one or more substitutions selected from the group consisting of: G7V, G7E, G7E, G7D, W15S, N19G, N19S, Q22A, R26L, N29G, A31S, W48Y, S52V, Q53V, Q53L, T74V, T74L, R90L, K108V, F113E, R116Q, V 117G, N126G, N126A, N128E, G133D, A139S, W140H, T141C, T141G, F145A, P146L, Q151L, K156F, K156Q, K156L, K156S, K156G, R158S, T165I, D166S, Q169L, S170V, R171A, R181G K185G, A186D, Y203W, Y203L, N215C, L217S, F245G, L312F, L312V, G337V, S342A, P350Q, D387L, N418G, and G435N, wherein these positions correspond to amino acid positions in the amino acid sequence as defined in SEQ ID NO:3, and wherein the one or more substitutions provide an IF ≥ 1.6 for a measure of specific activity in the standard A detergent composition and an IF ≥ 1.0 for a measure of stability.

[0115] In another embodiment, the variant includes substitutions at one or more locations selected from the group consisting of: H1, H2, D3, G4, T5, G7, T8, I9, M10, Q11, Y12, F13, E14, W15, N16, P18, N19, Q22, W24, N25, R26, L27, H28, N29, N30, A31, Q32, N33, L34, K35, N36, A37, G38, I39, T40, A41, I42, W43, I44, P45, P46, A47, W48, G50, T51, S52, Q53, N54, D55, G59, A60, Y61, L63, Y64. L66, E68, F69, N70, Q71, K72, G73, T74, V75, R76, T77, K78, T81, K82, A83, E8 4. L85, E86, R87, A88, I89, R90, S91, L92, K93, N95, G96, I97, Q98, V99, Y100, V103, H107, K108, G109, D112, F113, E115, R116, V117, A119, V120, E121, V1 22. N123, Q125, N126, R127, N128, Q129, E130, V131, S132, G133, T134, Y135, I137, E138, A139, W140, T141, G142, F143, N144, F145, P146, G147, G149, N1 50, Q151, S153, F155, K156, W157, R158, W159, Y160, H161, F162, D163, G164, T165, D166, W167, D168, Q169, S170, R171, Q172, L173, A174, N175, R176, I1 77. Y178, K179, F180, R181, G184, K185, A186, W187, D188, W189, E190, V191, D192, T193, E194, N195, G196, N197, Y198, D199, Y200, M202, Y203, A204, D2 05, V206, M208, D209, H210, P211, E212, V213, I214, N215, L217, N218, R219, V222, W223, Y224, A225, N226, T227, L228, N229, L230, D231, F233, V238, I2 41. K242, F245, M246, W249, L250, H252, V253, R254, G255, Q256, T257, G258,K259, N260, L261, F262, A263, V264, Y267, W268, K269, N270, D271, G273, A274, E276, Y278, L279, S280, K281, T282, N283, W284 , T285, M286, S287, A288, V291, P292, L293, H294, Y295, N296, L297, Y298, Q299, A300, S301, N302, S303, S304, G305, N306, Y307 , M309, L312, L313, N314, G315, T316, V318, Q319, R320, H321, P322, A325, V326, T327, V329, N331, P336, G337, E341, S342, V34 4. Q345, G346, W347, F348, K349, P350, L351, A352, T355, L357, T358, E360, Q361, G362, Y363, Q365, V366, Y368, G369, D370, Y37 2. G373, I374, P375, S376, D377, V379, P380, S381, Y382, R383, Q384, I386, D387, P388, L389, L390, K391, A392, Q394, Q395, Y3 96. A397, R400, H402, D403, F405, D406, H407, W408, V410, I411, T414, R415, E416, N418, A419, S420, H421, S424, G425, A427, T4 28, I429, M430, S431, G435, G436, S437, K438, W439, M440, Y441, V442, R444, Q445, K446, A447, G448, V450, H452, D453, M454, T455, G456, N457, S459, G460, T463, I464, N465, Q466, D467, W469, H471, F473, N475, G476, G477, V481, and W482, where these positions correspond to as shown in SEQ The amino acid positions in the amino acid sequence specified in ID NO:3, and wherein the one or more substitutions provide an IF of ≥1.2 for a measure of specific activity in the standard X detergent composition and optionally an IF of ≥1.0 for a measure of stability.

[0116] In one embodiment, the variant includes one or more substitutions selected from the group consisting of: H1V, H1G, H1S, H1C, H2L, H2T, H2M, H2E, H2P, H2G, D3C, D3A, G4Q, G4D, T5L, T5Q, T5G, T5E, T5V, G7F, G7R, G7V, G7E, G7E, G7D, T8A, T8S, I9C, I9M, I9L, M10L, Q11C, Q11L, Q11V, Y12V, Y12A, Y12F, F13Y, F13I, E14L, E14M, E14S, W15G, W15F, W15S, N16G, N16Q, P18L, N19 Y, N19T, N19G, N19S, N19V, Q22N, Q22C, Q22S, Q22M, Q22T, Q22E, Q22A, W24H, W24F, N25V, N25S, N25G, N25E, N25E, R26G, R26D, R26M, R26I, R26S, R26L, R2 6W, R26A, R26F, L27A, L27M, H28A, H28E, H28I, N29C, N29G, N29L, N30S, N30H , N30L, N30T, N30E, A31W, A31V, A31S, Q32D, Q32G, Q32L, Q32A, N33F, N33A, N3 3R, L34F, L34Y, K35L, K35E, K35W, K35Q, N36R, N36Q, N36A, N36E, A37R, A37G , A37T, A37S, G38N, I39L, I39M, T40Q, T40D, T40A, T40E, A41E, A41N, A41M, A 41Q, A41D, I42V, I42A, W43R, I44T, P45A, P46G, A47S, A47G, W48G, W48L, W48 C, W48I, W48M, W48P, W48Q, W48Y, W48F, G50P, G50S, G50N, T51S, T51G, T51P, T 51L, T51C, S52M, S52V, S52I, S52L, Q53G, Q53V, Q53L, N54G, N54K, N54V, D55 V, D55I, V56L, V56M, G59E, G59A, A60S, A60W, A60L, A60G, A60T, Y61F, L63V, L 63P, L63I, L63H, L63K, Y64W, Y64S, Y64V,, Y64I, Y64H, Y64M, Y64C, Y64R, Y6 4G, Y64L, Y64A, L66M, E68D, F69H, F69W, N70G, N70A, N70T, N70S, N70E, Q71N,Q71E、Q71D、Q71I、Q71V、K72Y、K72R、G73V、G73L、G73E、T74V、T74L、V75T、V75G、R76S、R76G、R76A、T77A、T77S、K78L、K78T、K78A、T81G、T81W、、T81L、K82A、A83G、A83E、E84F、E84D、L85V、E86L、E86D、R87M、R87A、R87E、A88S、A88G、A88V、I89A、I89H、I89P、R90L、R90G、S91A、S91E、S91G、L92C、K93W、K93G、K93L、N95L、N95E、N95W、N95C、N95S、N95G、G96Q、I97M、I97V、Q98V、Q98L、Q98C、Q98M、Q98T、V99I、V99L、V99S、V99T、Y100W、Y100V、Y100F、V103G、H107Q、K108V、G109A、G109S、D112E、F113M、F113V、F113L、F113C、F113E、F113D、F113Y、E115C、E115A、R116N、R116L、R116I、R116Q、R116D、R116T、R116G、R116E、R116S、R116C、R116V、R116F、V117C、V117L、V117S、V117W、V117A、V117G、V117E、A119Y、V120I、V120D、V120L、E121P、E121G、E121V、E121M、E121D、V122E、V122P、V122I、N123G、N123A、N123E、N123W、N123T、N123P、Q125E、Q125T、N126E、N126G、N126A、R127G、R127S、R127C、R127W、R127L、R127V、R127F、R127Y、N128S、N128E、N128C、Q129L、Q129C、Q129E、E130C、V131L、V131E、S132A、S132H、S132P、S132C、S132E、S132D、A132N、G133S、G133T、G133L、G133A、G133V、G133I、G133E、G133D、T134V、T134D、T134E、Y135L、Y135V、Y135S、Y135R、Y135T、Y135W、Y135K、Y135G、Y135P、Y135M、Y135D、Y135H、Y135E、Y135E、I137A、I137S、I137G、E138A、A139M、A139S、W140Y、W140S、W140H、W140A、T141S、T141M、T141C、T141G、G142D、G142E、F143M、F143L、N144C、F145C、F145S、F145A、P146S、P146V、P146L、P146C、G147V、G147P、G147D、G147S、G147I、G147L、G147C、G149C、N150C、N150D、N150E、Q151L、Q151T、Q151C、S153C、F155H、F155T、F155L、K156Y、K156V、K156A、K156F、K156D、K156Q、K156L、K156S、K156W、K156P、K156E、K156C、K156G、W157L、R158M、R158A、R158G、R158Q、R158S、R158W、R158L、R158T、R158V、R158E、R158Y、W159N、W159G、W159T、W159C、W159E、W159D、W159H、Y160Q、Y160M、Y160T、Y160A、Y160S、Y160V、Y160G、Y160I、Y160L、Y160E、H161C、H161N、F162M、F162L、F162Y、F162I、D163S、D163K、D163A、D163R、D163L、G164S、G164T、G164C、T165L、T165M、T165S、T165G、T165I、D166T、D166S、D166N、W167Y、W167G、W167D、D168M、D168S、D168G、Q169L、S170A、S170V、R171F、R171S、R171K、R171A、R171V、Q172L、Q172E、Q172S、L173I、L173G、L173N、L173M、L173S、L173Y、L173V、A174V、A174G、N175L、N175V、N175I、N175G、R176E、R176W、R176G、R176M、I177L、I177S、I177G、Y178V、Y178R、Y178K、K179L、K179C、K179E、K179S、K179W、K179A、K179G、K179T、K179F、F180A、F180L、R181K、R181P、R181S、R181A、R181V、R181Q、R181G、R181E、G184L、G184R、G184V、G184D、K185A、K185D、K185N、K185G、K185V、A186S、A186C、A186D、W187G、D188Q、D188G、D188R、D188M、D188L、D188V、W189R、W189A、W189G、W189C、E190D、E190N、E190A、V191M、V191A、D192E、D192Q、D192S、T193S、T193K、T193V、T193C、T193P、E194S、E194D、E194L、E194Q、N195R、N195A、G196A、G196V、N197G、N197V、N197Y、N197C、N197D、Y198W、D199G、D199S、D199A、Y200W、Y200F、Y200C、Y200V、Y200T、Y200L、Y200A、Y200G、Y200S、M202T、Y203F、Y203G、Y203C、Y203I、Y203Q、Y203H、Y203W、Y203T、Y203L、Y203M、Y203S、Y203V、A204P、A204R、A204C、A204H、A204W、A204F、A204L、A204Y、A204D、A204G、D205C、V206H、V206Q、V206P、M208R、M208P、M208L、M208C、D209C、D209S、D209M、D209E、D209H、D209F、D209Y、D209V、H210F、H210V、H210A、H210W、H210L、H210G、H210D、H210E、H210N、P211G、P211W、P211C、E212G、E212F、E212M、E212D、E212I、V213C、V213M、V213T、V213G、V213W、I214P、N215D、N215C、N215V、L217V、L217C、L217P、L217T、L217G、L217S、N218S、R219L、R219H、R219W、R219S、R219I、R219A、R219M、R219T、R219E、R219Y、R219D、R219C、V222E、V222F、V222C、W223Y、Y224L、A225K、N226C、N226Q、T227S、T227L、T227M、T227C、L228A、L228F、N229T、N229S、L230S、L230A、L230T、L230V、F233A, F233L, V238C, V238W, I241L, K242A, K242C, K242Y, K242P, K242W, K242G, K242L, K242R, K242S, K242E, K242Q, F245C, F245V, F245D, F245G, M246 L, M246F, W249L, L250C, L250W, L250T, L250A, H252C, H252E, H252W, V253A, V253L, V253S, V253W, R254Q, R254S, G255C, G255E, G255L, Q256P, T257V, G25 8P、G258V、G258C、G258I、G258L、K259L、K259V、K259Q、K259I、N260W、N260E 、N260D、N260C、L261G、L261D、F262Q、F262L、F262E、F262R、F262P、F262V、A 263L, A263C, V264T, V264L, Y267V, Y267L, W268V, K269W, K269L, N270D, N270A, N270M, D271E, D271V, D271C, G273W, G273R, G273V, A274S, A274D, A274E E276R, Y278G, Y278S, L279Q, L279V, L279G, S280C, K281L, K281A, K281W, K281G, T282G, T282A, T282C, T282R, T282I, N283G, N283E, N283Q, N283V, N283 A、W284T、W284I、W284S、W284E、W284C、W284V、W284A、T285E、T285F、T285S、 M286L、M286D、S287V、S287Q、S287T、S287E、S287P、S287L、S287W、S287C、A28 8L, A288Y, V291T, P292A, P292Q, P292L, P292R, L293A, L293E, L293T, L293G, H294G, H294A, Y295H, Y295S, Y295F, N296A, N296Y, N296M, N296F, L297S, L 297W, Y298V, Y298P, Q299S, Q299L, Q299R, Q299I, A300V, S301A, S301R, S301Q, N302I, N302T, N302M, S303I, S303E, S304I, S304P, G305V, G305L, G305SN306V, Y307F, Y307W, M309L, L312F, L312V, L313S, N314Y, N314F, N314D, G315I, G315V, T316K, T316S, V318C, V318L, V318E, V318A, Q319L, Q319I, Q319 A、R320V、R320L、R320C、H321T、H321E、H321V、H321C、P322E、P322C、A325V、 A325S、V326I、T327V、T327L、T327S、V329A、V329S、N331C、N331V、N331I、P33 6M, P336E, P336D, P336L, P336R, G337T, G337C, G337V, G337M, W439R, W439N, E341C, E341D, E341I, S342G, S342T, S342A, V344S, V344L, Q345K, Q345C, Q 345V, Q345L, Q345I, G346H, G346A, G346R, G346L, G346N, G346Q, G346V, W347L, F348Q, F348S, F348C, K349G, K349A, K349Y, K349L, K349S, P350S, P350L P350G, P350Q, L351W, L351M, L351S, A352S, A352C, T355V, T355A, L357C, L357T, L357S, T358V, T358F, E360S, E360D, E360C, Q361V, Q361P, Q361R, Q361 A、Q361E、G362Y、G362R、G362C、G362N、G362M、G362V、Y363L、Y363Q、Y363I、 Y363A、Q365D、V366L、V366A、Y368M、Y368T、Y368H、Y368I、Y368L、Y368A、Y36 8S、Y368Q、Y368V、G369K、G369S、G369A、G369E、G369R、G369I、D370A、D370T 、Y372S、Y372I、Y372L、Y372G、G373C、G373S、I374Y、P375L、P375A、P375T、S 376A, S376N, S376P, S376R, S376W, S376L, D377C, D377L, D377V, V379L, V379F, V379P, P380L, P380V, P380G, P380E, P380A, P380I, S381N, S381V, S381LS381A、S381I、Y382I、Y382A、Y382E、Y382V、Y382L、R383G、R383S、R383P、Q384A、Q384P、Q384L、Q384H、Q384R、Q384Y、I386T、D387H、D387Q、D387V、D387L、P388G、P388T、P388M、P388W、P388V、L389S、L389H、L390V、L390S、L390F、L390I、L390G、K391L、K391Y、K391A、A392I、Q394S、Q394R、Q395W、Q395E、Q395K、Q395N、Q395L、Q395R、Y396T、Y396L、A397C、A397G、R400L、R400C、R400K、R400S、R400M、H402L、H402V、H402E、H402M、H402A、H402S、H402P、H402Y、D403E、F405G、F405C、F405V、F405I、F405P、F405W、D406E、H407C、H407D、W408R、W408P、W408C、W408Y、W408G、W408D、V410I、V410Q、I411A、I411L、I411V、T414A、T414V、R415F、R415L、E416C、E416V、N418L、N418M、N418A、N418C、N418G、A419M、A419E、A419C、S420E、S420T、S420V、H421V、H421C、H421A、H421R、S424C、S424A、G425A、A427V、T428L、T428N、T428A、T428G、I429V、I429A、M430L、M430R、S431A、G435N、G435E、G436M、G436E、G436L、G436T、S437G、S437Q、S437L、K438T、W439E、W439C、W439H、M440L、Y441N、Y441H、Y441Q、V442I、、R444A、R444S、R444L、R444V、R444P、Q445L、Q445S、Q445C、Q445E、K446G、K446Q、A447G、A447Q、A447E、A447T、G448S、V450T、V450A、H452A、H452T、D453E、M454R、M454L、M454V、T455L、T455S、G456Y、G456V、G456R、N457V、N457I、N457G, N457R, S459E, S459Q, S459V, S459C, S459P, S459M, S459T, G460S, G460D, G46 0E, T463C, T463V, T463L, T463D, T463H, I464V, N465G, N465L, N465R, N465Q, Q466L, Q 466C, Q466E, D467C, D467V, W469M, W469E, W469T, H471Q, F473Y, F473C, F473R, N475T, N475H, G476Y, G477Q, G477F, G477K, G477L, V481Q, V481S, W482F, wherein these positions correspond to amino acid positions in the amino acid sequence as illustrated in SEQ ID NO:3, and wherein one or more substitutions provide an IF of ≥1.2 for a measure of specific activity in the standard X detergent composition and optionally an IF of ≥1.0 for a measure of stability.

[0117] In one embodiment, the variant includes substitutions at one or more positions selected from the group consisting of: H1, H2, D3, G4, T5, G7, I9, Q11, F13, E14, N16, N19, Q22, W24, N25, R26, N29, A31, Q32, L34, N36, A37, A41, P45, P46, W48, G50, T51, S52, Q53, G59, A60, Y61, L63, Y64, F69, N70, Q71, K72, G73, T74, V75, R76, T77, K82, R87, I89, R90, S91, L92, K93, N95, Q98, V99. Y100, K108, G109, D112, F113, R116, V117, A119, V120, E121, V122, N123, N1 26. R127, N128, Q129, E130, V131, S132, G133, T134, Y135, I137, A139, W140, T141, G142, F143, N144, F145, P146, G147, G149, N150, Q151, F155, K156, R1 58. W159, Y160, D163, T165, D166, W167, D168, Q169, R171, Q172, L173, A174, N175, R176, I177, Y178, K179, F180, R181, G184, K185, A186, W187, D188, E1 94. D199, Y200, M202, Y203, A204, D205, H210, E212, N215, L217, R219, W223, N226, L230, V238, I241, K242, K245, R254, G258, N260, F262, A263, K269, N2 70. D271, A274, L279, K281, N283, W284, T285, S287, P292, L293, N296, Q299, S301, G305, N306, L312, N314, G315, V318, Q319, R320, H321, P322, A325, G3 37. W439, E341, S342, V344, Q345, G346, F348, K349, P350, L357, E360, Q361, G362, Y363, Y368, G369, Y372, I374, P375, S376, D377, V379, P380, S381, Y3 82. Q384, D387, P388, Q394, Q395, A397, R400, H402, W408, V410, R415, N418,H421, G425, S431, G435, G436, W439, R444, Q445, A447, G456, S459, N465, W469, G477, and V481, wherein these positions correspond to amino acid positions in the amino acid sequence as illustrated in SEQ ID NO:3, and wherein substitutions at one or more of these positions provide an IF of ≥1.4 for a measure of specific activity in the standard X detergent composition and optionally an IF of at least ≥1.0 for a measure of stability.

[0118] In one embodiment, the variant includes one or more substitutions selected from the group consisting of: H1G, H1C, H2G, D3A, G4D, T5Q, T5E, T5V, G7F, G7E, G7R, G7V, G7E, G7D, I9M, Q11V, F13I, E14M, N16G, N16Q, N19Y, N19T, N19G, N19S, N19V, Q22N, Q22C, Q22S, Q22M, Q22A, W24F, N25G, N25E, N25E, R26G, R26D, R26I, R26S, R26L, R26W, R26A, R26F, N29G, N29L, A31W, A 31S, Q32G, Q32L, Q32A, L34Y, N36E, A37G, A41Q, P45A, P46G, W48G, W48C, W48 I, W48M, W48P, W48Q, G50S, G50N, T51G, T51C, S52V, S52I, S52L, Q53L, Q53G, Q53V, G59A, A60S, A60W, A60L, A60G, A60T, Y61F, L63V, L63H, L63K, Y64W, Y6 4S, Y64V, Y64I, Y64H, Y64M, Y64C, Y64R, Y64G, Y64L, Y64A, F69W, N70A, N70T, N70S, N70E, Q71N, Q71D, Q71I, Q71V, K72Y, K72R, G73V, G73L, G73E, T74L, V7 5G, R76A, T77S, K82A, R87A, I89A, I89P, R90L, R90G, S91A, S91E, S91G, L92C , K93W, K93G, N95W, N95C, N95G, Q98L, Q98C, Q98M, Q98T, V99S, V99T, Y100W, Y100V, Y100F, K108V, G109A, G109S, D112E, F113M, F113V, F113L, F113C, F11 3E, F113D, R116N, R116L, R116I, R116Q, R116D, R116T, R116G, R116E, R116S , R116C, R116V, R116F, V117C, V117S, V117W, V117A, V117G, V117E, A119Y, V 120D, V120L, E121V, E121M, E121D, V122E, N123G, N123A, N123E, N123W, N12 3T, N123P, N126E, N126G, N126A, R127G, R127S, R127C, R127W, R127L, R127V,R127F、R127Y、N128C、N128S、N128E、Q129E、E130C、V131L、V131E、S132A、S132H、S132P、S132C、S132E、S132D、G133S、G133V、G133I、G133E、G133D、T134V、T134D、T134E、Y135V、Y135S、Y135R、Y135T、Y135W、Y135K、Y135G、Y135P、Y135M、Y135D、Y135H、Y135E、I137A、I137S、I137G、A139S、W140Y、W140H、T141M、T141C、T141G、T141S、G142D、G142E、F143M、N144C、F145C、F145A、P146C、P146L、G147V、G147P、G147D、G147S、G147I、G147L、G147C、G149C、N150C、N150D、N150E、Q151T、Q151L、F155H、F155L、K156Y、K156V、K156A、K156D、K156Q、K156W、K156P、K156E、K156C、K156F、K156L、K156S、K156G、R158M、R158A、R158G、R158Q、R158S、R158W、R158L、R158T、R158V、R158E、R158Y、W159N、W159G、W159T、W159C、W159E、W159D、Y160Q、Y160T、Y160S、Y160V、Y160G、Y160I、Y160L、Y160E、D163S、D163K、D163L、T165M、T165G、T165I、D166S、W167Y、W167D、D168M、D168S、D168G、Q169L、R171F、R171S、R171K、R171V、R171A、Q172S、Q172E、L173G、L173N、L173M、L173S、L173Y、L173V、A174G、N175L、N175V、N175I、N175G、R176E、R176G、R176M、I177G、Y178V、Y178R、Y178K、K179L、K179C、K179E、K179S、K179W、K179A、K179G、K179T、K179F、F180L、R181K、R181P、R181S、R181V、R181Q、R181E、R181A、R181G、G184V、G184D、K185A、K185D、K185N、K185V、K185G、A186C、A186S、A186D、W187G、D188Q、D188G、D188M、D188L、D188V、E194Q、D199G、D199S、D199A、Y200W、Y200F、Y200C、Y200V、Y200T、Y200L、Y200A、Y200G、Y200S、M202T、Y203I、Y203Q、Y203H、Y203T、Y203M、Y203V、Y203L、A204R、A204H、A204W、A204F、A204L、A204Y、A204D、A204G、D205C、H210F、H210A、H210W、H210L、H210G、H210D、H210E、E212F、E212M、E212D、E212G、E212I、N215D、N215V、N215C、L217C、L217P、R219H、R219I、R219M、R219T、R219E、R219Y、R219D、R219C、W223Y、N226Q、N226C、L230V、V238C、V238W、I241L、K242C、K242Y、K242P、K242W、K242G、K242L、K242R、K242S、K242E、K242A、K242Q、F245C、F245V、F245D、F245G、R254Q、R254S、G258L、N260D、F262Q、F262L、F262E、F262R、F262P、F262V、A263L、A263C、K269L、K269W、N270D、N270A、N270M、D271C、A274S、A274D、A274E、L279Q、L279G、K281W、K281G、N283Q、N283V、N283A、W284C、W284V、W284A、T285F、T285S、S287V、S287Q、S287P、S287L、S287W、S287C、P292R、L293G、N296M、N296F、Q299S、Q299I、S301Q、G305V、G305L、N306V、L312F、L312V、N314F、G315I、G315V、V318A、Q319A、R320L、R320C、H321E、H321V、H321C、P322E、P322C、A325V、A325S、G337C、G337V、G337M、E341D、E341I、S342A、V344L、Q345C、G346Q、G346V、F348Q、F348S、F348C, K349L, K349S, P350S, P350L, P350G, P350Q, L357T, E360C, Q361A, Q361E, G362R, G362C, G362N, G362M, G362V, Y363I, Y363A, Y368I, Y368L, Y368V, G369A, G 369R, Y372L, Y372G, I374Y, P375T, S376L, D377V, V379F, V379P, V379L, P380G, P 380I, S381N, S381L, S381A, S381I, S381V, Y382V, Q384R, Q384Y, D387L, P388W, Q3 94S, Q394R, Q395R, A397G, R400M, H402L, H402S, H402P, W408P, W408C, W408Y, V4 10Q, R415L, N418C, N418G, H421R, G425A, S431A, G435N, G435E, G436M, G436E, G43 6T, W439H, W439R, W439N, R444V, Q445C, Q445E, A447E, A447T, G456R, S459T, N465R, N465Q, W469M, W469E, G477Q, G477F, G477K, G477L, and V481S, wherein these positions correspond to amino acid positions in the amino acid sequence as illustrated in SEQ ID NO:3, and wherein one or more substitutions provide an IF of ≥1.4 for a measure of specific activity in a standard X detergent composition and optionally an IF of ≥1.0 for a measure of stability.

[0119] In one embodiment, the variant includes substitutions at one or more positions selected from the group consisting of: G7, F13, N16, N19, R26, N29, A31, P46, G50, T51, S52, Q53, G59, A60, Y64, N70, Q71, K72, G73, T74, R90, K108, G109, F113, R116, V117, A119 , E121, V122, N123, N126, R127, E130, V131, S132, G133, T134, Y135, I137, T141, G142, F143, N144, P146, G147, G149, N150, F155, K156, R158, W159, Y160, D163, T165, W167, D168, Q169, R1 71. Q172, L173, N175, Y178, K179, R181, K185, A186, W187, D188, E194, Y200, Y203, A204, D20 5. H210, N215, R219, W223, V238, K242, K245, G258, F262, K269, N270, A274, L279, K281, W284, T285, S287, Q299, L312, H321, A325, G337, E341, S342, F348, K349, P350, Q361, G362, Y368, G369, D377, V379, P380, S381, Q384, V410, N418, G435, Q445, G456, N465, and G477, wherein these positions correspond to amino acid positions in the amino acid sequence as defined in SEQ ID NO:3, and wherein the substitutions at one or more of these positions provide an IF of ≥1.6 for a measure of specific activity in the standard X detergent composition and optionally an IF of ≥1.0 for a measure of stability.

[0120] In one embodiment, the variant includes one or more substitutions selected from the group consisting of: G7V, G7E, G7D, F13I, N16Q, N19T, N19G, N19S, N19V, R26A, N29G, A31S, P46G, G50N, T51G, T51C, S52I, S52L, Q53L, Q53G, Q53V, G59A, A60S, A60W, A60G, Y64V, Y64M, Y64C, Y64R, Y64G, Y64L, Y64A, N70E, Q71V, K72R, G73L, G73E, T74L, R90G, K108V, G109S, F113V, F11 3C, F113E, F113D, R116I, R116Q, R116D, R116T, R116G, R116E, R116S, R116C , R116V, R116F, V117A, V117G, V117E, A119Y, E121D, V122E, N123T, N126G, R 127W, R127L, R127F, R127Y, E130C, V131L, V131E, S132H, S132P, S132C, S13 2E, S132D, G133E, G133D, T134V, T134D, T134E, Y135S, Y135T, Y135W, Y135K, Y135G, Y135P, Y135M, Y135D, Y135E, I137S, I137G, T141G, G142D, G142E, N1 44C, P146C, P146L, G147S, G147L, G149C, N150D, N150E, F155L, K156Y, K156 A. K156D, K156Q, K156W, K156P, K156E, K156C, K156F, K156L, K156S, K156G, R158A, R158G, R158Q, R158S, R158W, R158L, R158T, R158V, R158E, R158Y, W15 9T, W159C, W159E, W159D, Y160S, Y160V, Y160I, Y160L, Y160E, D163L, T165I , W167D, D168G, Q169L, R171V, Q172S, L173V, N175L, N175V, N175I, N175G, Y 178K, K179A, K179G, K179T, K179F, R181V, R181Q, R181E, R181G, K185A, K18 5D, K185N, K185V, K185G, A186S, W187G, D188V, E194Q, Y200C, Y200T, Y200L,Y200A, Y200G, Y200S, Y203M, Y203V, Y203L, A204L, A204D, A204G, D205C, H210 F, H210A, H210W, H210L, H210G, H210D, H210E, N215V, R219Y, R219D, R219C, W2 23Y, V238W, K242R, K242S, K242E, K242A, K242Q, F245D, F245G, G258L, F262Q, F262E, F262R, F262P, F262V, K269L, N270D, N270M, A274E, L279G, K281G, W284A , T285F, S287V, S287W, S287C, Q299I, L312F, L312V, H321V, H321C, A325S, G33 7V, G337M, E341I, S342A, F348Q, F348S, K349L, K349S, P350L, P350G, Q361E, G 362R, G362V, Y368L, Y368V, G369A, D377V, V379P, P380I, S381L, S381A, S381I, Q384Y, V410Q, N418G, G435E, Q445E, G456R, N465R, and G477L, wherein these positions correspond to amino acid positions in the amino acid sequence as illustrated in SEQ ID NO:3, and wherein the one or more substitutions provide an IF of ≥1.6 for a measure of specific activity in a standard X detergent composition and optionally an IF of ≥1.0 for a measure of stability.

[0121] Variants according to the invention have further demonstrated improved stability in liquid detergent compositions. Thus, in one embodiment, the variant exhibits improved stability, such as improved storage stability.

[0122] In one embodiment, the variant includes substitutions at one or more positions selected from the group consisting of: H1, H2, D3, T5, G7, I9, Q11, E14, W15, N16, V17, N19, Q22, H23, W24, N25, R26, H28, N29, N30, A31, N33, L34, K35, N36, A37, G38, I39, T40, A41, I42, I44, A47, W48, K49, T51, S52, Q53, N54, D55, V56, G59, A60, L63, E68, G73, T74, V75, R76, T77, T81, K82, A83, E84 , E86, I89, R90, S91, L92, N95, G96, I97, Q98, V99, Y100, V103, H107, K108, G 109, G110, A111, D112, F113, T114, R116, V117, Q118, V120, V122, N123, P124 , N126, N128, Q129, V131, G133, Q136, I137, E138, A139, W140, T141, G142, F 143, N144, F145, P146, G149, Q151, S153, S154, F155, K156, R158, Y160, H161 , T165, D166, Q169, S170, R171, Q172, L173, A174, N175, R176, Y178, K179, R 181, G184, K185, A186, T193, N195, G196, Y198, Y203, D205, V206, D209, H210 , E212, V213, I214, N215, L217, N218, R219, W220, G221, V222, W223, N226, T 227, L228, D231, F233, V238, H240, K242, S244, F245, R247, D248, W249, L250 , G251, H252, V253, T257, G258, K259, N260, L261, V264, W268, K269, L272, G 273, L275, E276, N277, L279, S280, K281, T285, M286, A288, P292, L293, H294 , Y295, N296, L297, Q299, A300, N302, S303, S304, G305, N306, R310, N311, L3 12. L313, G315, T316, V318, Q319, R320, H321, S323, V326, T327, F328, V329,T334, P336, G337, E338, E341, S342, V344, Q345, G346, W347, F348, P350 , L351, A354, T355, L357, T358, E360, Q361, Y363, Q365, V366, F367, Y368 , G369, Y372, G373, I374, P375, S376, D377, V379, P380, S381, Y382, R38 3. Q384, I386, D387, P388, L389, L390, K391, A392, Q394, Q395, Y396, A39 7. Y398, R400, H402, D403, Y404, F405, D406, D409, V410, T414, R415, E416, N418, A419, S420, H421, P422, S424, G425, L426, A427, M430, S431, D432, G435, W439, Y441, Q445, K446, A447, G448, V450, W451, H452, M454, G456, S459, T463, N465, D467, F473, N475, G476, and W482; where these positions correspond to as shown in SEQ The amino acid positions of the amino acid sequence specified in ID NO:3, wherein the one or more substitutions provide an IF of ≥1.0 for a measure of stability.

[0123] In one embodiment, the variant includes one or more substitutions selected from the group consisting of: H1V, H1L, H2T, D3R, D3Y, D3W, D3S, D3F, T5L, T5F, T5W, G7F, G7V, G7E, G7E, G7D, G7L, G7A, G7S, G7N, I9M, I9L, Q11L, Q11V, Q11M, E14Q, E14F, W15G, W15S, W15I, W15H , N16R, V17D, V17G, V17E, V17Q, V17A, V17T, V17L, N19G, N19S, Q22C, Q22A, Q22Y, H23W, W24F, N25V, N25 R, N25L, N25T, R26L, R26E, H28E, H28R, H28M, H28W, H28K, N29G, N29K, N29V, N29W, N29A, N30S, N30R, A3 1S, A31G, A31R, N33D, L34Y, K35L, N36V, N36P, N36W, N36T, A37E, G38M, G38Y, G38K, I39L, I39V, T40Q, T 40A, T40S, A41Q, I42V, I42L, I44T, I44L, I44V, A47G, A47T, W48C, W48I, W48H, W48A, W48Y, W48F, K49Q, T51A, S52M, S52V, S52C, S52F, S52A, S52D, S52K, S52N, S52E, S52T, Q53V, Q53L, Q53M, N54T, N54L, N54I , D55G, V56L, G59E, G59S, A60V, A60F, L63C, L63G, L63A, E68S, E68M, E68C, E68T, E68V, E68Q, E68Y, G73C T74V, T74L, T74N, V75T, V75E, R76V, T77V, T81G, K82G, A83Q, A83R, A83S, A83F, A83V, E84M, E84W, E86W, E86R, I89A, I89L, R90L, S91A, S91G, S91V, S91T, L92C, L92F, N95E, N95S, N95G, N95D, N95R, G 96R, I97V, I97L, Q98V, Q98L, Q98F, Q98W, Q98P, Q98H, V99G, V99E, Y100W, V103A, V103S, V103T, H10 7M, K108V, G109D, G109F, G110C, A111S, D112V, D112F, D112C, D112K, F113M, F113V, F113E, F113K,F113Y、T114S、T114V、T114L、R116Q、R116K、R116W、V117G、V117I、V117M、Q118V、Q118G、Q118E、Q118F、Q118R、V120C、V120A、V120N、V122L、N123K、N123V、P124R、P124G、N126G、N126A、N126R、N126T、N128S、N128E、N128P、N128G、N128A、Q129E、V131I、G133A、G133D、G133R、Q136E、Q136W、Q136G、I137V、E138A、E138Y、E138T、E138L、A139S、A139G、A139V、A139T、A139P、W140H、T141S、T141C、T141G、G142R、G142A、F143M、F143L、F143I、N144M、N144K、N144G、N144R、F145A、F145N、F145Q、F145T、F145H、P146L、P146M、P146T、G149Y、G149L、G149A、Q151L、Q151I、S153G、S153T、S154M、S154E、S154A、S154V、F155W、K156F、K156Q、K156L、K156S、K156G、K156N、K156T、R158S、Y160W、Y160R、Y160H、H161C、T165I、T165V、T165E、D166S、Q169L、Q169S、Q169V、Q169F、S170A、S170V、S170Q、S170W、S170G、S170F、S170T、S170L、R171A、R171M、Q172L、Q172E、Q172C、Q172R、Q172G、Q172W、L173I、L173A、A174V、A174L、A174M、A174K、N175G、N175S、R176Y、R176M、R176T、R176K、Y178W、K179L、R181A、R181G、R181D、G184D、K185G、A186S、A186D、A186T、A186Q、A186V、A186H、T193S、T193D、T193R、T193G、N195T、N195F、G196P、Y198A、Y198G、Y203W、Y203L、D205R、D205A、D205L、V206A、V206S、V206K、D209S、D209M、D209V、D209Q、D209L、D209I、H210Q、H210N、E212G、E212I、E212Y、E212R、E212C、E212K、V213C、V2 13W、V213R、V213F、I214V、N215C、N215T、L217S、L217A、L217W、N218S、N218 D、N218R、N218L、N218M、R219L、W220L、W220I、W220P、W220E、W220R、W220A、 W220S、G221S、G221A、V222E、V222L、W223F、W223L、N226C、N226M、N226E、N22 6K, N226R, T227L, T227Y, T227R, L228F, L228M, D231P, F233T, F233P, F233M, V238A, H240W, K242A, S244Q, F245G, F245A, R247Y, R247G, R247M, D248G, D 248R, W249F, L250C, L250F, L250Y, G251K, G251M, G251Q, H252C, H252T, H252F, H252V, V253A, V253L, T257V, G258P, G258F, G258D, K259L, K259A, K259S K259D, K259C, N260W, N260S, N260R, N260G, L261T, V264L, W268Y, K269W, K269Q, K269R, L272M, G273E, L275G, L275E, L275A, L275R, L275K, E276L, N277 D, L279Q, L279M, S280R, S280E, S280D, K281A, T285W, T285M, T285P, M286L, M286C, A288V, P292L, P292G, L293T, L293G, L293Y, L293Q, H294W, H294Q, H29 4S、Y295I、Y295A、Y295F、N296S、N296K、N296H、L297W、L297H、Q299S、Q299L 、Q299R、Q299A、Q299H、Q299P、A300C、N302S、S303A、S303M、S304G、S304L、S 304F, S304A, S304M, G305Q, G305F, G305I, N306V, N306L, N306D, N306S, N306T, R310A, R310T, N311L, N311R, N311C, N311Y, L312F, L312V, L312I, L313ML313I、G315V、G315T、G315R、G315F、T316S、T316C、T316G、V318L、Q319P、Q3 19H、Q319D、Q319F、R320V、R320C、R320A、R320S、H321T、H321G、H321R、H321 S, S323N, V326G, V326A, V326C, V326T, T327V, T327L, T327Q, T327E, F328M, F328C, V329S, V329C, V329I, T334A, T334S, P336L, P336W, P336S, P336K, G33 7V、G337F、E338T、E338A、E338H、E341P、E341M、E341F、E341K、E341S、E341T 、S342A、V344L、Q345L、Q345W、Q345N、G346R、G346S、W347L、W347R、W347V、W 347C, F348P, F348M, P350S, P350Q, P350D, P350E, L351W, L351H, L351Q, A354C, T355L, T355Y, T355F, L357H, T358L, E360G, E360A, E360M, Q361V, Q361G Q361D, Q361S, Y363L, Y363R, Y363V, Q365M, Q365L, Q365T, Q365C, Q365S, V366I, V366C, F367L, F367S, F367A, Y368H, G369L, Y372A, G373T, G373Q, G373 W, G373R, I374Y, I374S, I374M, I374L, P375E, P375S, P375V, P375R, S376R, S376L, S376V, S376I, S376Q, S376F, S376E, S376T, D377S, D377G, D377T, V37 9L、V379S、P380V、P380Y、S381V、Y382K、Y382R、R383V、Q384S、Q384V、Q384T 、Q384G、Q384E、I386L、I386M、I386W、I386V、D387Q、D387L、P388S、P388R、P 388K, P388L, L389A, L389V, L389F, L389M, L390V, L390T, L390N, L390H, L390C, K391Q, A392I, A392M, A392T, Q394N, Q394L, Q394T, Q394M, Q395E, Q395KQ395R, Q395V, Q395S, Q395M, Y396R, Y396W, A397S, Y398S, Y398C, Y398N, Y398K, R400E, R400I, H402L, H402 D, H402R, H402T, D403P, D403Q, D403I, Y404K, Y404F, Y404W, F405G, F405A, D406V, D406T, D406M, D406S, D40 9G, V410L, V410E, T414A, R415A, R415Q, E416V, E416L, E416A, E416Q, N418M, N418G, N418R, N418F, A419M, A 419G, A419V, A419L, A419Q, A419R, S420M, H421T, P422Y, P422H, P422D, P422R, S424L, G425V, L426S, L426A, A427S, M430L, M430A, M430T, S431A, S431T, S431F, D432Q, D432P, G435N, G435M, G435I, G435S, W439E, W439 A. W439T, W439R, W439N, Y441K, Q445R, K446G, K446T, K446P, A447R, A447S, G448R, V450T, V450Q, V450R, W45 1L, H452C, H452G, H452Y, H452R, H452V, M454Q, M454S, G456Y, G456K, G456L, S459E, S459Q, S459R, T463M, T463A, T463Q, N465L, N465R, D467C, F473E, F473G, F473T, F473V, N475G, G476F, G476Y, and W482F, wherein the numbering is based on the amino acid sequence as defined in SEQ ID NO:3, and wherein one or more substitutions provide an IF ≥ 1.0 for a measure of stability.

[0124] In one embodiment, the variant includes substitutions at one or more locations selected from the group consisting of: H1, H2, D3, T5, G7, I9, E14, W15, N16, V17, Q22, H23, W24, N25, R26, H28, N29, N33, N36, A37, G38, I39, T40, A41, I42, I44, W48, S52, Q53, N54, D55, V56, G59, A60, L63, E68, G73, T74, V75, R76, T81, A83, E86, R90, S91, L92, N95, I97, Q98, V99, Y100, V103, H107. K108, G109, G110, A111, D112, F113, T114, R116, V117, Q118, V120, V122, N1 23. P124, N126, N128, Q129, V131, G133, Q136, I137, E138, A139, W140, T141, G142, F143, N144, F145, P146, G149, Q151, S153, S154, F155, K156, Y160, T1 65. D166, Q169, S170, R171, Q172, L173, A174, N175, R176, K179, R181, K185, A186, T193, N195, G196, Y198, Y203, D205, V206, D209, H210, E212, V213, I2 14. N215, L217, N218, W220, G221, W223, N226, T227, L228, D231, F233, V238, H240, K242, S244, R247, D248, L250, G251, H252, T257, G258, K259, N260, L2 61. K269, L272, G273, L275, E276, N277, L279, K281, T285, M286, A288, P292, L293, H294, Y295, N296, L297, Q299, A300, N302, S303, S304, G305, N306, R3 10. N311, L312, L313, G315, T316, Q319, R320, H321, T327, T334, P336, E338, E341, S342, V344, G346, W347, F348, P350, L351, A354, T355, T358, E360, Q3 61. Y363, Q365, F367, Y368, G369, G373, I374, P375, S376, D377, V379, S381,Y382, Q384, I386, D387, P388, L389, L390, K391, A392, Q394, Q395, Y396, A397, Y398, R400, H402, D403, Y404, F405, D406, D409, V410, R415, E416, N418, A419, S420, P422, S424, G425, L426, A427, M430, S431, D432, G435, W439, Y441, Q445, K446, A447, V450, H452, M454, G456, S459, F473, and N475; where these positions correspond to SEQ The amino acid positions in the amino acid sequence as defined in ID NO:3, wherein the one or more substitutions provide an improvement factor of ≥1.2 for measures of stability and optionally an improvement factor of ≥1.0 for measures of specific activity in detergent compositions of Standard A and / or Standard X.

[0125] In one embodiment, the variant includes one or more substitutions selected from the group consisting of: H1V, H1L, H2T, D3R, D3Y, D3W, D3S, D3F, T5L, T5F, T5W, G7F, G7V, G7E, G7E, G7D, G7L, G7A, I9L, E14Q, W15I, N16R, V17D, V17G, V17E, V17Q, V17A, V17T, V17L, Q22C, Q22A, Q22Y, H23W, W24F, N25V, N25R, N25L, N25T, R26L, R26E, H28E, H28R, H28M, H28W, H28K, N29G, N29 V, N29W, N33D, N36V, N36P, N36W, A37E, G38Y, G38K, I39L, I39V, T40Q, T40A, T40S, A41Q, I42L, I44T, W48C, W48I, W48H, W48A, W48Y, W48F, S52M, S52V, S52 C. S52F, S52A, S52D, S52K, S52N, S52E, S52T, Q53V, Q53L, Q53M, N54T, N54L, N54I, D55G, V56L, G59E, G59S, A60V, A60F, L63C, L63G, L63A, E68S, E68M, E68 C. E68T, E68V, E68Q, E68Y, G73C, T74V, T74L, V75T, V75E, R76V, T81G, A83Q, A83R, A83S, A83F, A83V, E86W, E86R, R90L, S91G, S91T, L92C, N95E, N95S, N95 G, N95D, N95R, I97L, Q98V, Q98L, Q98W, Q98P, Q98H, V99G, V99E, Y100W, V103 S, V103T, H107M, K108V, G109D, G109F, G110C, A111S, D112F, D112C, D112K, F 113M, F113V, F113E, F113K, T114S, T114V, T114L, R116Q, R116K, V117G, V11 7I, V117M, Q118V, Q118G, Q118E, Q118F, Q118R, V120N, V122L, N123K, N123V, P124R, P124G, N126T, N128S, N128E, N128P, N128G, N128A, Q129E, V131I, G13 3A, G133D, G133R, Q136E, Q136W, Q136G, I137V, E138A, E138Y, E138T, E138L,A139S、A139G、A139V、A139T、A139P、W140H、T141S、T141C、T141G、G142R、G142A、F143M、F143L、F143I、N144M、N144K、N144G、N144R、F145A、F145N、F145Q、F145T、F145H、P146L、P146M、P146T、G149Y、G149L、G149A、Q151L、S153G、S153T、S154M、S154E、S154A、S154V、F155W、K156F、K156Q、K156L、K156S、K156G、K156N、K156T、Y160H、T165I、D166S、Q169L、Q169S、Q169V、Q169F、S170A、S170V、S170Q、S170W、S170G、S170F、S170T、S170L、R171A、R171M、Q172L、Q172E、Q172C、Q172R、Q172G、Q172W、L173I、L173A、A174V、A174L、A174M、A174K、N175G、N175S、R176Y、R176M、R176T、R176K、K179L、R181A、R181G、R181D、K185G、A186S、A186D、A186T、A186Q、A186V、T193S、T193D、T193R、T193G、N195T、N195F、G196P、Y198G、Y203W、Y203L、D205R、D205A、D205L、V206A、V206S、V206K、D209S、D209M、D209V、D209Q、D209L、D209I、H210N、E212G、E212I、E212Y、E212R、E212C、E212K、V213C、V213W、V213R、V213F、I214V、N215C、N215T、L217S、L217A、L217W、N218S、N218D、N218R、N218L、N218M、W220L、W220I、W220P、W220E、W220R、W220A、W220S、G221A、W223L、N226C、N226M、N226E、N226K、N226R、T227L、T227Y、T227R、L228F、D231P、F233T、F233P、F233M、V238A、H240W、K242A、S244Q、R247Y、R247G、R247M、D248G、D248R、L250C、L250F、L250Y, G251K, G251M, G251Q, H252C, H252T, H252F, H252V, T257V, G258D, K259L, K259A, K259S, K259D, K259C, N260W, N260S, N260R, L261T, K269W, K269 Q, K269R, L272M, G273E, L275G, L275E, L275A, L275R, L275K, E276L, N277D, L279Q, L279M, K281A, T285W, T285M, T285P, M286L, M286C, A288V, P292L, P29 2G, L293T, L293G, L293Y, L293Q, H294W, H294Q, H294S, Y295A, N296S, N296K, N296H, L297W, L297H, Q299S, Q299L, Q299R, Q299A, Q299H, Q299P, A300C, N 302S、S303M、S304G、S304L、S304F、S304A、S304M、G305F、G305I、N306V、N30 6L、N306D、N306S、N306T、R310A、R310T、N311L、N311R、N311C、N311Y、L312F、 L312V, L312I, L313M, L313I, G315V, G315T, G315R, G315F, T316S, T316C, T316G, Q319P, Q319H, Q319D, Q319F, R320V, H321T, H321G, H321R, H321S, T327 V, T327L, T327E, T334A, T334S, P336L, P336W, P336S, P336K, E338A, E338H, E341P, E341M, E341F, E341K, E341S, S342A, V344L, G346S, W347L, W347R, W34 7V、W347C、F348M、P350Q、L351W、L351H、L351Q、A354C、T355Y、T355F、T358L 、E360G、E360A、E360M、Q361S、Y363L、Y363R、Y363V、Q365S、F367L、F367S、F 367A, Y368H, G369L, G373T, G373Q, G373W, G373R, I374Y, I374S, I374M, I374L, P375E, P375S, P375V, P375R, S376R, S376V, S376I, S376Q, S376F, S376ES376T, D377G, D377T, V379L, V379S, S381V, Y382K, Y382R, Q384S, Q384V, Q384T, Q384G, Q384E, I386W, I386V , D387Q, D387L, P388S, P388R, P388K, P388L, L389A, L389V, L389F, L389M, L390V, L390T, L390N, L390H, L390 C. K391Q, A392I, A392M, A392T, Q394N, Q394L, Q394T, Q394M, Q395E, Q395K, Q395R, Y396R, Y396W, A397S, Y39 8S, Y398C, Y398N, Y398K, R400E, R400I, H402R, H402T, D403I, Y404K, Y404F, Y404W, F405G, F405A, D406S, D40 9G, V410L, V410E, R415Q, E416V, E416L, E416A, E416Q, N418M, N418G, N418R, N418F, A419M, A419G, A419V, A4 19L, A419Q, A419R, S420M, P422Y, P422H, P422D, P422R, S424L, G425V, L426S, L426A, A427S, M430L, M430A, M 430T, S431A, S431T, S431F, D432Q, D432P, G435N, G435M, G435I, G435S, W439E, W439A, Y441K, Q445R, K446G, K446P, A447S, V450R, H452V, M454Q, M454S, G456Y, S459Q, F473E, F473G, F473T, F473V, and N475G, wherein the numbering is based on the amino acid sequence as defined in SEQ ID NO:3, and wherein the one or more substitutions provide variants having an IF of ≥1.2 for measures of stability and optionally an IF of ≥1.0 for measures of specific activity in standard A and / or standard X detergent compositions.

[0126] In one embodiment, the variant includes one or more substitutions selected from the group consisting of: H1V, H2T, T5L, G7F, G7V, G7E, G7E, G7D, I9L, E14F, Q22C, Q22A, W24F, N25V, R26L, H28E, N29G, N29A, I39L, T40Q, T40A, A41Q, I44T, W48C, W48I, W48Y, W48F, T51A, S52M, S52V, Q53V, Q53L, V56L, G59E, T74V, T74L, V75T, R90L, S91G, L92C, N95E, N95S, N95G, Q98V, Q9 8L, Y100W, K108V, F113M, F113V, F113E, F113Y, R116Q, V117G, N128S, N128E , Q129E, G133A, G133D, E138A, A139S, W140H, T141S, T141C, T141G, F143M, F 143L, F145A, P146L, Q151L, K156F, K156Q, K156L, K156S, K156G, T165I, D16 6S, Q169L, S170A, S170V, R171A, Q172L, Q172E, L173I, A174V, N175G, K179L, R181A, R181G, G184D, K185G, A186S, A186D, A186H, T193S, Y203W, Y203L, D2 09S, D209M, D209V, H210N, E212G, E212I, V213C, V213W, N215C, L217S, N218 S, N226C, T227L, L228F, K242A, L250C, H252C, T257V, K259L, N260W, N260G, K269W, L279Q, K281A, M286L, P292L, L293T, L293G, Y295F, L297W, Q299S, Q29 9L, Q299R, N306V, L312F, L312V, G315V, T316S, R320V, H321T, S323N, T327V , T327L, P336L, E341T, S342A, V344L, W347L, P350Q, L351W, Y363L, Y368H, I 374Y, S376R, V379L, S381V, D387Q, D387L, L390V, A392I, Q395E, Q395K, Q39 5R, F405G, E416V, N418M, N418G, A419M, M430L, S431A, G435N, W439E, W439R,W439N, K446G, G456Y, S459Q, and G476Y, wherein the numbering is based on the amino acid sequence as defined in SEQ ID NO:3, and wherein the one or more substitutions provide variants having an IF of ≥1.2 for measures of stability and an IF of ≥1.0 for measures of specific activity in detergent compositions of standard A and / or standard X.

[0127] In one embodiment, the variant includes substitutions at one or more locations selected from the group consisting of: H1, D3, G7, N16, V17, Q22, H28, N33, N36, T40, W48, S52, Q53, N54, D55, V56, G59, A60, V75, R76, N95, V99, G109, A111, D112, F113, T114, R116, V117, Q118, V122, N123, P124, N126, N1 28. V131, Q136, I137, E138, A139, T141, G142, F143, N144, F145, P146, G149, Q151, S153, S154, K156, Y160, T165, D 166, Q169, S170, R171, Q172, A174, R176, R181, K185, A186, T193, N195, Y203, D205, V206, D209, E212, V213, N215, N 218, W220, G221, N226, F233, K242, D248, L250, G251, H252, G258, N260, K269, G273, E276, N277, T285, P292, L293, H294, N296, Q299, S304, G305, N306, R310, N311, L312, Q319, R320, H321, T327, T334, P336, E338, E341, G346, W347, L351, A354, Y363, G373, I374, P375, S376, D377, V379, S381, Y382, Q384, P388, L389, L390, Q394, Q395, Y398, R400, H402, Y404, V410, E416, N418, A419, S420, P422, M430, S431, G435, Q445, A447, M454, F473, and N475; wherein these positions correspond to amino acid positions in the amino acid sequence as defined in SEQ ID NO:3, and the one or more substitutions provide an improvement factor of ≥1.5 for measures of stability and optionally an improvement factor of ≥1.0 for measures of specific activity in detergent compositions of standard A and / or standard X.

[0128] In one embodiment, the variant includes one or more substitutions selected from the group consisting of: H1V, H1L, D3S, D3F, G7F, G7E, G7A, N16R, V17D, V17G, V17E, V17Q, V17A, V17T, V17L, Q22A, Q22Y, H28E, H28M, H28W, H28K, N33D, N36V, N36P, N36W, T40Q, T40S, W48C, W48I, W48H, W48A, S52M, S52V, S52C, S52F, S52A, S52D, S52K, S52N, S52E, S52T, Q53V, Q53L, Q53M, N 54T, N54L, N54I, D55G, V56L, G59E, A60V, A60F, V75T, R76V, N95E, N95S, N95 G, N95D, N95R, V99G, V99E, G109D, G109F, A111S, D112K, F113K, T114L, R116Q , R116K, V117G, V117I, V117M, Q118V, Q118G, Q118E, Q118F, Q118R, V122L, N 123K, N123V, P124G, N126T, N128S, N128E, N128P, N128G, N128A, V131I, Q136 E, Q136W, Q136G, I137V, E138T, E138L, A139S, A139G, A139V, A139T, A139P, T141S, T141G, G142R, G142A, F143L, N144G, N144R, F145A, F145N, F145Q, F14 5T, F145H, P146L, P146M, P146T, G149Y, G149L, G149A, Q151L, S153T, S154M , S154E, S154A, S154V, K156F, K156L, K156S, K156G, Y160H, T165I, D166S, Q1 69L, Q169S, Q169V, Q169F, S170A, S170V, S170Q, S170W, S170G, S170F, S170 T, S170L, R171A, R171M, Q172L, Q172E, Q172C, Q172R, Q172G, Q172W, A174K, R 176Y, R176M, R176T, R176K, K179L, R181A, R181G, R181D, K185G, A186S, A18 6D, A186T, A186Q, A186V, T193S, T193D, T193R, T193G, N195T, N195F, Y203W,Y203L、D205A、D205L、V206A、V206S、V206K、D209S、D209M、D209V、D209Q、D2 09L、D209I、E212G、E212I、E212C、E212K、V213W、V213R、V213F、N215C、N215 T、N218D、N218R、N218L、N218M、W220L、W220I、W220P、W220E、W220R、W220A、 W220S、G221A、N226C、N226M、N226E、N226K、N226R、F233T、F233P、F233M、K24 2A, D248G, D248R, L250Y, G251K, G251M, G251Q, H252F, H252V, G258D, N260W, N260R, K269W, G273E, E276L, N277D, T285M, T285P, P292L, P292G, L293G, L 293Q、H294S、N296H、Q299L、Q299R、Q299P、S304G、S304L、S304F、S304A、S30 4M、G305F、G305I、N306V、N306L、N306D、N306S、N306T、R310T、N311C、N311Y、 L312F, L312V, Q319H, Q319D, Q319F, R320V, H321R, H321S, T327V, T327E, T334S, P336L, P336W, P336S, P336K, E338A, E338H, E341M, E341F, E341K, E341 S、G346S、W347L、W347R、W347V、W347C、L351H、L351Q、A354C、Y363R、Y363V、 G373Q、G373W、G373R、I374Y、I374S、I374M、I374L、P375E、P375S、P375V、P37 5R, S376R, S376F, S376E, S376T, D377T, V379L, V379S, S381V, Y382R, Q384E, P388S, P388R, P388K, P388L, L389A, L389V, L389F, L389M, L390V, L390C, Q 394N、Q394L、Q394T、Q394M、Q395R、Y398K、R400E、R400I、H402T、Y404F、Y40 4W、V410E、E416Q、N418M、N418G、N418R、N418F、A419M、A419L、A419Q、A419R、S420M, P422Y, P422H, P422D, P422R, M430L, S431A, S431F, G435S, Q445R, A447S, M454S, F473V, and N475G, wherein the numbering is based on the amino acid sequence as defined in SEQ ID NO:3, and wherein the one or more substitutions provide variants having an IF of ≥1.5 for measures of stability and optionally an IF of ≥1.0 for measures of specific activity in detergent compositions of standard A and / or standard X.

[0129] In one embodiment, the variant includes one or more substitutions selected from the group consisting of: H1V, G7F, G7E, Q22A, H28E, T40Q, W48C, W48I, S52M, S52V, Q53V, Q53L, V56L, G59E, V75T, N95E, N95S, N95G, R116Q, V117G, N128S, N128E, A139S, T141S, T141G, F143L, F145A, P146L, Q151L, K156F, K156L, K156S, K156G, T165I, D166S, Q169L, S170A, S170V, R171A, Q172L, Q172E, K 179L, R181A, R181G, K185G, A186S, A186D, T193S, Y203W, Y203L, D209S, D209M, D209V, E212G, E212I, V213W, N215C, N226C, K242A, N260W, K269W, P292L, L293G, Q299L, Q299R, N306V, L312F, L312V, R320V, T327V, P336L, W347L, I374Y, S376R, V379L, S381V, L390V, Q395R, N418M, N418G, A419M, M430L, and S431A, wherein the numbering is based on the amino acid sequence as defined in SEQ ID NO:3, and wherein the one or more substitutions provide variants having an IF of ≥1.5 for measures of stability and an IF of ≥1.0 for measures of specific activity in detergent compositions of standard A and / or standard X.

[0130] In one embodiment, the variant includes substitutions at one or more positions selected from the group consisting of: H1, H2, T5, G7, I9, Q11, E14, W15, N19, Q22, W24, N25, R26, H28, N29, N30, A31, L34, K35, I39, T40, A41, I42, I44, A47, W48, T51, S52, Q53, V56, G59, T74, V75, I89, R90, S91, L92, N95, I97, Q98. Y100, K108, F113, R116, V117, N126, N128, Q129, G133, A139, W140, T141, F143, F145, P146, Q151, K156, R158, H161, T16 5. D166, Q169, S170, R171, Q172, L173, A174, N175, K179, R181, G184, K185, A186, T193, Y203, D209, H210, E212, V213, N 215, L217, N218, R219, V222, N226, T227, L228, K242, K245, L250, H252, V253, T257, G258, K259, N260, V264, K269, L279 , K281, M286, P292, L293, Y295, L297, Q299, N306, L312, G315, T316, V318, R320, H321, S323, T327, V329, P336, G337, E3 41, S342, V344, Q345, G346, W347, P350, L351, Q361, Y363, Y368, I374, S376, V379, P380, S381, D387, L390, A392, Q395, H402, F405, T414, E416, N418, A419, M430, S431, G435, W439, K446, V450, G456, S459, N465, G476, and W482; wherein these positions correspond to amino acid positions in the amino acid sequence as defined in SEQ ID NO:3, and the one or more substitutions provide an improvement factor of ≥1.0 for measures of stability and an improvement factor of ≥1.0 for measures of specific activity in standard A detergent compositions.

[0131] In a specific embodiment, the variant includes one or more substitutions selected from the group consisting of: H1V, H2T, T5L, G7F, G7V, G7E, G7E, G7D, I9M, I9L, Q11L, Q11V, E14F, W15G, W15S, N19G, N19S, Q22C, Q22A, W24F, N25V, R26L, H28E, N29G, N29A, N30S, A31S, L34Y, K35L, I39L, T40Q, T40A, A41Q, I42V, I44T, A47G, W48C, W48I, W48Y, W48F, T51A, S52M, S5 2V, Q53V, Q53L, V56L, G59E, T74V, T74L, V75T, I89A, R90L, S91A, S91G, L9 2C, N95E, N95S, N95G, I97V, Q98V, Q98L, Y100W, K108V, F113M, F113V, F11 3E, F113Y, R116Q, V117G, N126G, N126A, N128S, N128E, Q129E, G133A, G13 3D, E138A, A139S, W140H, T141S, T141C, T141G, F143M, F143L, F145A, P146 L, Q151L, K156F, K156Q, K156L, K156S, K156G, R158S, H161C, T165I, D166 S, Q169L, S170A, S170V, R171A, Q172L, Q172E, L173I, A174V, N175G, K179 L, R181A, R181G, G184D, K185G, A186S, A186D, T193S, Y203W, Y203L, D209 S, D209M, D209V, H210N, E212G, E212I, V213C, V213W, N215C, L217S, N218 S, R219L, V222E, N226C, T227L, L228F, K242A, F245G, L250C, H252C, V253 A. V253L, T257V, G258P, K259L, N260W, N260G, V264L, K269W, L279Q, K281 A. M286L, P292L, L293T, L293G, Y295F, L297W, Q299S, Q299L, Q299R, N306 V, L312F, L312V, G315V, T316S, V318L, R320V, R320C, H321T, S323N, T327VT327L, V329S, P336L, G337V, S342A, V344L, Q345L, G346R, W347L, P350S, P350Q, L351W, Q361V, Y36 3L, Y368H, Y372A, I374Y, S376R, S376L, V379L, P380V, S381V, D387Q, D387L, L390V, A392I, Q395E, Q395K, Q395R, H402L, F405G, T414A, E416V, N418M, N418G, A419M, M430L, S431A, G435N, W439E, W439R, W439N, K446G, V450T, G456Y, S459E, S459Q, N465L, N465R, D467C, G476Y, and W482F, wherein the numbers correspond to the amino acid positions in the amino acid sequence as defined in SEQ ID NO:3, and wherein these substitutions provide variants having an IF of ≥1.0 for measures of stability and an IF of ≥1.0 for measures of specific activity in standard A detergent compositions.

[0132] In one embodiment, the variant includes substitutions at one or more positions selected from the group consisting of: H1, H2, T5, G7, I9, Q11, N19, Q22, W24, N25, R26, H28, N29, N30, A31, L34, K35, I39, T40, A41, I42, I44, A47, W48, T51, S52, Q53, V56, G59, T74, V75, T81, I89, R90, S91, L92, N95, I97, Q98, Y1 00, K108, F113, R116, V117, N126, N128, Q129, G133, E138, A139, W140, T141, F143, F145, P146, Q151, K156, R158, H161 , T165, D166, Q169, S170, R171, Q172, L173, A174, N175, K179, R181, G184, K185, A186, T193, E194, Y203, D209, H210, E 212, V213, N215, L217, N218, R219, V222, N226, T227, L228, K242, K245, L250, H252, V253, T257, G258, K259, N260, V26 4. K269, L279, K281, M286, P292, L293, Y295, L297, Q299, N306, L312, G315, T316, V318, R320, H321, S323, T327, V329, P336, G337, E341, S342, V344, Q345, G346, W347, P350, Q361, Y363, I374, S376, V379, P380, S381, D387, L390, A392, Q395, H402, F405, E416, N418, A419, S431, G435, W439, K446, V450, G456, S459, N465, D467, G476, and W482, wherein these positions correspond to amino acid positions in the amino acid sequence as defined in SEQ ID NO:3, and wherein the one or more substitutions provide an IF of ≥1.0 for a measure of specific activity in the standard X detergent composition and an IF of ≥1.0 for a measure of stability.

[0133] In one embodiment, the variant includes one or more substitutions selected from the group consisting of: H1V, H2T, T5L, G7F, G7E, G7V, G7E, G7D, I9M, I9L, Q11L, Q11V, N19G, N19S, Q22C, Q22A, W24F, N25V, R26L, H28E, N29G, N29A, N30S, A31S, L34Y, K35L, I39L, T40A, T40Q, A41Q, I42V, I44T, A47G, W48C, W48I, W48Y, W48F, T51A, S52V, S52M, Q53L, Q53V, V56L, G59E, T74 L, T74V, V75T, T81G, I89A, R90L, S91A, S91G, L92C, N95G, N95E, N95S, I97V, Q98V, Q98L, Y100W, K108V, F113M, F113V, F113E, F113Y, R116Q, V117G, N126G , N126A, N128S, N128E, Q129E, G133A, G133D, E138A, A139S, W140H, T141C, T 141G, T141S, F143M, F143L, F145A, P146L, Q151L, K156Q, K156F, K156L, K156 S, K156G, R158S, H161C, T165I, D166S, Q169L, S170A, S170V, R171A, Q172E, Q172L, L173I, A174V, N175G, K179L, R181A, R181G, G184D, K185G, A186S, A18 6D, A186H, T193S, Y203L, Y203W, D209S, D209M, D209V, H210N, E212G, E212I , V213C, V213W, N215C, L217S, N218S, R219L, V222E, N226C, T227L, L228F, K2 42A, F245G, L250C, H252C, V253A, V253L, T257V, G258P, K259L, N260W, V264 L, K269W, L279Q, K281A, M286L, P292L, L293T, L293G, Y295F, L297W, Q299S, Q 299L, Q299R, N306V, L312F, L312V, G315V, T316S, V318L, R320C, R320V, H32 1T, S323N, T327L, T327V, V329S, P336L, G337V, S342A, V344L, Q345L, G346R,W347L, P350S, P350Q, Q361V, Y363L, I374Y, S376R, S376L, V379L, P380V, S381V, D387Q, D387L, L390V, A392I, Q395E, Q395K, Q395R, H402L, F405G, E416V, N418M, N418G, A419M, S431A, G435N, W439E, W439R, W439N, K446G, V450T, G456Y, S459Q, N465L, N465R, D467C, G476, and W482F, where these positions correspond to SEQ ID. The amino acid positions in the amino acid sequence specified in NO:3, wherein the one or more substitutions provide an IF of ≥1.0 for a measure of specific activity in the standard X detergent composition and an IF of ≥1.0 for a measure of stability.

[0134] In one embodiment, the variant includes substitutions at one or more positions selected from the group consisting of: H1, G7, Q22, N25, R26, H28, N29, W48, S52, Q53, V56, G59, T74, V75, R90, N95, Q98, K108, F113, R116, V117, N128, Q129, G133, A139, W140, T141, F143, F145, P146, Q151, K156, T165, Q169, S170, R171, Q 172, A174, K179, R181, K185, A186, T193, Y203, D209, E212, N215, L217, N226, K242, K259, N260, K269, K281, P292, L293, L297, N306, L312, G315, R320, H321, P336, V344, P350, Y363, Y368, S381, D387, A392, Q395, E416, N418, and S431; where these positions correspond to SEQ The amino acid positions in the amino acid sequence as defined in ID NO:3, wherein one or more substitutions provide an improvement factor of ≥1.3 for measures of stability and an improvement factor of ≥1.3 for measures of specific activity in standard A detergent compositions.

[0135] In one embodiment, the variant includes one or more substitutions selected from the group consisting of: H1V, G7F, G7V, G7E, G7E, Q22A, N25V, R26L, H28E, N29G, W48C, W48I, W48F, S52M, S52V, Q53V, Q53L, V56L, G59E, T74L, V75T, R90L, N95E, N95S, N95G, Q98V, Q98L, K108V, F113V, R116Q, V117G, N128S, N128E, Q129E, G133A, G133D, A139S, W140H, T141S, T141 C. T141G, F143M, F143L, F145A, P146L, Q151L, K156F, K156Q, K156L, K156S, K156G, T165I, D166S, Q 169L, S170A, S170V, R171A, Q172L, Q172E, A174V, K179L, R181A, R181G, K185G, A186S, A186D, T19 3S, Y203W, Y203L, D209S, D209M, D209V, E212I, N215C, L217S, N226C, K242A, K259L, N260W, K269W, K281A, P292L, L293T, L293G, L297W, N306V, L312F, L312V, G315V, R320V, H321T, P336L, V344L, P350Q, Y363L, Y368H, S381V, D387L, A392I, Q395K, Q395R, E416V, N418M, N418G, and S431A, wherein the numbering is based on the amino acid sequence as defined in SEQ ID NO:3, and wherein the one or more substitutions provide variants having an IF of ≥1.3 for measures of stability and an IF of ≥1.3 for measures of specific activity in standard A detergent compositions.

[0136] In one embodiment, the variant includes substitutions at one or more positions selected from the group consisting of: H1, T5, G7, Q22, N25, R26, N29, W48, S52, Q53, T74, R90, N95, Q98, K108, F113, R116, V117, N128, Q129, G133, A139, W140, T141, F143, F145, P146, Q151, K156, T165, D166, Q169, S1 70, R171, Q172, A174, K179, R181, K185, A186, Y203, E212, N215, N226, K242, N260, K269, L279, K281, L293, Q299, N306, L312, G315, R320, V344, P350, Y363, I374, V379, S381, D387, A392, Q395, E416, N418, and S431, wherein these positions correspond to amino acid positions in the amino acid sequence as defined in SEQ ID NO:3, and wherein the substitutions at one or more of these positions provide an IF of ≥1.3 for a measure of specific activity in the standard X detergent composition and an IF of ≥1.3 for a measure of stability.

[0137] In one embodiment, the variant includes one or more substitutions selected from the group consisting of: H1V, T5L, G7F, G7E, G7V, G7E, Q22A, N25V, R26L, N29G, W48C, W48I, S52V, S52M, Q53L, Q53V, T74L, R90L, N95G, N95E, N95S, Q98V, Q98L, K1 08V, F113V, R116Q, V117G, N128S, N128E, Q129E, G133A, G133D, A139S, W140H, T141C, T141 G, T141S, F143M, F145A, P146L, Q151L, K156Q, K156F, K156L, K156S, K156G, T165I, D166S, Q169L, S170A, S170V, R171A, Q172E, Q172L, A174V, K179L, R181A, R181G, K185G, A186S, A1 86D, Y203L, Y203W, E212G, E212I, N215C, N226C, K242A, N260W, K269W, L279Q, K281A, L293 T, L293G, Q299S, Q299L, N306V, L312F, L312V, G315V, R320V, V344L, P350Q, Y363L, I374Y, V379L, S381V, D387L, A392I, Q395K, Q395R, E416V, N418M, N418G, and S431A, wherein these positions correspond to amino acid positions in the amino acid sequence as defined in SEQ ID NO:3, and wherein the one or more substitutions provide an IF of ≥1.3 for a measure of specific activity in the standard X detergent composition and an IF of ≥1.3 for a measure of stability.

[0138] In one embodiment, the variant includes substitutions at one or more positions selected from the group consisting of: H1, G7, Q22, S52, Q53, G59, R116, V117, N128, A139, T141, F143, F145, P146, Q151, K156, T165, D166, Q169, S170, R171, R181, K185, A186, Y203, E212, N215, K242, K269, N306, L312, and N418, wherein the numbering is based on the amino acid sequence as defined in SEQ ID NO:3, and wherein the substitutions at one or more positions provide an IF ≥ 1.5 for a measure of specific activity in a standard A detergent composition and an IF ≥ 1.5 for a measure of stability.

[0139] In one embodiment, the variant includes one or more substitutions selected from the group consisting of: H1V, G7E, Q22A, S52V, Q53V, Q53L, G59E, R116Q, V117G, N128S, N128E, A139S, T141S, T141G, F143L, F145A, P146L, Q151L, K156F, K156L, K 156S, K156G, T165I, D166S, Q169L, S170A, S170V, R171A, R181A, R181G, K185G, A186S, A186D, Y203W, Y203L, E212I, N215C, K242A, K269W, N306V, L312F, L312V, and N418G, wherein the numbering is based on the amino acid sequence as defined in SEQ ID NO:3, and wherein the substitution at one or more positions provides an IF ≥ 1.5 for a measure of specific activity in a standard A detergent composition and an IF ≥ 1.5 for a measure of stability.

[0140] In one aspect, the present invention relates to an α-amylase variant, wherein the variant is a mature form of an α-amylase having amylase activity, and includes substitutions at one or more positions selected from the group consisting of: E14, D20, Q22, G59, R116, A119, S132, G133, Y135, K156, R158, W159, Y160, K185, W187, Y202, D236, V238, K242, G337, E341, Y368, G369, D377, Y382, and K445; wherein these positions correspond to amino acids in the amino acid sequence as defined in SEQ ID NO:3; and wherein the substitution provides at least one improved property selected from the group consisting of: improved washing performance, improved detergent stability, and improved thermal stability.

[0141] As used herein, the term "thermal stability" refers to the ability of an enzyme to retain its activity after exposure to elevated temperatures. The thermal stability of an enzyme (such as α-amylase or α-amylase variants) is given by its half-life (tday) in minutes, hours, or days (during which the enzyme activity is halved under defined conditions). 1 / 2 The half-life can be measured by measuring the residual α-amylase activity after exposure to elevated temperatures (i.e., stimulation).

[0142] In another aspect, the present invention relates to an α-amylase variant, wherein the variant is a mature form of an α-amylase having amylase activity, and comprises substitutions at one or more positions selected from the group consisting of: E14, D20, Q22, G59, R116, A119, S132, G133, Y135, K156, R158, W159, Y160, K185, W187, Y202, D236, V238, K242, G337, E341, Y368, G369, D377, Y382, and K445, wherein these positions correspond to amino acids in the amino acid sequence specified in SEQ ID NO:3; and wherein the substitution provides a pH An α-amylase variant having an improvement factor of ≥1.0 for specific activity and ≥1.0 for stability and thermal stability in the detergent composition at 8.0 and 25°C.

[0143] In one embodiment, the variant includes one or more substitutions selected from the group consisting of: D377V, W159D, K242E, R158Y, A119Y, D236P, R158E, Y135D, W187G, Y382L, R158V, G369I, G337M, K156G, W159E, Y160E, R158T, G59A, Y202S, K185V, S132D, Q22A, K156E, R116F, K185G, K242S, G133D, G369R, Y382E, D20R, Y368L, E341I, K445E, Y203V, V238W, and E14M, wherein these positions correspond to SEQ ID. The positions of amino acids in the amino acid sequence as explained in NO:3.

[0144] In one embodiment, the amino acid substituted at that specific position is selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, provided that the substituted amino acid is different from a naturally occurring amino acid at that specific position.

[0145] As used herein, “naturally occurring amino acid” means any amino acid from the groups A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y found in the parental α-amylase before any modification (e.g., in the wild-type amino acid sequence of the α-amylase).

[0146] In some embodiments, the variant may include paired deletions within a specific loop of the α-amylase, which have been found to further stabilize the α-amylase. Thus, in one embodiment, the variant further includes paired deletions of amino acid residues corresponding to R181, G182, D183, and G184 of the amino acid sequence as defined in SEQ ID NO:3, provided that the paired deletions are at the positions corresponding to G182 and D183 of the amino acid sequence as defined in SEQ ID NO:3 when the amino acid at the position corresponding to R181 and / or G184 is substituted.

[0147] As used herein, the term "paired deletion" refers to a deletion at two separate positions. Such positions may be adjacent to each other, but are not limited to such adjacent pairs. Thus, a paired deletion may also be the deletion of one amino acid and another amino acid, the other amino acid being up to three amino acids further downstream or upstream of the first deletion. Thus, in one embodiment, the variant comprises paired deletions of amino acid residues selected from the group consisting of: R181+G182, R181+D183, R181+G184, G182+D183, G182+G182, and D183+G184, wherein the numbering is based on the amino acid sequence as illustrated in SEQ ID NO:3.

[0148] In a specific embodiment, the paired deletions are at positions corresponding to positions G182 and D183 of the amino acid sequence as described in SEQ ID NO:3.

[0149] In another aspect, the present invention relates to α-amylase variants, said variants being mature forms having amylase activity, and comprising a) deletions and / or substitutions at two or more positions corresponding to the amino acid sequences defined in SEQ ID NO:3, namely R181, G182, D183, and G184; and b) substitutions at one or more positions selected from the group consisting of: H1, H2, D3, G4, T5, G7, T8, I9, M10, Q11, Y12, F13, E14, W15, N16, V17, P18, N19, D20, Q22, H23, W24, N25, R26, L27, H28, N29, N30, A31, Q32, N33, L34, K35, N36, A37, G38, I39, T40, A41, I 42. W43, I44, P45, P46, A47, W48, K49, G50, T51, S52, Q53, N54, D55, V56 , G59, A60, Y61, L63, Y64, L66, E68, F69, N70, Q71, K72, G73, T74, V75, R7 6. T77, K78, T81, K82, A83, E84, L85, E86, R87, A88, I89, R90, S91, L92, K 93, N95, G96, I97, Q98, V99, Y100, V103, H107, K108, G109, G110, A111, D 112, F113, T114, E115, R116, V117, Q118, A119, V120, E121, V122, N123 , P124, Q125, N126, R127, N128, Q129, E130, V131, S132, G133, T134, Y13 5. Q136, I137, E138, A139, W140, T141, G142, F143, N144, F145, P146, G 147, G149, N150, Q151, S153, S154, F155, K156, W157, R158, W159, Y160, H161, F162, D163, G164, T165, D166, W167, D168, Q169, S170, R171, Q17 2. L173, A174, N175, R176, I177, Y178, K179, F180, R181, G184, K185, A1 86. W187, D188, W189, E190, V191, D192, T193, E194, N195, G196, N197, Y 198, D199, Y200, M202, Y203, A204, D205, V206, M208, D209, H210, P211,E212, V213, I214, N215, L217, N218, R219, W220, G221, V222, W223, Y224, A225, N226, T227, L228, N229, L230, D231, F233, V238, H240, I241, K242, S244 F245, M246, R247, D248, W249, L250, G251, H252, V253, R254, G255, Q256, T257, G258, K259, N260, L261, F262, A263, V264, Y267, W268, K269, N270, D271 L272, G273, A274, L275, E276, N277, Y278, L279, S280, K281, T282, N283, W284, T285, M286, S287, A288, V291, P292, L293, H294, Y295, N296, L297, Y298 Q299, A300, S301, N302, S303, S304, G305, N306, Y307, M309, R310, N311, L312, L313, N314, G315, T316, V318, Q319, R320, H321, P322, S323, A325, V326 T327, F328, V329, N331, T334, P336, G337, E338, E341, S342, V344, Q345, G346, W347, F348, K349, P350, L351, A352, A354, T355, L357, T358, E360, Q361 G362, Y363, Q365, V366, F367, Y368, G369, D370, Y372, G373, I374, P375, S376, D377, V379, P380, S381, Y382, R383, Q384, I386, D387, P388, L389, L390 K391、A392、Q394、Q395、Y396、A397、Y398、R400、H402、D403、Y404、F405、D4 06、H407、W408、D409、V410、I411、T414、R415、E416、N418、A419、S420、H421、 P422、S424、G425、L426、A427、T428、I429、M430、S431、D432、G435、G436、S4 37、K438、W439、M440、Y441、V442、R444、Q445、K446、A447、G448、V450、W451、H452, D453, M454, T455, G456, N457, S459, G460, T463, I464, N465, Q466, D467, W469, H471, F473, N475, G476, G477, V481, and W482; where these positions correspond to the amino acid positions in the amino acid sequence described in SEQ ID NO:3.

[0150] In one embodiment, the variant includes a) pairwise deletions of amino acid residues selected from the group consisting of: R181+G182, R181+D183, R181+G184, G182+D183, G182+G182, and D183+G184, wherein the numbering is based on SEQ ID The amino acid sequence described in NO:3, and b) are selected from one or more substitutions of the following group, which consists of: H1V, H1L, H1G, H1S, H1C, H2T, H2L, H2M, H2E, H2P, H2G, D3R, D3Y, D3W, D3S, D3F, D3C, D3A, G4Q, G4D, G4R, T5L, T5F, T5W, T5Q, T5G, T5E, T5V, G7F, G7V, G7E, G7E, G7D, G7L, G7A, G7R, G7S, G7N, T8A, T8S, I9C, I9M, I9L, M10L, Q11 C, Q11L, Q11V, Q11M, Y12V, Y12A, Y12F, F13Y, F13I, E14Q, E14L, E14M, E14S, E14F, W15G, W15S, W15I, W15F, W15H, N16R, N16G, N16Q, V17 D, V17G, V17E, V17Q, V17A, V17T, V17L, P18L, N19G, N19S, N19Y, N19T, N19V, D20R, Q22C, Q22A, Q22Y, Q22N, Q22S, Q22M, Q22T, Q22E, H23W , W24H, W24F, N25V, N25R, N25L, N25T, N25S, N25G, N25E, N25E, R26L, R26E, R26G, R26D, R26M, R26I, R26S, R26W, R26A, R26F, L27A, L27M , H28A, H28E, H28R, H28M, H28W, H28K, H28I, N29G, N29K, N29V, N29W, N29C, N29L, N29A, N30S, N30R, N30H, N30L, N30T, N30E, A31W, A31S , A31G, A31R, A31V, Q32D, Q32G, Q32L, Q32A, N33F, N33A, N33R, N33D, L34F, L34Y, K35L, K35E, K35W, K35Q, N36R, N36Q, N36A, N36E, N36V , N36P, N36W, N36T, A37R, A37G, A37T, A37S, A37E, G38N, G38M, G38Y, G38K, I39M, I39L, I39V, T40D, T40E, T40Q, T40A, T40S, A41E, A41N,A41M、A41Q、A41D、I42A、I42V、I42L、W43R、I44T、I44L、I44V、P45A、P46G、A47S、A47G、A47T、W48G、W48L、W48C、W48I、W48H、W48A、W48M、W48P、W48Q、W48Y、W48F、K49Q、G50P、G50S、G50N、T51S、T51G、T51P、T51L、T51C、T51A、S52M、S52V、S52C、S52F、S52A、S52D、S52K、S52N、S52E、S52T、S52I、S52L、Q53V、Q53L、Q53M、Q53G、N54G、N54K、N54V、N54T、N54L、N54I、N54S、D55G、D55V、D55I、V56L、V56M、V56T、G59E、G59S、G59A、A60V、A60F、A60S、A60W、A60L、A60G、A60T、Y61F、L63V、L63P、L63I、L63H、L63K、L63C、L63G、L63A、Y64W、Y64S、Y64V、Y64I、Y64H、Y64M、Y64C、Y64R、Y64G、Y64L、Y64A、L66M、E68D、E68S、E68M、E68C、E68T、E68V、E68Q、E68Y、F69H、F69W、N70G、N70A、N70T、N70S、N70E、Q71N、Q71E、Q71D、Q71I、Q71V、K72Y、K72R、G73V、G73C、G73L、G73E、T74V、T74L、T74N、V75T、V75E、V75G、R76S、R76G、R76A、R76V、T77A、T77S、T77V、K78L、K78T、K78A、T81W、T81L、T81G、K82A、K82G、A83G、A83E、A83Q、A83R、A83S、A83F、A83V、E84F、E84D、E84M、E84W、L85V、E86L、E86D、E86W、E86R、R87M、R87A、R87E、A88S、A88G、A88V、I89H、I89P、I89A、I89L、R90L、R90G、S91E、S91A、S91G、S91V、S91T、L92C、L92F、K93W、K93G、K93L、N95L、N95W、N95C、N95E、N95S、N95G、N95D、N95R、G96Q、G96R、I97M、I97V、I97L、Q98C、Q98M、Q98T、Q98V、Q98L、Q98F、Q98W、Q98P、Q98H、V99I、V99L、V99S、V99T、V99G、V99E、Y100V、Y100F、Y100W、V103G、V103A、V103S、V103T、H107Q、H107M、K108V、G109A、G109D、G109F、G109S、G110C、A111S、D112V、D112F、D112C、D112K、D112E、F113L、F113M、F113V、F113E、F113K、F113C、F113D、F113Q、F113Y、T114S、T114V、T114L、E115C、E115A、R116N、R116L、R116I、R116D、R116T、R116G、R116E、R116S、R116C、R116V、R116Q、R116K、R116F、R116H、R116W、V117C、V117L、V117S、V117W、V117A、V117G、V117I、V117M、V117E、Q118V、Q118G、Q118E、Q118F、Q118R、A119Y、A119W、V120I、V120D、V120L、V120C、V120A、V120N、E121P、E121G、E121V、E121M、E121D、V122E、V122P、V122L、V122I、V122F、N123G、N123A、N123E、N123W、N123T、N123P、N123K、N123V、P124R、P124G、Q125E、Q125T、N126E、N126G、N126A、N126R、N126T、R127G、R127S、R127C、R127W、R127L、R127V、R127F、R127Y、N128C、N128S、N128E、N128P、N128G、N128A、Q129L、Q129C、Q129E、E130C、V131L、V131E、V131I、S132A、S132H、S132P、S132C、S132E、S132D、A132Q、S132N、G133S、G133T、G133L、G133V、G133I、G133E、G133A、G133D、G133R、T134V、T134D、T134E、Y135L、Y135V、Y135S、Y135R、Y135T、Y135W、Y135K、Y135G、Y135P、Y135M、Y135D、Y135H、Y135F、Y135E、Q136E、Q136W、Q136G、I137A、I137S、I137G、I137V、E138A、E138Y、E138T、E138L、A139M、A139S、A139G、A139V、A139T、A139P、W140Y、W140S、W140A、W140H、T141M、T141S、T141C、T141G、G142D、G142E、G142R、G142A、F143M、F143L、F143I、N144C、N144M、N144K、N144G、N144R、F145C、F145S、F145A、F145N、F145Q、F145T、F145H、P146S、P146V、P146C、P146L、P146M、P146T、G147V、G147P、G147D、G147S、G147I、G147L、G147C、G149C、G149Y、G149L、G149A、N150C、N150D、N150E、Q151T、Q151C、Q151L、A151I、S153C、S153G、S153T、S154M、S154E、S154A、S154V、F155H、F155T、F155L、F155W、K156Y、K156V、K156A、K156D、K156W、K156P、K156E、K156C、K156F、K156Q、K156L、K156S、K156G、K156N、K156T、W157L、R158M、R158A、R158G、R158Q、R158W、R158L、R158T、R158V、R158E、R158Y、R158S、W159N、W159G、W159T、W159C、W159E、W159D、W159S、W159H、Y160Q、Y160M、Y160T、Y160A、Y160S、Y160V、Y160G、Y160I、Y160L、Y160E、Y160W、Y160R、Y160H、H161N、H161C、F162M、F162L、F162Y、F162I、D163S、D163K、D163A、D163R、D163L、G164S、G164T、G164C、T165L、T165M、T165S、T165G、T165I、T165V、T165E、D166T、D166N、D166S、W167Y、W167G、W167D、W167FD168M、D168S、D168G、Q169A、Q169L、Q169S、Q169V、Q169F、Q169E、S170A、S170V、S170Q、S170W、S170G、S170F、S170T、S170L、R171F、R171S、R171K、R171V、R171A、R171M、Q172S、Q172L、Q172E、Q172C、Q172R、Q172G、Q172W、Q172K、L173G、L173N、L173M、L173S、L173Y、L173V、L173I、L173A、L173F、A174G、A174V、A174L、A174M、A174K、A174S、N175L、N175V、N175I、N175G、N175S、R176E、R176W、R176G、R176M、R176Y、R176M、R176T、R176K、I177L、I177S、I177G、Y178V、Y178R、Y178K、Y178W、K179C、K179E、K179S、K179W、K179A、K179G、K179T、K179F、K179L、F180A、F180L、R181K、R181P、R181S、R181V、R181Q、R181E、R181A、R181G、R181D、G184L、G184R、G184V、G184T、G184D、K185A、K185D、K185N、K185V、K185G、A186C、A186S、A186D、A186T、A186Q、A186V、A186N、A186E、A186H、W187G、D188Q、D188G、D188R、D188M、D188L、D188V、W189R、W189A、W189G、W189C、E190D、E190N、E190A、V191M、V191A、D192E、D192Q、D192S、T193K、T193V、T193C、T193P、T193S、T193D、T193R、T193G、E194S、E194D、E194L、E194Q、N195R、N195A、N195T、N195F、G196A、G196V、G196P、N197G、N197V、N197Y、N197C、N197D、Y198W、Y198A、Y198G、D199G、D199S、D199A、Y200W、Y200F、Y200C、Y200V、Y200T、Y200L、Y200A、Y200G、Y200S、M202T、M202L、Y203F、Y203G、Y203C、Y203I、Y203Q、Y203H、Y203T、Y203M、Y203S、Y203V、Y203W、Y203L、A204P、A204R、A204C、A204H、A204W、A204F、A204L、A204Y、A204D、A204G、D205C、D205R、D205A、D205L、V206H、V206Q、V206P、V206A、V206S、V206K、V206L、V206I、V206M、V206F、V206Y、M208R、M208P、M208L、M208C、D209C、D209E、D209H、D209F、D209Y、D209S、D209M、D209V、D209Q、D209L、D209I、H210F、H210V、H210A、H210W、H210L、H210G、H210D、H210E、H210Q、H210N、P211G、P211W、P211C、E212F、E212M、E212D、E212G、E212I、E212Y、E212R、E212C、E212K、V213M、V213T、V213G、V213C、V213W、V213R、V213F、I214P、I214V、N215D、N215V、N215C、N215T、L217V、L217C、L217P、L217T、L217G、L217S、L217A、L217W、N218S、N218D、N218R、N218L、N218M、R219H、R219W、R219S、R219I、R219A、R219M、R219T、R219E、R219Y、R219D、R219C、R219L、W220L、W220I、W220P、W220E、W220R、W220A、W220S、G221S、G221A、V222F、V222C、V222E、V222L、W223Y、W223F、W223L、Y224L、A225K、N226Q、N226C、N226M、N226E、N226K、N226R、T227S、T227M、T227C、T227L、T227Y、T227R、L228A、L228F、L228M、N229T、N229S、L230S、L230A、L230T、L230V、D231E、D231C、D231G、D231P、F233A、F233L、F233T、F233P、F233M、V238C、V238W、V238A、H240W、I241L、K242C、K242Y、K242P、K242W、K242G、K242L、K242R、K242S、K242E、K242A、K242H、S244Q、F245C、F245V、F245D、F245G、F245A、M246L、M246F、M246V、M246I、R247Y、R247G、R247M、D248G、D248R、W249L、 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L293T, L293G, L293Y, L293Q, H294G, H294AH294W, H294Q, H294S, Y295H, Y295S, Y295I, Y295A, Y295F, N296A, N296Y, N296M, N296F, N296S, N296K, N296H, L297S, L 297W, L297H, Y298V, Y298P, Q299I, Q299S, Q299L, Q299R, Q299A, Q299H, Q299P, A300V, A300C, S301A, S301R, S301Q, N3 02I、N302T、N302M、N302S、S303I、S303E、S303A、S303M、S3 04I、S304P、S304G、S304L、S304F、S304A、S304M、G305V、G30 5L, G305S, G305Q, G305F, G305I, N306V, N306L, N306D, N306S, N306T, Y307F, Y307W, M309L, R310A, R310T, N311L, N311R N311C, N311Y, L312F, L312V, L312I, L313S, L313M, L313I, N314Y, N314F, N314D, G315I, G315V, G315T, G315R, G315F T316K, T316S, T316C, T316G, V318C, V318E, V318A, V318L, Q319L, Q319I, Q319A, Q319P, Q319H, Q319D, Q319F, R320L, R3 20V、R320C、R320A、R320S、H321E、H321V、H321C、H321T、H3 21G、H321R、H321S、P322E、P322C、S323N、A325V、A325S、V32 6I、V326G、V326A、V326C、V326T、T327S、T327V、T327L、T327 Q、T327E、F328M、F328C、V329A、V329S、V329C、V329I、N331C N331V, N331I, T334A, T334S, P336M, P336E, P336D, P336R, P336L, P336W, P336S, P336K, G337T, G337C, G337M, G337V, G337F, E338T E338A、E338H、E341C、E341D、E341I、E341P、E341M、E341F、E341K、 E341S、E341T、S342G、S342T、S342A、V344S、V344L、Q345K、Q345C、Q345V、Q345I、Q345L、Q345W、Q345N、G346H、G346A、G346L、G346N、G346Q、G3 46V、G346R、G346S、W347L、W347R、W347V、W347C、F348Q、F348S、F348C、F348 P、F348M、K349G、K349A、K349Y、K349L、K349S、P350L、P350G、P350S、P350Q、 P350D、P350E、L351M、L351S、L351W、L351H、L351Q、A352S、A352C、A354C、T35 5V、T355A、T355L、T355Y、T355F、L357C、L357T、L357S、L357H、T358V、T358F 、T358S、T358L、E360S、E360D、E360C、E360G、E360A、E360M、Q361P、Q361R、Q 361A, Q361E, Q361V, Q361G, Q361D, Q361S, G362Y, G362R, G362C, G362N, G362M, G362V, Y363Q, Y363I, Y363A, Y363L, Y363R, Y363V, Q365D, Q365M, Q365L Q365T, Q365C, Q365S, V366L, V366A, V366I, V366C, F367L, F367S, F367A, Y368M, Y368T, Y368I, Y368L, Y368A, Y368S, Y368Q, Y368V, Y368H, G369K, G36 9S、G369A、G369E、G369R、G369I、G369L、D370A、D370T、Y372S、Y372I、Y372L 、Y372G、Y372A、G373C、G373S、G373T、G373Q、G373W、G373R、I374Y、I374S、I3 74M, I374L, P375L, P375A, P375T, P375E, P375S, P375V, P375R, S376A, S376N, S376P, S376W, S376R, S376L, S376V, S376I, S376Q, S376F, S376E, S376T, D 377C, D377L, D377V, D377S, D377G, D377T, V379F, V379P, V379L, V379S, P380L, P380G, P380E, P380A, P380I, P380V, P380Y, S381N, S381L, S381A, S381IS381V、Y382I、Y382A、Y382E、Y382V、Y382L、Y382K、Y382R、R383G、R383S、R383P、R383V、Q384A、Q384P、Q384L、Q384H、Q384R、Q384Y、Q384S、Q384V、Q384T、Q384G、Q384E、I386T、I386L、I386M、I386W、I386V、D387H、D387V、D387Q、D387L、P388G、P388T、P388M、P388W、P388V、P388S、P388R、P388K、P388L、L389S、L389H、L389A、L389V、L389F、L389M、L390S、L390F、L390I、L390G、L390V、L390T、L390N、L390H、L390C、K391L、K391Y、K391A、K391Q、A392I、A392M、A392T、Q394S、Q394R、Q394N、Q394L、Q394T、Q394M、Q395W、Q395N、Q395L、Q395E、Q395K、Q395R、Q395V、Q395S、Q395M、Y396T、Y396L、Y396R、Y396W、A397C、A397G、A397S、Y398S、Y398C、Y398N、Y398K、R400L、R400C、R400K、R400S、R400M、R400E、R400I、H402V、H402E、H402M、H402A、H402S、H402P、H402Y、H402L、H402D、H402R、H402T、D403E、D403P、D403Q、D403I、Y404K、Y404F、Y404W、F405C、F405V、F405I、F405P、F405W、F405G、F405A、D406E、D406V、D406T、D406M、D406S、H407C、H407D、W408R、W408P、W408C、W408Y、W408G、W408D、D409G、V410I、V410Q、V410L、V410E、I411A、I411L、I411V、T414V、414A、R415F、R415L、R415A、R415Q、E416C、E416V、E416L、E416A、E416Q、N418L、N418A、N418C、N418M、N418G、N418R、N418F、A419E、A419C、A419M、A419G、A419V、A419L、A419Q、A419R、S420E、S420T、S420V、S420M、H421V、H421C、H421A、H421R、H421T、P422Y、P422H、P422D、P422R、S424C、S424A、S424L、G425A、G425V、L426S、L426A、A427V、A427S、T428L、T428N、T428A、T428G、I429V、I429A、M430R、M430L、M430A、M430T、S431A、S431T、S431F、D432Q、D432P、G435E、G435N、G435M、G435I、G435S、G436M、G436E、G436L、G436T、S437G、S437Q、S437L、K438T、W439C、W439H、W439E、W439A、W439R、W439T、W439N、W439T、W439R、W439N、M440L、Y441N、Y441H、Y441Q、Y441K、V442I、R444A、R444S、R444L、R444V、R444P、Q445L、Q445S、Q445C、Q445E、Q445R、K446Q、K446G、K446T、K446P、A447G、A447Q、A447E、A447T、A447R、A447S、G448S、G448R、V450A、V450T、V450Q、V450R、W451L、H452A、H452T、H452C、H452G、H452Y、H452R、H452V、D453E、M454R、M454L、M454V、M454Q、M454S、T455L、T455S、G456V、G456R、G456Y、G456K、G456L、N457V、N457I、N457G、N457R、S459V、S459C、S459P、S459M、S459T、S459E、S459Q、S459R、G460S、G460D、G460E、T463C、T463V、T463L、T463D、T463H、T463M、T463A、T463Q、I464V、N465G、N465Q、N465L、N465R、Q466L、Q466C、Q466E、D467V、D467C、W469M、W469E、W469T、W469N、H471Q、F473Y、F473C、F473R、F473E、F473G、F473T、F473V、N475T、N475H、N475G、G476F、G476K、G476Y、G477Q、G477F, G477K, G477L, V481Q, V481S, and W482F, where the numbers are based on the amino acid sequence described in SEQ ID NO:3.

[0151] Variants according to the invention may further include one or more additional changes at one or more (e.g., several) other locations (compared to those described above).

[0152] These amino acid alterations can be minor, i.e., conserved amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions typically of 1–30 amino acids; small amino or carboxyl terminus extensions, such as N-terminal methionine residues; small linker peptides of up to 20–25 residues; or small extensions that facilitate purification by altering net charge or another function, such as polyhistidine segments, antigenic epitopes, or binding domains.

[0153] Examples of conserved substitutions are found in the following group: 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 generally do not alter specific reactivity are known in the art and are described, for example, by H. Neurath and RL Hill, 1979, in *The Proteins*, Academic Press, New York. Common substitutes 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.

[0154] Alternatively, these amino acid alterations can have the following properties: change the physicochemical properties of the peptide. For example, amino acid alterations can improve the thermal stability of the peptide, change its substrate specificity, or change its optimal pH.

[0155] Essential amino acids in peptides can be identified using procedures 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, a single alanine mutation is introduced at each residue in the molecule, and the α-amylase activity of the resulting mutant molecule is tested to identify amino acid residues critical to molecular activity. See also Hilton et al., 1996, J. Biol. Chem. 271:4699-4708. Alternatively, mutations in amino acids at hypothetical contact sites can be combined with physical analysis of the structure, such as by techniques like NMR, crystallography, electron diffraction, or photoaffinity labeling, to determine the enzyme's active site or other biological interactions. See, for example, 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 identity of essential amino acids can also be inferred from comparisons with related peptides.

[0156] In one embodiment, the variant includes modifications in positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0157] In one embodiment, the variant includes substitutions at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0158] In any of the embodiments disclosed herein, stability may be determined as described in Example [Stability], and specific activity may be determined as described in Example [Specific Activity], when using standard A detergent composition or standard X detergent composition.

[0159] The variants according to the invention may consist of 400 to 485 amino acids, such as 410 to 485, 425 to 485, and 440 to 485 amino acids.

[0160] Polynucleotides

[0161] The present invention also relates to polynucleotides encoding variants of the invention. Specifically, the present invention relates to polynucleotides encoding variants comprising changes at one or more of the following positions: H1, H2, D3, G4, T5, G7, T8, I9, M10, Q11, Y12, F13, E14, W15, N16, V17, P18, N19, D20, Q22, H23, W24, N25, R26, L27, H28, N29, N30, A31, Q32, N33, L34, K35, N36, A37, G38, I39, T40, A41, I42, W43, I44, P45, P46, A47, W48, K49, G50, T51, S52, Q 53. N54, D55, V56, G59, A60, Y61, L63, Y64, L66, E68, F69, N70, Q71, K72, G7 3. T74, V75, R76, T77, K78, T81, K82, A83, E84, L85, E86, R87, A88, I89, R90 , S91, L92, K93, N95, G96, I97, Q98, V99, Y100, V103, H107, K108, G109, G11 0. A111, D112, F113, T114, E115, R116, V117, Q118, A119, V120, E121, V122, N123, P124, Q125, N126, R127, N128, Q129, E130, V131, S132, G133, T134, Y 135, Q136, I137, E138, A139, W140, T141, G142, F143, N144, F145, P146, G1 47. G149, N150, Q151, S153, S154, F155, K156, W157, R158, W159, Y160, H16 1. F162, D163, G164, T165, D166, W167, D168, Q169, S170, R171, Q172, L173, A174, N175, R176, I177, Y178, K179, F180, R181, G184, K185, A186, W187, D 188, W189, E190, V191, D192, T193, E194, N195, G196, N197, Y198, D199, Y2 00, M202, Y203, A204, D205, V206, M208, D209, H210, P211, E212, V213, I21 4. N215, L217, N218, R219, W220, G221, V222, W223, Y224, A225, N226, T227,L228, N229, L230, D231, F233, V238, H240, I241, K242, S244, F245, M246, R247, D248, W249, L250, G251, H252, V253, R254, G255, Q256, T257, G258, K259 N260, L261, F262, A263, V264, Y267, W268, K269, N270, D271, L272, G273, A274, L275, E276, N277, Y278, L279, S280, K281, T282, N283, W284, T285, M286 S287, A288, V291, P292, L293, H294, Y295, N296, L297, Y298, Q299, A300, S301, N302, S303, S304, G305, N306, Y307, M309, R310, N311, L312, L313, N314 G315, T316, V318, Q319, R320, H321, P322, S323, A325, V326, T327, F328, V329, N331, T334, P336, G337, E338, E341, S342, V344, Q345, G346, W347, F348 K349, P350, L351, A352, A354, T355, L357, T358, E360, Q361, G362, Y363, Q365, V366, F367, Y368, G369, D370, Y372, G373, I374, P375, S376, D377, V379 P380, S381, Y382, R383, Q384, I386, D387, P388, L389, L390, K391, A392, Q394, Q395, Y396, A397, Y398, R400, H402, D403, Y404, F405, D406, H407, W408 D409、V410、I411、T414、R415、E416、N418、A419、S420、H421、P422、S424、G4 25、L426、A427、T428、I429、M430、S431、D432、G435、G436、S437、K438、W439、 M440、Y441、V442、R444、Q445、K446、A447、G448、V450、W451、H452、D453、M4 54、T455、G456、N457、S459、G460、T463、I464、N465、Q466、D467、W469、H471、F473, N475, G476, G477, V481, and W482, where these positions correspond to the amino acid positions in the amino acid sequence described in SEQ ID NO:3.

[0162] As used herein, the term "encoding polynucleotide" refers to a polynucleotide that encodes a mature polypeptide having α-amylase activity. In one respect, the polypeptide-encoding sequence is the nucleotide sequence set forth in SEQ ID NO:1.

[0163] In one embodiment, the polynucleotide encoding the variant according to the invention has at least 80%, for example at least 85%, for example at least 90%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, but less than 100% sequence similarity to the polynucleotide of SEQ ID NO:1.

[0164] Nucleic acid constructs

[0165] The present invention also relates to nucleic acid constructs comprising a polynucleotide operably linked to one or more control sequences encoding a variant of the invention, the one or more control sequences guiding the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences. Therefore, specifically, the present invention relates to nucleic acid constructs comprising a polypeptide encoding a variant comprising a polypeptide corresponding to SEQ ID [SEQ ID]. The positions of the amino acid sequences specified in NO:3 are: H1, H2, D3, G4, T5, G7, T8, I9, M10, Q11, Y12, F13, E14, W15, N16, V17, P18, N19, D20, Q22, H23, W24, N25, R26, L27, H28, N29, N30, A31, Q32, N33, L34, K35, N36, A37, G38, I39, T40, A41, I42, W43, I44, P45, P46, A47, W48, K49, G50, T51, S52, Q53, N54. , D55, V56, G59, A60, Y61, L63, Y64, L66, E68, F69, N70, Q71, K72, G73, T74, V75, R76, T77, K78, T81, K82, A83, E84, L85, E86, R87, A88 , I89, R90, S91, L92, K93, N95, G96, I97, Q98, V99, Y100, V103, H107, K108, G109, G110, A111, D112, F113, T114, E115, R116, V117, Q1 18. A119, V120, E121, V122, N123, P124, Q125, N126, R127, N128, Q129, E130, V131, S132, G133, T134, Y135, Q136, I137, E138, A139, W140, T141, G142, F143, N144, F145, P146, G147, G149, N150, Q151, S153, S154, F155, K156, W157, R158, W159, Y160, H161, F162, D16 3. G164, T165, D166, W167, D168, Q169, S170, R171, Q172, L173, A174, N175, R176, I177, Y178, K179, F180, R181, G184, K185, A186, W 187, D188, W189, E190, V191, D192, T193, E194, N195, G196, N197, Y198, D199, Y200, M202, Y203, A204, D205, V206, M208, D209, H210,P211, E212, V213, I214, N215, L217, N218, R219, W220, G221, V222, W223, Y224, A225, N226, T227, L228, N229, L230, D231, F233, V238, H240, I241, K242 S244, F245, M246, R247, D248, W249, L250, G251, H252, V253, R254, G255, Q256, T257, G258, K259, N260, L261, F262, A263, V264, Y267, W268, K269, N270 D271, L272, G273, A274, L275, E276, N277, Y278, L279, S280, K281, T282, N283, W284, T285, M286, S287, A288, V291, P292, L293, H294, Y295, N296, L297 Y298, Q299, A300, S301, N302, S303, S304, G305, N306, Y307, M309, R310, N311, L312, L313, N314, G315, T316, V318, Q319, R320, H321, P322, S323, A325 V326, T327, F328, V329, N331, T334, P336, G337, E338, E341, S342, V344, Q345, G346, W347, F348, K349, P350, L351, A352, A354, T355, L357, T358, E360 Q361, G362, Y363, Q365, V366, F367, Y368, G369, D370, Y372, G373, I374, P375, S376, D377, V379, P380, S381, Y382, R383, Q384, I386, D387, P388, L389 L390、K391、A392、Q394、Q395、Y396、A397、Y398、R400、H402、D403、Y404、F4 05、D406、H407、W408、D409、V410、I411、T414、R415、E416、N418、A419、S420、 H421、P422、S424、G425、L426、A427、T428、I429、M430、S431、D432、G435、G4 36、S437、K438、W439、M440、Y441、V442、R444、Q445、K446、A447、G448、V450、Changes in one or more positions of W451, H452, D453, M454, T455, G456, N457, S459, G460, T463, I464, N465, Q466, D467, W469, H471, F473, N475, G476, G477, V481, and W482, wherein the polypeptide is operatively linked to one or more control sequences.

[0166] As used herein, the term "nucleic acid construct" refers to a single-stranded or double-stranded nucleic acid molecule isolated from a naturally occurring gene, modified in a manner not normally found in nature to contain segments of nucleic acid, or synthesized, comprising one or more control sequences. Nucleic acids include DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding polypeptides. Nucleic acids can be single-stranded or double-stranded and can be chemically modified. The terms "nucleic acid" and "polynucleotide" are used interchangeably but serve the same meaning and purpose. Because the genetic code is degenerate, more than one codon can be used to encode a specific amino acid, and the compositions and methods of the present invention comprise nucleotide sequences encoding specific amino acid sequences. Unless the context otherwise indicates, nucleic acids are written from left to right in a 5' to 3' direction; amino acid sequences are written from left to right in an amino-to-carboxyl direction.

[0167] As used herein, the term "operably linked" refers to a configuration in which a control sequence is positioned relative to the coding sequence of a polynucleotide so that the control sequence directs the expression of the coding sequence.

[0168] The polynucleotide can be manipulated in a variety of ways to provide expression of the variant. Depending on the expression vector, manipulation of the polynucleotide prior to insertion into the vector may be desirable or necessary. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

[0169] The control sequence can be a promoter, which is a polynucleotide recognized by the host cell for the expression of that polynucleotide. The promoter includes a transcriptional control sequence that mediates the expression of that variant. The promoter can be any polynucleotide that exhibits transcriptional activity in the host cell, including mutant, truncated, and heterozygous promoters, and can be derived from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to that of the host cell.

[0170] As used herein, the term "promoter" refers to a regulatory sequence involved in binding to RNA polymerase to initiate gene transcription, and can be an inducible or constitutive promoter. Those skilled in the art will recognize possible promoters suitable for the specific nucleic acid construct used.

[0171] expression carrier

[0172] The present invention also relates to recombinant expression vectors comprising a polynucleotide encoding a variant of the invention, a promoter, and transcription and translation termination signals. Therefore, the present invention relates to expression vectors (optionally recombinant) comprising: encoding a polynucleotide corresponding to SEQ ID [SEQ ID]. The positions of the amino acid sequences specified in NO:3 are: H1, H2, D3, G4, T5, G7, T8, I9, M10, Q11, Y12, F13, E14, W15, N16, V17, P18, N19, D20, Q22, H23, W24, N25, R26, L27, H28, N29, N30, A31, Q32, N33, L34, K35, N36, A37, G38, I39, T40, A41, I42, W43, I44, P45, P46, A47, W48, K49, G50, T51, S52, Q53, N54, D55, V56, G59. A60, Y61, L63, Y64, L66, E68, F69, N70, Q71, K72, G73, T74, V75, R76, T77, K78, T81, K82, A83, E84, L85, E86, R87, A88, I89, R90, S91, L92, K9 3. N95, G96, I97, Q98, V99, Y100, V103, H107, K108, G109, G110, A111, D112, F113, T114, E115, R116, V117, Q118, A119, V120, E121, V122, N12 3. P124, Q125, N126, R127, N128, Q129, E130, V131, S132, G133, T134, Y135, Q136, I137, E138, A139, W140, T141, G142, F143, N144, F145, P1 46. ​​G147, G149, N150, Q151, S153, S154, F155, K156, W157, R158, W159, Y160, H161, F162, D163, G164, T165, D166, W167, D168, Q169, S170, R 171, Q172, L173, A174, N175, R176, I177, Y178, K179, F180, R181, G184, K185, A186, W187, D188, W189, E190, V191, D192, T193, E194, N195, G196, N197, Y198, D199, Y200, M202, Y203, A204, D205, V206, M208, D209, H210, P211, E212, V213, I214, N215, L217, N218, R219, W220, G221,V222, W223, Y224, A225, N226, T227, L228, N229, L230, D231, F233, V238, H240, I241, K242, S244, F245, M246, R247, D248, W249, L250, G251, H252, V253 R254, G255, Q256, T257, G258, K259, N260, L261, F262, A263, V264, Y267, W268, K269, N270, D271, L272, G273, A274, L275, E276, N277, Y278, L279, S280 K281, T282, N283, W284, T285, M286, S287, A288, V291, P292, L293, H294, Y295, N296, L297, Y298, Q299, A300, S301, N302, S303, S304, G305, N306, Y307 M309, R310, N311, L312, L313, N314, G315, T316, V318, Q319, R320, H321, P322, S323, A325, V326, T327, F328, V329, N331, T334, P336, G337, E338, E341 S342, V344, Q345, G346, W347, F348, K349, P350, L351, A352, A354, T355, L357, T358, E360, Q361, G362, Y363, Q365, V366, F367, Y368, G369, D370, Y372 G373, I374, P375, S376, D377, V379, P380, S381, Y382, R383, Q384, I386, D387, P388, L389, L390, K391, A392, Q394, Q395, Y396, A397, Y398, R400, H402 D403、Y404、F405、D406、H407、W408、D409、V410、I411、T414、R415、E416、N4 18、A419、S420、H421、P422、S424、G425、L426、A427、T428、I429、M430、S431、 D432、G435、G436、S437、K438、W439、M440、Y441、V442、R444、Q445、K446、A4 47、G448、V450、W451、H452、D453、M454、T455、G456、N457、S459、G460、T463、A variant polynucleotide with alterations at one or more positions in I464, N465, Q466, D467, W469, H471, F473, N475, G476, G477, V481, and W482, serving as a promoter and a signal for stopping transcription and translation.

[0173] As used herein, the term "expression vector" refers to a linear or circular DNA molecule that comprises polynucleotides encoding a variant and is operatively linked to a control sequence that provides for its expression.

[0174] Various nucleotides and control sequences can be linked together to produce a recombinant expression vector, which may include one or more suitable restriction sites to allow the insertion or substitution of a polynucleotide encoding the variant at such sites. Alternatively, the polynucleotide can be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into a suitable vector for expression. In generating the expression vector, the coding sequence is located within the vector, thus enabling operative linking of the coding sequence to a suitable control sequence for expression.

[0175] The recombinant expression vector can be any vector (e.g., plasmid or virus) that can readily undergo recombinant DNA procedures and induce polynucleotide expression. The choice of vector will typically depend on its compatibility with the host cell to which it will be introduced. The vector can be a linear or closed circular plasmid.

[0176] The vector can be a self-replicating vector, that is, a vector existing as an extrachromosomal entity whose replication is independent of chromosome replication, such as a plasmid, extrachromosomal element, microchromosome, or artificial chromosome. The vector can contain any means to ensure self-replication. Alternatively, the vector can be one that, when introduced into the host cell, is integrated into the genome and replicates along with one or more chromosomes in which it has been integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids (which together contain the total DNA of the genome to be introduced into the host cell), or transposons can be used.

[0177] The vector preferably contains one or more selective markers that allow for convenient selection of cells such as transformed cells, transfected cells, and transduced cells. A selective marker is a gene whose product provides resistance to biocides or viruses, heavy metal resistance, or auxotrophic prototrophs, etc.

[0178] Those skilled in the art will know which expression vector is most suitable for a specific expression system. Therefore, the present invention is not limited to any specific expression vector, but any expression vector including a polynucleotide encoding a variant according to the present invention is considered part of the present invention.

[0179] host cells

[0180] The present invention also relates to recombinant host cells comprising a polynucleotide operably linked to one or more control sequences encoding a variant of the invention, the one or more control sequences directing the generation of a variant of the invention. Therefore, the present invention relates to host cells (optionally recombinant) comprising: encoding a polynucleotide corresponding to SEQ ID NO. The amino acid sequences specified in IDNO:3 are: H1, H2, D3, G4, T5, G7, T8, I9, M10, Q11, Y12, F13, E14, W15, N16, V17, P18, N19, D20, Q22, H23, W24, N25, R26, L27, H28, N29, N30, A31, Q32, N33, L34, K35, N36, A37, G38, I39, T40, A41, I42, W43, I44, P45, P46, A47, W48, K49, G50, T51, S52, Q53, N54, D5 5. V56, G59, A60, Y61, L63, Y64, L66, E68, F69, N70, Q71, K72, G73, T74, V75, R76, T77, K78, T81, K82, A83, E84, L85, E86, R87, A88, I89, R90, S91, L92, K93, N95, G96, I97, Q98, V99, Y100, V103, H107, K108, G109, G110, A111, D112, F113, T114, E115, R116, V117, Q118, A119 , V120, E121, V122, N123, P124, Q125, N126, R127, N128, Q129, E130, V131, S132, G133, T134, Y135, Q136, I137, E138, A139, W140, T141 , G142, F143, N144, F145, P146, G147, G149, N150, Q151, S153, S154, F155, K156, W157, R158, W159, Y160, H161, F162, D163, G164, T165 , D166, W167, D168, Q169, S170, R171, Q172, L173, A174, N175, R176, I177, Y178, K179, F180, R181, G184, K185, A186, W187, D188, W189 , E190, V191, D192, T193, E194, N195, G196, N197, Y198, D199, Y200, M202, Y203, A204, D205, V206, M208, D209, H210, P211, E212, V213,I214, N215, L217, N218, R219, W220, G221, V222, W223, Y224, A225, N226, T227, L228, N229, L230, D231, F233, V238, H240, I241, K242, S244, F245, M246 R247, D248, W249, L250, G251, H252, V253, R254, G255, Q256, T257, G258, K259, N260, L261, F262, A263, V264, Y267, W268, K269, N270, D271, L272, G273 A274, L275, E276, N277, Y278, L279, S280, K281, T282, N283, W284, T285, M286, S287, A288, V291, P292, L293, H294, Y295, N296, L297, Y298, Q299, A300 S301, N302, S303, S304, G305, N306, Y307, M309, R310, N311, L312, L313, N314, G315, T316, V318, Q319, R320, H321, P322, S323, A325, V326, T327, F328 V329, N331, T334, P336, G337, E338, E341, S342, V344, Q345, G346, W347, F348, K349, P350, L351, A352, A354, T355, L357, T358, E360, Q361, G362, Y363 Q365, V366, F367, Y368, G369, D370, Y372, G373, I374, P375, S376, D377, V379, P380, S381, Y382, R383, Q384, I386, D387, P388, L389, L390, K391, A392 Q394、Q395、Y396、A397、Y398、R400、H402、D403、Y404、F405、D406、H407、W4 08、D409、V410、I411、T414、R415、E416、N418、A419、S420、H421、P422、S424、 G425、L426、A427、T428、I429、M430、S431、D432、G435、G436、S437、K438、W4 39、M440、Y441、V442、R444、Q445、K446、A447、G448、V450、W451、H452、D453、A variant polynucleotide with alterations at one or more positions of M454, T455, G456, N457, S459, G460, T463, I464, N465, Q466, D467, W469, H471, F473, N475, G476, G477, V481, and W482, operably linked to one or more control sequences that direct the generation of the variant.

[0181] As used herein, the term "host cell" refers to any cell type that is susceptible to transformation, transfection, transduction, etc., using nucleic acid constructs or expression vectors comprising the polynucleotides of the present invention. The term "host cell" also includes any offspring of a parent host cell that is inconsistent with the parent host cell due to mutations occurring during replication.

[0182] A construct or vector comprising multiple nucleotides is introduced into a host cell, thereby maintaining the construct or vector as a chromosomal integrase or as an autonomously replicating extrachromosomal vector, as previously described. The choice of host cell will depend to a large extent on the gene encoding the variant and its origin.

[0183] The host cell can be any cell that is useful in the recombinant-generated variants, such as prokaryotic or eukaryotic cells.

[0184] Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to: Bacillus, Clostridium, Enterococcus, Bacillus aeruginosa, Lactobacillus, Lactococcus, Marine Bacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to: Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Coliform, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0185] According to the method of the present invention

[0186] The present invention also relates to methods for generating variants, the methods comprising: (a) culturing host cells of the present invention under conditions suitable for expressing the variant; and (b) recovering the variant. Therefore, the present invention relates to methods for generating variants comprising, respectively, those corresponding to SEQ ID [SEQ ID]. The amino acid sequences specified in NO:3 are: H1, H2, D3, G4, T5, G7, T8, I9, M10, Q11, Y12, F13, E14, W15, N16, V17, P18, N19, D20, Q22, H23, W24, N25, R26, L27, H28, N29, N30, A31, Q32, N33, L34, K35, N36, A37, G38, I39, T40, A41, I42, W43, I44, P45, P46, A47, W48, K49, G50, T51, S52, Q53, N54, D55, V56, G59 , A60, Y61, L63, Y64, L66, E68, F69, N70, Q71, K72, G73, T74, V75, R76, T77, K78, T81, K82, A83, E84, L85, E86, R87, A88, I89, R90, S91, L92, K93, N95, G96, I97, Q98, V99, Y100, V103, H107, K108, G109, G110, A111, D112, F113, T114, E115, R116, V117, Q118, A119, V120, E121, V122, N123, P124, Q125, N126, R127, N128, Q129, E130, V131, S132, G133, T134, Y135, Q136, I137, E138, A139, W140, T141, G142, F143, N144, F14 5. P146, G147, G149, N150, Q151, S153, S154, F155, K156, W157, R158, W159, Y160, H161, F162, D163, G164, T165, D166, W167, D168, Q169, S1 70. R171, Q172, L173, A174, N175, R176, I177, Y178, K179, F180, R181, G184, K185, A186, W187, D188, W189, E190, V191, D192, T193, E194, N 195, G196, N197, Y198, D199, Y200, M202, Y203, A204, D205, V206, M208, D209, H210, P211, E212, V213, I214, N215, L217, N218, R219, W220,G221, V222, W223, Y224, A225, N226, T227, L228, N229, L230, D231, F233, V238, H240, I241, K242, S244, F245, M246, R247, D248, W249, L250, G251, H252 V253, R254, G255, Q256, T257, G258, K259, N260, L261, F262, A263, V264, Y267, W268, K269, N270, D271, L272, G273, A274, L275, E276, N277, Y278, L279 S280, K281, T282, N283, W284, T285, M286, S287, A288, V291, P292, L293, H294, Y295, N296, L297, Y298, Q299, A300, S301, N302, S303, S304, G305, N306 Y307, M309, R310, N311, L312, L313, N314, G315, T316, V318, Q319, R320, H321, P322, S323, A325, V326, T327, F328, V329, N331, T334, P336, G337, E338 E341, S342, V344, Q345, G346, W347, F348, K349, P350, L351, A352, A354, T355, L357, T358, E360, Q361, G362, Y363, Q365, V366, F367, Y368, G369, D370 Y372, G373, I374, P375, S376, D377, V379, P380, S381, Y382, R383, Q384, I386, D387, P388, L389, L390, K391, A392, Q394, Q395, Y396, A397, Y398, R400 H402、D403、Y404、F405、D406、H407、W408、D409、V410、I411、T414、R415、E4 16、N418、A419、S420、H421、P422、S424、G425、L426、A427、T428、I429、M430、 S431、D432、G435、G436、S437、K438、W439、M440、Y441、V442、R444、Q445、K4 46、A447、G448、V450、W451、H452、D453、M454、T455、G456、N457、S459、G460、A change in one or more positions of T463, I464, N465, Q466, D467, W469, H471, F473, N475, G476, G477, V481, and W482, wherein the method comprises the steps of: a) culturing host cells according to the invention under conditions suitable for expressing the variant; and b) recovering the variant.

[0187] These host cells are cultured in a nutrient medium suitable for producing the variant using methods known in the art. For example, the cells can be cultured by shake flask culture or by small-scale or large-scale fermentation (including continuous fermentation, batch fermentation, feed-feed fermentation, or solid-state fermentation) in a suitable medium and under conditions that allow for the expression and / or isolation of the variant in a laboratory or industrial fermenter. The culture occurs using procedures known in the art in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts. Suitable media are available from commercial suppliers or can be prepared according to publicly available compositions (e.g., in the catalogue of the U.S. Center for 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.

[0188] This variant can be detected using methods known in the art that are specific to peptides with α-amylase activity. These detection methods include, but are not limited to, the use of specific antibodies, the formation of enzyme products, or the disappearance of enzyme substrates. For example, enzyme assays can be used to determine the activity of this variant.

[0189] The variant can be recovered using methods known in the art. For example, the variant can be recovered from the nutrient medium through a variety of routine procedures, including but not limited to: collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

[0190] Variants can be purified to obtain substantially pure variants using a variety of procedures known in the art, including but not limited to: chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatographic focusing, and size exclusion chromatography), electrophoresis procedures (e.g., preparative isoelectric point focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, for example, Protein Purification, Janson and Ryden, editors, VCH Publishers, New York, 1989).

[0191] Alternatively, instead of recycling the variant, the host cell of the present invention expressing the variant is used as the source of the variant.

[0192] In a specific embodiment, the method for generating variants includes the following steps: a) introducing deletions and / or substitutions at two or more positions corresponding to positions 181, 182, 183, and 184 of the amino acid sequence as defined in SEQ ID NO:3; and b) introducing substitutions at one or more positions selected from the group consisting of: H1, H2, D3, G4, T5, G7, T8, I9, M10, Q11, Y12, F13, E14, W15, N16, V17, P18, N19, D20, Q22, H23, W24, N25, R26, L27, H28, N29, N30, A31, Q32, N33, L34, K35, N36, A37, G38, I39, T40, A41, I42, W43. I44, P45, P46, A47, W48, K49, G50, T51, S52, Q53, N54, D55, V56, G59, A60, Y61, L63, Y64, L66, E68, F69, N70, Q71, K72, G73, T74, V75, R76, T77, K78, T81, K82, A83, E84, L85, E86, R87, A88, I89, R90, S91, L92, K93, N95, G96, I97, Q98, V99, Y100, V103, H107, K108, G109, G110, A111, D112, F113, T114 , E115, R116, V117, Q118, A119, V120, E121, V122, N123, P124, Q125, N126 , R127, N128, Q129, E130, V131, S132, G133, T134, Y135, Q136, I137, E138 , A139, W140, T141, G142, F143, N144, F145, P146, G147, G149, N150, Q151 , S153, S154, F155, K156, W157, R158, W159, Y160, H161, F162, D163, G164 , T165, D166, W167, D168, Q169, S170, R171, Q172, L173, A174, N175, R176 , I177, Y178, K179, F180, R181, G184, K185, A186, W187, D188, W189, E190 , V191, D192, T193, E194, N195, G196, N197, Y198, D199, Y200, M202, Y203 ,A204,D205,V206,M208,D209,H210,P211,E212,V213,I214,N215,L217,N218, R219, W220, G221, V222, W223, Y224, A225, N226, T227, L228, N229, L230, D231, F233, V238, H240, I241, K242, S244, F245, M246, R247, D248, W249 L250, G251, H252, V253, R254, G255, Q256, T257, G258, K259, N260, L261, F262, A263, V264, Y267, W268, K269, N270, D271, L272, G273, A274, L275, E276 N277, Y278, L279, S280, K281, T282, N283, W284, T285, M286, S287, A288, V291, P292, L293, H294, Y295, N296, L297, Y298, Q299, A300, S301, N302, S303 S304, G305, N306, Y307, M309, R310, N311, L312, L313, N314, G315, T316, V318, Q319, R320, H321, P322, S323, A325, V326, T327, F328, V329, N331, T334 P336, G337, E338, E341, S342, V344, Q345, G346, W347, F348, K349, P350, L351, A352, A354, T355, L357, T358, E360, Q361, G362, Y363, Q365, V366, F367 Y368, G369, D370, Y372, G373, I374, P375, S376, D377, V379, P380, S381, Y382, R383, Q384, I386, D387, P388, L389, L390, K391, A392, Q394, Q395, Y396 A397、Y398、R400、H402、D403、Y404、F405、D406、H407、W408、D409、V410、I4 11、T414、R415、E416、N418、A419、S420、H421、P422、S424、G425、L426、A427、 T428、I429、M430、S431、D432、G435、G436、S437、K438、W439、M440、Y441、V4 42、R444、Q445、K446、A447、G448、V450、W451、H452、D453、M454、T455、G456、N457, S459, G460, T463, I464, N465, Q466, D467, W469, H471, F473, N475, G476, G477, V481, and W482; where these positions correspond to the amino acid positions in the amino acid sequence described in SEQ ID NO:3.

[0193] Furthermore, the present invention relates to a method for obtaining a variant, the method comprising introducing the following into a parental α-amylase having at least 80% sequence identity with the polypeptide of SEQ ID NO:4:

[0194] (a) Substitution and / or deletion at two or more positions in the parental α-amylase, said positions corresponding to positions 181, 182, 183, and 184 of the mature polypeptide of SEQ ID NO:3; and

[0195] (b) Substitution at one or more positions selected from the group consisting of the following items: H1, H2, D3, G4, T5, G7, T8, I9, M10, Q11, Y12, F13, E14, W15, N16, V17, P18, N19, D20, Q22, H23, W24, N25, R26, L27, H28, N29, N30, A31, Q32, N33, L34, K35, N36, A37, G38, I39, T40, A41, I42, W43, I44, P45, P46, A47, W48, K49, G50, T51, S52, Q53, N54, D55, V56, G59, A60, Y6 1. L63, Y64, L66, E68, F69, N70, Q71, K72, G73, T74, V75, R76, T77, K78, T81, K82, A83, E84, L85, E86, R87, A88, I89, R90, S91, L92, K93, N95, G96, I97, Q98 , V99, Y100, V103, H107, K108, G109, G110, A111, D112, F113, T114, E115, R1 16. V117, Q118, A119, V120, E121, V122, N123, P124, Q125, N126, R127, N128, Q129, E130, V131, S132, G133, T134, Y135, Q136, I137, E138, A139, W140, T1 41. G142, F143, N144, F145, P146, G147, G149, N150, Q151, S153, S154, F155, K156, W157, R158, W159, Y160, H161, F162, D163, G164, T165, D166, W167, D1 68. Q169, S170, R171, Q172, L173, A174, N175, R176, I177, Y178, K179, F180, R181, G184, K185, A186, W187, D188, W189, E190, V191, D192, T193, E194, N1 95, G196, N197, Y198, D199, Y200, M202, Y203, A204, D205, V206, M208, D209, H210, P211, E212, V213, I214, N215, L217, N218, R219, W220, G221, V222, W2 23. Y224, A225, N226, T227, L228, N229, L230, D231, F233, V238, H240, I241,K242, S244, F245, M246, R247, D248, W249, L250, G251, H252, V253, R254, G255, Q256, T257, G258, K259, N260, L261, F262, A263, V264, Y267, W268, K26 9, N270, D271, L272, G273, A274, L275, E276, N277, Y278, L279, S280, K281, T282, N283, W284, T285, M286, S287, A288, V291, P292, L293, H294, Y295, N 296, L297, Y298, Q299, A300, S301, N302, S303, S304, G305, N306, Y307, M309, R310, N311, L312, L313, N314, G315, T316, V318, Q319, R320, H321, P322 S323, A325, V326, T327, F328, V329, N331, T334, P336, G337, E338, E341, S342, V344, Q345, G346, W347, F348, K349, P350, L351, A352, A354, T355, L3 57, T358, E360, Q361, G362, Y363, Q365, V366, F367, Y368, G369, D370, Y372, G373, I374, P375, S376, D377, V379, P380, S381, Y382, R383, Q384, I386 D387, P388, L389, L390, K391, A392, Q394, Q395, Y396, A397, Y398, R400, H402, D403, Y404, F405, D406, H407, W408, D409, V410, I411, T414, R415, E41 6、N418、A419、S420、H421、P422、S424、G425、L426、A427、T428、I429、M430 、S431、D432、G435、G436、S437、K438、W439、M440、Y441、V442、R444、Q445、K 446、A447、G448、V450、W451、H452、D453、M454、T455、G456、N457、S459、G46 0、T463、I464、N465、Q466、D467、W469、H471、F473、N475、G476、G477、V481、And W482; where these positions correspond to the amino acid positions in the amino acid sequence described in SEQ ID NO:3,

[0196] The resulting variant has at least 80%, for example at least 85%, for example at least 90%, for example at least 95%, for example at least 97%, but less than 100% sequence identity with the mature polypeptide of SEQ ID NO:3 or 4, and the variant has α-amylase activity;

[0197] (c) Recycle the variant.

[0198] These variants can be prepared using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc.

[0199] Site-directed mutagenesis is a technique that introduces one or more (e.g., several) mutations at one or more designated sites in a polynucleotide encoding the parent.

[0200] Site-directed mutagenesis can be achieved in vitro via PCR involving the use of primers containing oligonucleotides with the desired mutation. Site-directed mutagenesis can also be performed in vitro via cassette mutagenesis, which involves cleavage by a restriction enzyme at a site in a plasmid containing a polynucleotide encoding the parent polypeptide, followed by the ligation of the oligonucleotide containing the mutation into the polynucleotide. Typically, the restriction enzyme used to digest the plasmid is the same as that used to digest the oligonucleotide, thus 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.

[0201] Site-directed mutagenesis can also be achieved in vivo using methods known in the art. See, for example, 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.

[0202] Any site-directed mutagenesis method can be used in this invention. Many commercially available kits exist for preparing variants. Those skilled in the art are well aware of such commercial kits and how to use them.

[0203] Synthetic gene construction requires the in vitro synthesis of designed polynucleotide molecules to encode polypeptides of interest. Gene synthesis can be performed using a variety of techniques, such as the multi-channel microchip-based technique described by Tian et al. (2004, Nature 432:1050-1054), and similar techniques in which oligonucleotides are synthesized and assembled on optically programmable microfluidic chips.

[0204] Single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested using known methods of mutagenesis, recombination, and / or truncation, followed by relevant screening procedures, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86:2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:10832-10837; US Patent 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).

[0205] Mutagenesis / reorganization methods can be combined with high-throughput, automated screening methods to detect the activity of cloned, mutagenesis-encoded peptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17:893-896). The mutagenesis-encoded DNA molecules encoding the active peptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow for the rapid determination of the importance of individual amino acid residues within the peptide.

[0206] Semi-synthetic gene construction is achieved through a combination of various methods, including synthetic gene construction, and / or site-directed mutagenesis, and / or random mutagenesis, and / or shuffling. Semi-synthetic construction typically utilizes the process of synthesizing polynucleotide fragments combined with PCR technology. Therefore, defined regions of the gene can be resynthesized, while other regions can be amplified using site-specific mutagenesis primers, and still others can be amplified using error-prone or non-error-prone PCR. The polynucleotide subsequence can then be shuffled.

[0207] The present invention also relates to a method for improving the performance (e.g., washing performance) of a parental α-amylase having an amino acid sequence having at least 65% sequence identity with SEQ ID NO:3 or 4, said method comprising the steps of: a) in SEQ ID NO:3 or 4, or an amino acid sequence having at least 65% sequence identity with it. Paired deletions and / or substitutions are introduced at two or more positions 181, 182, 183, and 184 of the amino acid sequence specified in NO:3; and b) substitutions are introduced at one or more positions selected from the group consisting of: H1, H2, D3, G4, T5, G7, T8, I9, M10, Q11, Y12, F13, E14, W15, N16, V17, P18, N19, D20, Q22, H23, W24, N25, R26, L27, H28, N29, N30, A31, Q32, N33, L34, K35, N36, A37, G38, I39, T 40. A41, I42, W43, I44, P45, P46, A47, W48, K49, G50, T51, S52, Q53, N54, D55, V56, G59, A60, Y61, L63, Y64, L66, E68, F69, N70, Q71, K72, G7 3. T74, V75, R76, T77, K78, T81, K82, A83, E84, L85, E86, R87, A88, I89, R90, S91, L92, K93, N95, G96, I97, Q98, V99, Y100, V103, H107, K108, G109, G110, A111, D112, F113, T114, E115, R116, V117, Q118, A119, V120, E121, V122, N123, P124, Q125, N126, R127, N128, Q129, E130, V13 1. S132, G133, T134, Y135, Q136, I137, E138, A139, W140, T141, G142, F143, N144, F145, P146, G147, G149, N150, Q151, S153, S154, F155, K1 56. W157, R158, W159, Y160, H161, F162, D163, G164, T165, D166, W167, D168, Q169, S170, R171, Q172, L173, A174, N175, R176, I177, Y178, K 179, F180, R181, G184, K185, A186, W187, D188, W189, E190, V191, D192, T193, E194, N195, G196, N197, Y198, D199, Y200, M202, Y203, A204,D205, V206, M208, D209, H210, P211, E212, V213, I214, N215, L217, N218, R219, W220, G221, V222, W223, Y224, A225, N226, T227, L228, N229, L230, D231 F233, V238, H240, I241, K242, S244, F245, M246, R247, D248, W249, L250, G251, H252, V253, R254, G255, Q256, T257, G258, K259, N260, L261, F262, A263 V264, Y267, W268, K269, N270, D271, L272, G273, A274, L275, E276, N277, Y278, L279, S280, K281, T282, N283, W284, T285, M286, S287, A288, V291, P292 L293, H294, Y295, N296, L297, Y298, Q299, A300, S301, N302, S303, S304, G305, N306, Y307, M309, R310, N311, L312, L313, N314, G315, T316, V318, Q319 R320, H321, P322, S323, A325, V326, T327, F328, V329, N331, T334, P336, G337, E338, E341, S342, V344, Q345, G346, W347, F348, K349, P350, L351, A352 A354, T355, L357, T358, E360, Q361, G362, Y363, Q365, V366, F367, Y368, G369, D370, Y372, G373, I374, P375, S376, D377, V379, P380, S381, Y382, R383 Q384, I386, D387, P388, L389, L390, K391, A392, Q394, Q395, Y396, A397, Y398, R400, H402, D403, Y404, F405, D406, H407, W408, D409, V410, I411, T414 R415、E416、N418、A419、S420、H421、P422、S424、G425、L426、A427、T428、I4 29、M430、S431、D432、G435、G436、S437、K438、W439、M440、Y441、V442、R444、Q445, K446, A447, G448, V450, W451, H452, D453, M454, T455, G456, N457, S459, G460, T463, I464, N465, Q466, D467, W469, H471, F473, N475, G476, G477, V481, and W482; wherein these positions correspond to the amino acid positions in the amino acid sequence specified in SEQ ID NO:3; the method thereby provides an α-amylase variant of the parental α-amylase, wherein the variant corresponds to the amino acid positions as specified in SEQ ID NO:3. The amino acid sequence as defined in NO:3 or 4 has at least 65%, for example, at least 70%, for example, at least 75%, for example, at least 80%, for example, at least 85%, for example, at least 90%, for example, at least 95%, for example, at least 97%, for example, at least 99%, but less than 100% sequence identity, and said variant has α-amylase activity and improved performance compared to said parental α-amylase.

[0208] The variant has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, but less than 100% sequence identity with the amino acid sequence described in SEQ ID NO:3 or 4, and the variant has α-amylase activity and improved performance compared with the parental α-amylase.

[0209] In one embodiment, the variant has at least 50%, for example at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100% activity of a parent polypeptide having the amino acid sequence of SEQ ID NO:3.

[0210] In one embodiment, its activity is determined according to Phadebas assay.

[0211] α-Amylase activity can be determined using Phadebas substrates (e.g., from McGill Life Sciences, Lund, Sweden). Phadebas tablets comprise interconnected starch polymers in the form of water-insoluble spherical microspheres. A blue dye is covalently bound to these microspheres. The interconnected starch polymers within the microspheres degrade at a rate proportional to α-amylase activity. As α-amylase degrades the starch polymers, the released blue dye is water-soluble, and the dye concentration can be determined by measuring absorbance at 620 nm. The concentration of blue dye is proportional to the α-amylase activity in the sample.

[0212] Dilute the variant sample to be analyzed in an activity buffer at the desired pH. Suspend both substrate tablets in 5 mL of activity buffer and mix on a magnetic stirrer. While mixing the substrate, transfer 150 μL to a microtiter plate (MTP) or PCR-MTP. Add 30 μL of diluted amylase sample to 150 μL of substrate and mix. Incubate at 37 °C for 15 min. Stop the reaction by adding 30 μL of 1M NaOH and mixing. Centrifuge the MTP at 4000 x g for 5 min. Transfer 100 μL to a fresh MTP and measure the absorbance at 620 nm.

[0213] The α-amylase sample should be diluted such that the absorbance at 620 nm is between 0 and 2.2, and within the linear range of the activity assay.

[0214] Therefore, in one embodiment, activity is determined by the following method, which includes the following steps:

[0215] a) Incubate the α-amylase variant according to the invention at 37°C for 15 minutes with stained amylose substrate; and

[0216] b) Measure absorbance at OD 620nm.

[0217] In another embodiment, activity is determined by the following method, which includes the following steps:

[0218] a) Incubate the α-amylase variant according to the invention at 37°C for 15 minutes with stained amylose substrate; and

[0219] b) Centrifuge the sample;

[0220] c) Transfer the supernatant to the reader plate, and

[0221] Absorbance was measured at OD 620nm.

[0222] On the other hand, the present invention relates to a method for improving the stability (e.g., stability in detergent compositions) and thermal stability of a parental α-amylase having an amino acid sequence having at least 65% sequence identity with SEQ ID NO:3 or 4, said method comprising the steps of: a) in SEQ ID NO:3 or 4, or having an amino acid sequence having at least 65% sequence identity with it. Paired deletions and / or substitutions are introduced at two or more positions 181, 182, 183, and 184 of the amino acid sequence specified in NO:3; and b) substitutions are introduced at one or more positions selected from the group consisting of: H1, H2, D3, G4, T5, G7, T8, I9, M10, Q11, Y12, F13, E14, W15, N16, V17, P18, N19, D20, Q22, H23, W24, N25, R26, L27, H28, N29, N30, A31, Q32, N33, L34, K35, N36, A37, G38. I39, T40, A41, I42, W43, I44, P45, P46, A47, W48, K49, G50, T51, S52, Q53, N54, D55, V56, G59, A60, Y61, L63, Y64, L66, E68, F69, N70, Q71 , K72, G73, T74, V75, R76, T77, K78, T81, K82, A83, E84, L85, E86, R87, A88, I89, R90, S91, L92, K93, N95, G96, I97, Q98, V99, Y100, V103, H107, K108, G109, G110, A111, D112, F113, T114, E115, R116, V117, Q118, A119, V120, E121, V122, N123, P124, Q125, N126, R127, N128, Q 129, E130, V131, S132, G133, T134, Y135, Q136, I137, E138, A139, W140, T141, G142, F143, N144, F145, P146, G147, G149, N150, Q151, S1 53. S154, F155, K156, W157, R158, W159, Y160, H161, F162, D163, G164, T165, D166, W167, D168, Q169, S170, R171, Q172, L173, A174, N17 5. R176, I177, Y178, K179, F180, R181, G184, K185, A186, W187, D188, W189, E190, V191, D192, T193, E194, N195, G196, N197, Y198, D199,Y200、M202、Y203、A204、D205、V206、M208、D209、H210、P211、E212、V213、I2 14、N215、L217、N218、R219、W220、G221、V222、W223、Y224、A225、N226、T227、 L228, N229, L230, D231, F233, V238, H240, I241, K242, S244, F245, M246, R247, D248, W249, L250, G251, H252, V253, R254, G255, Q256, T257, G258, K259 N260, L261, F262, A263, V264, Y267, W268, K269, N270, D271, L272, G273, A274, L275, E276, N277, Y278, L279, S280, K281, T282, N283, W284, T285, M286 S287, A288, V291, P292, L293, H294, Y295, N296, L297, Y298, Q299, A300, S301, N302, S303, S304, G305, N306, Y307, M309, R310, N311, L312, L313, N314 G315, T316, V318, Q319, R320, H321, P322, S323, A325, V326, T327, F328, V329, N331, T334, P336, G337, E338, E341, S342, V344, Q345, G346, W347, F348 K349, P350, L351, A352, A354, T355, L357, T358, E360, Q361, G362, Y363, Q365, V366, F367, Y368, G369, D370, Y372, G373, I374, P375, S376, D377, V379 P380, S381, Y382, R383, Q384, I386, D387, P388, L389, L390, K391, A392, Q394, Q395, Y396, A397, Y398, R400, H402, D403, Y404, F405, D406, H407, W408 D409、V410、I411、T414、R415、E416、N418、A419、S420、H421、P422、S424、G4 25、L426、A427、T428、I429、M430、S431、D432、G435、G436、S437、K438、W439、M440, Y441, V442, R444, Q445, K446, A447, G448, V450, W451, H452, D453, M454, T455, G456, N457, S459, G460, T463, I464, N465, Q466, D467, W469, H471, F473, N475, G476, G477, V481, and W482; wherein these positions correspond to the amino acid positions in the amino acid sequence specified in SEQ ID NO:3; the method thereby provides an α-amylase variant of the parental α-amylase, wherein the variant corresponds to SEQ ID NO:3. The amino acid sequence as defined in NO:3 or 4 has at least 80%, for example, at least 85%, for example, at least 90%, for example, at least 95%, for example, at least 97%, for example, at least 99%, but less than 100% sequence identity, and said variant has α-amylase activity and improved stability compared to said parental α-amylase.

[0223] In one embodiment, stability is determined based on Phadebas measurements.

[0224] Specifically, stability can be determined by measurements including the following steps:

[0225] a) Incubate the variant to be tested in a detergent composition such as standard detergent composition A at 43°C for 16 hours, and incubate another sample at 4°C for 16 hours.

[0226] b) Add the sample, including the variant to be tested, to the substrate.

[0227] c) Measure the absorbance at 620 nm, and

[0228] d) Calculate the % residual activity as the ratio between the sample incubated at 43°C and the sample incubated at 4°C.

[0229] Stability can be determined by other methods known in the art.

[0230] In one embodiment, the variant has at least 50%, for example at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100% activity of a parental α-amylase having the amino acid sequence of SEQ ID NO: 3 or 4.

[0231] Fermentation broth preparations or cell compositions

[0232] The present invention also relates to fermentation broth formulations or cell compositions comprising the polypeptides of the present invention. Therefore, in one embodiment, the fermentation broth formulation or cell composition comprises the following items: encoded in the positions corresponding to H1, H2, D3, G4, T5, G7, T8, I9, M10, Q11, Y12, F13, E14, W15, N16, V17, P18, N19, D20, Q22, H23, W24, N25, R26, L27, H28, N29, N30, A31, Q32, N33, L34, K35, N36, A37, G38, I39, T40, A41, I42, W43, I44, P45, P46, A47, W48, K49, G50, T51, S52, Q53, N54, D55. V56, G59, A60, Y61, L63, Y64, L66, E68, F69, N70, Q71, K72, G73, T74, V75, R76, T77, K78, T81, K82, A83, E84, L85, E86, R87, A88, I89, R90, S91, L92, K 93, N95, G96, I97, Q98, V99, Y100, V103, H107, K108, G109, G110, A111, D11 2. F113, T114, E115, R116, V117, Q118, A119, V120, E121, V122, N123, P124 , Q125, N126, R127, N128, Q129, E130, V131, S132, G133, T134, Y135, Q136 , I137, E138, A139, W140, T141, G142, F143, N144, F145, P146, G147, G149, N150, Q151, S153, S154, F155, K156, W157, R158, W159, Y160, H161, F162, D 163, G164, T165, D166, W167, D168, Q169, S170, R171, Q172, L173, A174, N1 75. R176, I177, Y178, K179, F180, R181, G184, K185, A186, W187, D188, W1 89. E190, V191, D192, T193, E194, N195, G196, N197, Y198, D199, Y200, M20 2. Y203, A204, D205, V206, M208, D209, H210, P211, E212, V213, I214, N215 , L217, N218, R219, W220, G221, V222, W223, Y224, A225, N226, T227, L228,N229, L230, D231, F233, V238, H240, I241, K242, S244, F245, M246, R247, D248, W249, L250, G251, H252, V253, R254, G255, Q256, T257, G258, K259, N260 L261, F262, A263, V264, Y267, W268, K269, N270, D271, L272, G273, A274, L275, E276, N277, Y278, L279, S280, K281, T282, N283, W284, T285, M286, S287 A288, V291, P292, L293, H294, Y295, N296, L297, Y298, Q299, A300, S301, N302, S303, S304, G305, N306, Y307, M309, R310, N311, L312, L313, N314, G315 T316, V318, Q319, R320, H321, P322, S323, A325, V326, T327, F328, V329, N331, T334, P336, G337, E338, E341, S342, V344, Q345, G346, W347, F348, K349 P350, L351, A352, A354, T355, L357, T358, E360, Q361, G362, Y363, Q365, V366, F367, Y368, G369, D370, Y372, G373, I374, P375, S376, D377, V379, P380 S381, Y382, R383, Q384, I386, D387, P388, L389, L390, K391, A392, Q394, Q395, Y396, A397, Y398, R400, H402, D403, Y404, F405, D406, H407, W408, D409 V410、I411、T414、R415、E416、N418、A419、S420、H421、P422、S424、G425、L4 26、A427、T428、I429、M430、S431、D432、G435、G436、S437、K438、W439、M440、 Y441、V442、R444、Q445、K446、A447、G448、V450、W451、H452、D453、M454、T4 55、G456、N457、S459、G460、T463、I464、N465、Q466、D467、W469、H471、F473、A variant polynucleotide with alterations at one or more positions of N475, G476, G477, V481, and W482, wherein these positions correspond to SEQ ID. The amino acid positions in the amino acid sequence specified in NO:3; encoding the positions H1, H2, D3, G4, T5, G7, T8, I9, M10, Q11, Y12, F13, E14, W15, N16, V17, P18, N19, D20, Q22, H23, W24, N25, R26, L27, H28, N29, N30, A31, Q32, N33, L34, K35, N36, A37, G38, I39, T40, A41, I42, W43, I44, P45, P46, A47, W48, K49, G50, T51, S52, Q53, N54, D5 5. V56, G59, A60, Y61, L63, Y64, L66, E68, F69, N70, Q71, K72, G73, T74, V75, R76, T77, K78, T81, K82, A83, E84, L85, E86, R87, A88, I89, R90, S91 , L92, K93, N95, G96, I97, Q98, V99, Y100, V103, H107, K108, G109, G110 ,A111,D112,F113,T114,E115,R116,V117,Q118,A119,V120,E121,V12 2. N123, P124, Q125, N126, R127, N128, Q129, E130, V131, S132, G133, T 134, Y135, Q136, I137, E138, A139, W140, T141, G142, F143, N144, F145 , P146, G147, G149, N150, Q151, S153, S154, F155, K156, W157, R158, W1 59. Y160, H161, F162, D163, G164, T165, D166, W167, D168, Q169, S170, R 171, Q172, L173, A174, N175, R176, I177, Y178, K179, F180, R181, G184 , K185, A186, W187, D188, W189, E190, V191, D192, T193, E194, N195, G19 6. N197, Y198, D199, Y200, M202, Y203, A204, D205, V206, M208, D209, H 210, P211, E212, V213, I214, N215, L217, N218, R219, W220, G221, V222,W223, Y224, A225, N226, T227, L228, N229, L230, D231, F233, V238, H240, I241, K242, S244, F245, M246, R247, D248, W249, L250, G251, H252, V253, R254 G255, Q256, T257, G258, K259, N260, L261, F262, A263, V264, Y267, W268, K269, N270, D271, L272, G273, A274, L275, E276, N277, Y278, L279, S280, K281 T282, N283, W284, T285, M286, S287, A288, V291, P292, L293, H294, Y295, N296, L297, Y298, Q299, A300, S301, N302, S303, S304, G305, N306, Y307, M309 R310, N311, L312, L313, N314, G315, T316, V318, Q319, R320, H321, P322, S323, A325, V326, T327, F328, V329, N331, T334, P336, G337, E338, E341, S342 V344, Q345, G346, W347, F348, K349, P350, L351, A352, A354, T355, L357, T358, E360, Q361, G362, Y363, Q365, V366, F367, Y368, G369, D370, Y372, G373 I374, P375, S376, D377, V379, P380, S381, Y382, R383, Q384, I386, D387, P388, L389, L390, K391, A392, Q394, Q395, Y396, A397, Y398, R400, H402, D403 Y404、F405、D406、H407、W408、D409、V410、I411、T414、R415、E416、N418、A4 19、S420、H421、P422、S424、G425、L426、A427、T428、I429、M430、S431、D432、 G435、G436、S437、K438、W439、M440、Y441、V442、R444、Q445、K446、A447、G4 48、V450、W451、H452、D453、M454、T455、G456、N457、S459、G460、T463、I464、Nucleic acid constructs of variants with altered positions at one or more of the following locations: N465, Q466, D467, W469, H471, F473, N475, G476, G477, V481, and W482, wherein these positions correspond to SEQ ID. The amino acid positions in the amino acid sequence specified in NO:3; or encoding the positions H1, H2, D3, G4, T5, G7, T8, I9, M10, Q11, Y12, F13, E14, W15, N16, V17, P18, N19, D20, Q22, H23, W24, N25, R26, L27, H28, N29, N30, A31, Q32, N33, L34, K35, N36, A37, G38, I39, T40, A41, I42, W43, I44, P45, P46, A47, W48, K49, G50, T51, S52, Q5 3. N54, D55, V56, G59, A60, Y61, L63, Y64, L66, E68, F69, N70, Q71, K72, G73, T74, V75, R76, T77, K78, T81, K82, A83, E84, L85, E86, R87, A88 , I89, R90, S91, L92, K93, N95, G96, I97, Q98, V99, Y100, V103, H107, K108, G109, G110, A111, D112, F113, T114, E115, R116, V117, Q118, A1 19. V120, E121, V122, N123, P124, Q125, N126, R127, N128, Q129, E130, V131, S132, G133, T134, Y135, Q136, I137, E138, A139, W140, T141, G142, F143, N144, F145, P146, G147, G149, N150, Q151, S153, S154, F155, K156, W157, R158, W159, Y160, H161, F162, D163, G164, T165, D16 6. W167, D168, Q169, S170, R171, Q172, L173, A174, N175, R176, I177, Y178, K179, F180, R181, G184, K185, A186, W187, D188, W189, E190, V 191, D192, T193, E194, N195, G196, N197, Y198, D199, Y200, M202, Y203, A204, D205, V206, M208, D209, H210, P211, E212, V213, I214, N215,L217, N218, R219, W220, G221, V222, W223, Y224, A225, N226, T227, L228, N229, L230, D231, F233, V238, H240, I241, K242, S244, F245, M246, R247, D248 W249, L250, G251, H252, V253, R254, G255, Q256, T257, G258, K259, N260, L261, F262, A263, V264, Y267, W268, K269, N270, D271, L272, G273, A274, L275 E276, N277, Y278, L279, S280, K281, T282, N283, W284, T285, M286, S287, A288, V291, P292, L293, H294, Y295, N296, L297, Y298, Q299, A300, S301, N302 S303, S304, G305, N306, Y307, M309, R310, N311, L312, L313, N314, G315, T316, V318, Q319, R320, H321, P322, S323, A325, V326, T327, F328, V329, N331 T334, P336, G337, E338, E341, S342, V344, Q345, G346, W347, F348, K349, P350, L351, A352, A354, T355, L357, T358, E360, Q361, G362, Y363, Q365, V366 F367, Y368, G369, D370, Y372, G373, I374, P375, S376, D377, V379, P380, S381, Y382, R383, Q384, I386, D387, P388, L389, L390, K391, A392, Q394, Q395 Y396、A397、Y398、R400、H402、D403、Y404、F405、D406、H407、W408、D409、V4 10、I411、T414、R415、E416、N418、A419、S420、H421、P422、S424、G425、L426、 A427、T428、I429、M430、S431、D432、G435、G436、S437、K438、W439、M440、Y4 41、V442、R444、Q445、K446、A447、G448、V450、W451、H452、D453、M454、T455、Expression vectors of variants with altered positions at one or more of the following positions: G456, N457, S459, G460, T463, I464, N465, Q466, D467, W469, H471, F473, N475, G476, G477, V481, and W482, wherein these positions correspond to amino acid positions in the amino acid sequence specified in SEQ ID NO:3. The fermentation broth product may further include additional components for the fermentation process, such as cells (including host cells containing genes encoding the polypeptides of the present invention for producing the polypeptides of interest), cell debris, biomass, fermentation medium, and / or fermentation products. In some embodiments, the composition is a cell-killing whole culture medium comprising one or more organic acids, killed cells and / or cell debris, and a culture medium.

[0233] As used herein, the term "fermentation broth" refers to a preparation produced by cell fermentation that undergoes little or no recovery and / or purification. For example, fermentation broth is produced when a microbial culture is incubated to saturation under carbon-limited conditions that allow protein synthesis (e.g., expression by enzymes of the host cell) and secretion of proteins into the cell culture medium. Fermentation broth may contain unfractionated or fractionated contents of the fermentation material obtained at the end of fermentation. Typically, fermentation broth is unfractionated and includes used culture medium and cell debris remaining after, for example, removal of microbial cells (e.g., filamentous fungal cells) by centrifugation. In some embodiments, fermentation broth contains used cell culture medium, extracellular enzymes, and viable and / or non-viable microbial cells.

[0234] In one embodiment, the fermentation broth formulation and cell composition comprise a first organic acid component (comprising at least one 1-5 carbon organic acid and / or its salt) and a second organic acid component (comprising at least one 6- or more carbon organic acid and / or its salt). In a specific embodiment, the first organic acid component is acetic acid, formic acid, propionic acid, its salt, or a mixture of two or more of the foregoing; and the second organic acid component is benzoic acid, cyclohexanecarboxylic acid, 4-methylvaleric acid, phenylacetic acid, its salt, or a mixture of two or more of the foregoing.

[0235] In one embodiment, the composition comprises one or more organic acids and optionally further comprises killed cells and / or cell debris. In one embodiment, the killed cells and / or cell debris are removed from the cell-killing whole culture medium to provide a composition free of these components.

[0236] These fermentation broth formulations or cell compositions may further include preservatives and / or antimicrobial (e.g., bacteriostatic) agents, including but not limited to sorbitol, sodium chloride, potassium sorbate, and other reagents known in the art.

[0237] The cell-killing whole culture or composition may include the ungraded contents of the fermentation material obtained at the end of fermentation. Typically, the cell-killing whole culture or composition includes used culture medium and cell debris present after microbial cells (e.g., filamentous fungal cells) have been grown to saturation and incubated under carbon-limited conditions to allow protein synthesis. In some embodiments, the cell-killing whole culture or composition includes used cell culture medium, extracellular enzymes, and killed filamentous fungal cells. In some embodiments, methods known in the art can be used to permeate and / or lyse the microbial cells present in the cell-killing whole culture or composition.

[0238] The whole culture medium or cell composition described herein is typically a liquid, but may include insoluble components such as killed cells, cell debris, culture medium components, and / or one or more insoluble enzymes. In some embodiments, insoluble components may be removed to provide a clear liquid composition.

[0239] The whole fermentation broth formulations and cell compositions of the present invention can be produced by the methods described in WO 90 / 15861 or WO 2010 / 096673.

[0240] Enzyme composition

[0241] The present invention also relates to compositions comprising variations according to the invention. Therefore, the present invention relates to compositions comprising variations including changes in one or more of the following positions: H1, H2, D3, G4, T5, G7, T8, I9, M10, Q11, Y12, F13, E14, W15, N16, V17, P18, N19, D20, Q22, H23, W24, N25, R26, L27, H28, N29, N30, A31, Q32, N33, L34, K35, N36, A37, G38, I39, T40, A41, I42, W43, I44, P45, P46, A47, W48, K49, G50, T51, S52, Q53, N54 , D55, V56, G59, A60, Y61, L63, Y64, L66, E68, F69, N70, Q71, K72, G73, T74, V75, R76, T77, K78, T81, K82, A83, E84, L85, E86, R87, A88, I89, R90, S91, L 92, K93, N95, G96, I97, Q98, V99, Y100, V103, H107, K108, G109, G110, A111 , D112, F113, T114, E115, R116, V117, Q118, A119, V120, E121, V122, N123, P 124, Q125, N126, R127, N128, Q129, E130, V131, S132, G133, T134, Y135, Q1 36. I137, E138, A139, W140, T141, G142, F143, N144, F145, P146, G147, G14 9. N150, Q151, S153, S154, F155, K156, W157, R158, W159, Y160, H161, F162 , D163, G164, T165, D166, W167, D168, Q169, S170, R171, Q172, L173, A174, N175, R176, I177, Y178, K179, F180, R181, G184, K185, A186, W187, D188, W 189, E190, V191, D192, T193, E194, N195, G196, N197, Y198, D199, Y200, M2 02, Y203, A204, D205, V206, M208, D209, H210, P211, E212, V213, I214, N21 5. L217, N218, R219, W220, G221, V222, W223, Y224, A225, N226, T227, L228,N229, L230, D231, F233, V238, H240, I241, K242, S244, F245, M246, R247, D248, W249, L250, G251, H252, V253, R254, G255, Q256, T257, G258, K259, N260 L261, F262, A263, V264, Y267, W268, K269, N270, D271, L272, G273, A274, L275, E276, N277, Y278, L279, S280, K281, T282, N283, W284, T285, M286, S287 A288, V291, P292, L293, H294, Y295, N296, L297, Y298, Q299, A300, S301, N302, S303, S304, G305, N306, Y307, M309, R310, N311, L312, L313, N314, G315 T316, V318, Q319, R320, H321, P322, S323, A325, V326, T327, F328, V329, N331, T334, P336, G337, E338, E341, S342, V344, Q345, G346, W347, F348, K349 P350, L351, A352, A354, T355, L357, T358, E360, Q361, G362, Y363, Q365, V366, F367, Y368, G369, D370, Y372, G373, I374, P375, S376, D377, V379, P380 S381, Y382, R383, Q384, I386, D387, P388, L389, L390, K391, A392, Q394, Q395, Y396, A397, Y398, R400, H402, D403, Y404, F405, D406, H407, W408, D409 V410、I411、T414、R415、E416、N418、A419、S420、H421、P422、S424、G425、L4 26、A427、T428、I429、M430、S431、D432、G435、G436、S437、K438、W439、M440、 Y441、V442、R444、Q445、K446、A447、G448、V450、W451、H452、D453、M454、T4 55、G456、N457、S459、G460、T463、I464、N465、Q466、D467、W469、H471、F473、N475, G476, G477, V481, and W482; these positions correspond to the amino acid positions in the amino acid sequence described in SEQ ID NO:3.

[0242] In one embodiment, the composition includes the following variant:

[0243] a) Deletion and / or substitution at two or more positions corresponding to the amino acid sequences as illustrated in SEQ ID NO:7 or 10, namely positions R181, G182, D183, and G184.

[0244] b) Substitution at one or more positions selected from the group consisting of the following items: H1, H2, D3, G4, T5, G7, T8, I9, M10, Q11, Y12, F13, E14, W15, N16, V17, P18, N19, D20, Q22, H23, W24, N25, R26, L27, H28, N29, N30, A31, Q32, N33, L34, K35, N36, A37, G38, I39, T40, A41, I42, W43, I44, P45, P46, A47, W48, K49, G50, T51, S52, Q53, N54, D55, V56, G59, A60, Y61 , L63, Y64, L66, E68, F69, N70, Q71, K72, G73, T74, V75, R76, T77, K78, T81, K 82, A83, E84, L85, E86, R87, A88, I89, R90, S91, L92, K93, N95, G96, I97, Q98, V99, Y100, V103, H107, K108, G109, G110, A111, D112, F113, T114, E115, R11 6. V117, Q118, A119, V120, E121, V122, N123, P124, Q125, N126, R127, N128, Q 129, E130, V131, S132, G133, T134, Y135, Q136, I137, E138, A139, W140, T14 1. G142, F143, N144, F145, P146, G147, G149, N150, Q151, S153, S154, F155, K156, W157, R158, W159, Y160, H161, F162, D163, G164, T165, D166, W167, D1 68. Q169, S170, R171, Q172, L173, A174, N175, R176, I177, Y178, K179, F180, R181, G184, K185, A186, W187, D188, W189, E190, V191, D192, T193, E194, N1 95, G196, N197, Y198, D199, Y200, M202, Y203, A204, D205, V206, M208, D209, H210, P211, E212, V213, I214, N215, L217, N218, R219, W220, G221, V222, W2 23. Y224, A225, N226, T227, L228, N229, L230, D231, F233, V238, H240, I241,K242, S244, F245, M246, R247, D248, W249, L250, G251, H252, V253, R254, G255, Q256, T257, G258, K259, N260, L261, F262, A263, V264, Y267, W268, K26 9, N270, D271, L272, G273, A274, L275, E276, N277, Y278, L279, S280, K281, T282, N283, W284, T285, M286, S287, A288, V291, P292, L293, H294, Y295, N 296, L297, Y298, Q299, A300, S301, N302, S303, S304, G305, N306, Y307, M309, R310, N311, L312, L313, N314, G315, T316, V318, Q319, R320, H321, P322 S323, A325, V326, T327, F328, V329, N331, T334, P336, G337, E338, E341, S342, V344, Q345, G346, W347, F348, K349, P350, L351, A352, A354, T355, L3 57, T358, E360, Q361, G362, Y363, Q365, V366, F367, Y368, G369, D370, Y372, G373, I374, P375, S376, D377, V379, P380, S381, Y382, R383, Q384, I386 D387, P388, L389, L390, K391, A392, Q394, Q395, Y396, A397, Y398, R400, H402, D403, Y404, F405, D406, H407, W408, D409, V410, I411, T414, R415, E41 6、N418、A419、S420、H421、P422、S424、G425、L426、A427、T428、I429、M430 、S431、D432、G435、G436、S437、K438、W439、M440、Y441、V442、R444、Q445、K 446、A447、G448、V450、W451、H452、D453、M454、T455、G456、N457、S459、G46 0、T463、I464、N465、Q466、D467、W469、H471、F473、N475、G476、G477、V481、And W482; wherein these positions correspond to the amino acid positions in the amino acid sequence as illustrated in SEQ ID NO:3. Preferably, these compositions are enriched for this variant. As used herein, the term "enriched" means that the α-amylase activity of the composition has been increased, for example, with an enrichment factor of at least 1.1.

[0245] These compositions may include variants of the invention as the main enzyme component, such as single-component compositions. In another embodiment, the composition further includes at least one additional active component.

[0246] As used herein, the term "active component" refers to any biological or non-biological molecule that is active in itself. For example, an active component is an enzyme.

[0247] Therefore, in one embodiment, the additional active component is an enzyme, such as a protease, lipase, cellulose, pectic acid lyase, and mannanase. Therefore, these compositions may include a variety of enzymatic activities, such as one or more (e.g., several) enzymes selected from the group consisting of: hydrolases, isomerases, ligases, lyases, oxidoreductases, or transferases, such as α-galactosidase, α-glucosidase, aminopeptidase, amylase, β-galactosidase, β-glucosidase, β-xylosidase, glycosylase, carboxypeptidase, catalase, cellobiase, cellulase, chitosanase, keratinase, cyclodextrin glucosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucosylamylase, invertase, laccase, lipase, lichinase, mannosidase, polysaccharidase, oxidase, pectinase, peroxidase, phytase, polyphenol oxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.

[0248] In one embodiment, the composition is a detergent composition, such as a liquid or powder detergent composition.

[0249] In one embodiment, the composition is a liquid laundry or liquid dishwashing composition, such as an automatic dishwashing (ADW) liquid detergent composition, or a powdered laundry detergent, such as a soap bar, or a powdered dishwashing composition, such as an ADW unit dose detergent composition.

[0250] The selection of other components is within the capabilities of those skilled in the art and includes conventional ingredients, including the exemplary non-limiting components described below. For fabric care, the selection of components may include considerations such as the type of fabric to be cleaned, the type and / or extent of the stain, the temperature at which cleaning is performed, and the formulation of the detergent product. Although the components mentioned below are categorized under a general heading according to their specific functionality, this is not to be construed as limiting, as a component may include additional functionality as would be understood by one of ordinary skill in the art.

[0251] In one embodiment of the invention, variations of the invention may be added to the detergent composition in amounts corresponding to the following: 0.001-100 mg of protein per liter of detergent liquid, for example, 0.01-100 mg of protein, preferably 0.005-50 mg of protein, more preferably 0.01-25 mg of protein, even more preferably 0.05-10 mg of protein, most preferably 0.05-5 mg of protein, and even most preferably 0.01-1 mg of protein. The term "protein" is to be understood herein to include variations according to the invention.

[0252] Compositions for use in automated dishwashing (ADW) may, for example, include 0.0001%-50%, such as 0.001%-20%, such as 0.01%-10%, such as 0.05%-5% of enzyme protein by weight of the composition.

[0253] The composition used in laundry granulation may, for example, include 0.0001%-50%, such as 0.001%-20%, such as 0.01%-10%, such as 0.05%-5% of the enzyme protein by weight of the composition.

[0254] Compositions for use in laundry detergents may, for example, include 0.0001%-10%, such as 0.001%-7%, or such as 0.1%-5%, of enzyme protein by weight of the composition.

[0255] These variants of the invention, as well as other active components such as additional enzymes, can be stabilized using conventional stabilizers, such as polyols (e.g., propylene glycol or glycerol), sugars or sugar alcohols, lactic acid, boric acid or boric acid derivatives (e.g., aromatic borate esters), or phenylboronic acid derivatives (e.g., 4-formylphenylboronic acid), and the compositions can be formulated as described in, for example, WO92 / 19709 and WO 92 / 19708.

[0256] In some markets, different washing conditions and, in themselves, different types of detergents are used. This is disclosed, for example, in EP 1 025 240. For instance, low detergent concentration systems are used in Asia (Japan), medium detergent concentration systems are used in the United States, and high detergent concentration systems are used in Europe.

[0257] Low detergent concentration systems contain detergents in which less than approximately 800 ppm of detergent components are present in the wash water. Japanese detergents are typically considered low detergent concentration systems because they have approximately 667 ppm of detergent components present in the wash water.

[0258] Medium detergent concentration systems contain detergents with detergent components present in wash water at concentrations between approximately 800 ppm and approximately 2000 ppm. North American detergents are generally considered medium detergent concentration systems because they have approximately 975 ppm of detergent components present in wash water.

[0259] High detergent concentration systems contain detergents with more than approximately 2000 ppm of detergent components in the wash water. European detergents are generally considered high detergent concentration systems because they contain approximately 4500-5000 ppm of detergent components in the wash water.

[0260] Latin American detergents are typically high-foaming phosphate-based detergents, and the range of detergents used in Latin America can fall between medium and high detergent concentrations, as their detergent components in the wash water range from 1500 ppm to 6000 ppm. Such detergent compositions are examples of the present invention.

[0261] Variations of the present invention may also be incorporated into detergent formulations disclosed in WO 97 / 07202, which is incorporated herein by reference.

[0262] Examples of preferred uses of the compositions of the present invention are given herein. The dosage of the composition and other conditions for using the composition can be determined based on methods known in the art.

[0263] Specifically, the compositions according to the invention further include chelating agents.

[0264] As used herein, the term "chelating agent" refers to chemicals that form molecules with certain metal ions, passivating these ions and thus preventing them from reacting with other elements. Therefore, a chelating agent can be defined as a binding agent that inhibits chemical activity by forming chelates. Chelation is the formation or presence of two or more separate bonds between a ligand and a single central atom. The ligand can be any organic compound, silicate, or phosphate. In this context, the term "chelating agent" includes chelants, chelating agents, complexing agents, or sequestering agents that can form water-soluble complexes with metal ions such as calcium and magnesium. Chelation describes the enhanced affinity of a chelating ligand for a metal ion relative to the affinity of a similar set of non-chelating ligands for the same metal. Chelating agents have the ability to bind to metal ions, particularly calcium (Ca2+) ions, and are widely used in detergents and compositions generally used for washing, such as laundry or dishwashing. However, chelating agents themselves exhibit inhibitory activity of enzymes. In this application, the term chelating agent is used interchangeably with "complexing agent" or "chelant".

[0265] Since most α-amylases are calcium sensitive, the presence of a chelating agent can affect enzyme activity. The calcium sensitivity of α-amylase can be determined by incubating a given α-amylase in the presence of a strong chelating agent and analyzing the effect of this incubation on the activity of the α-amylase. Calcium-sensitive α-amylases will lose most or all of their activity during incubation. The chelating agent may be present in the composition in an amount from 0.0001 wt% to 20 wt%, preferably from 0.01 wt% to 10 wt%, more preferably from 0.1 wt% to 5 wt%.

[0266] Non-limiting examples of chelating agents are EDTA, DTMPA, HEDP, and citrate. Therefore, in one embodiment, the composition comprises a variant according to the invention and a chelating agent such as EDTA, DTMPA, HEDP, or citrate.

[0267] As used herein, the term "EDTA" refers to ethylenediaminetetraacetic acid, which falls under the definition of a "strong chelating agent".

[0268] As used herein, the term "DTMPA" refers to diethylenetriaminepenta (methylenephosphonic acid). DTMPA can inhibit the scaling of carbonate, sulfate, and phosphate ions.

[0269] As used herein, the term "HEDP" refers to hydroxy-ethanediphosphonic acid, which falls under the definition of "strong chelating agent".

[0270] The chelate effect (or chelating effect) describes the enhanced affinity of a chelating ligand for a metal ion relative to the affinity of a similar set of non-chelating ligands for the same metal. However, the strength of the chelate effect can be determined by various types of determinations or measurements, thereby allowing chelating agents to be differentiated and ranked according to their chelate effect (or strength).

[0271] In the assay, chelating agents can bind free calcium ions (Ca) at pH 8.0. 2+ The ability to reduce the concentration of ) from 2.0 mM to 0.10 mM or less can be characterized, for example, by using a test based on the method described by MKNagarajan et al., JAOCS, Vol. 61, no. 9 (September 1984), pp. 1475-1478.

[0272] For reference, the chelating agent and EDTA at the same concentration of the chelating agent have the effect of removing free calcium ions (Ca) at pH 1. 2+ If a chelating agent has the same ability to reduce its concentration from 2.0 mM to 0.10 mM, then it is called a strong chelating agent.

[0273] The compositions of the present invention can be in any suitable form, such as strips, homogeneous tablets, tablets having two or more layers, bags having one or more compartments, conventional or compressed powders, granules, pastes, gels, or conventional, compressed or concentrated liquids. Various detergent formulations are available, such as layers (same or different phases), bags, and forms for mechanical feeding devices.

[0274] The bag can be configured as a single compartment or multiple compartments. It can have any form, shape, and material suitable for preserving the composition, for example, preventing the composition from being released from the bag before contact with water. The bag is made of a water-soluble membrane encapsulating the inner volume. The inner volume can be divided into compartments containing the bag. Preferred membranes are polymeric materials that form membranes or sheets, preferably polymers. Preferred polymers, copolymers, or derivatives thereof are selected from polyacrylates and water-soluble acrylate copolymers, methylcellulose, carboxymethylcellulose, sodium dextrin, ethylcellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, maltodextrin, polymethyl acrylates, and most preferably polyvinyl alcohol copolymers and hydroxypropyl methylcellulose (HPMC). Preferably, the polymer level in the membrane, such as PVA, is at least about 60%. Preferred average molecular weights will typically be from about 20,000 to about 150,000. The membrane can also be a blend composition comprising a hydrolyzable and water-soluble polymer blend, such as polylactic acid and polyvinyl alcohol (known under trade reference number M8630, such as that sold by Chris Craft In. Prod., Gary, Indiana, USA), plus plasticizers, such as glycerin, ethylene glycol, propylene glycol, sorbitol, and mixtures thereof. These bags may include solid laundry cleaning compositions or portions thereof and / or liquid cleaning compositions or portions thereof separated by a water-soluble membrane. Chambers for liquid components may be structurally different from those containing solid components. Reference: (US2009 / 0011970A1).

[0275] Detergent components can be physically separated from each other by compartments in different layers of water-soluble pouches or tablets. This avoids negative storage interactions between components. The different dissolution profiles of each compartment in the washing solution can also cause delayed dissolution of selected components.

[0276] Non-unit-volume liquid or gel detergents may be aqueous, typically containing at least 20% and up to 95% water by weight, such as up to about 70%, about 65%, about 55%, about 45%, or about 35%. Other types of liquids, including but not limited to alkanols, amines, glycols, ethers, and polyols, may be included in aqueous liquids or gels. Aqueous liquid or gel detergents may contain from 0-30% organic solvents. Liquid or gel detergents may be non-aqueous.

[0277] Another form of composition is as follows: soap bars, such as laundry soap bars, which can be used for hand washing clothes, fabrics, and / or textiles. As used herein, the term "soap bar" refers to a bar including clothing bars, soap bars, combo bars, synthetic bars, and detergent bars. The types of bars are generally distinguished by the type of surfactant they contain, and the term laundry soap bar includes those containing soaps derived from fatty acids and / or synthetic soaps. Laundry soap bars have a physical form that is solid at room temperature, rather than liquid, gel, or powder. As used herein, the term "solid" means a physical form that does not change significantly over time, i.e., if a solid object (such as a laundry soap bar) is placed in a container, the solid object will not change in order to fill the container in which it is placed. When a bar is solid, it is typically in the form of a bar, but it can also be other solid shapes such as round or oval.

[0278] The soap bar may also include complexing agents such as EDTA and HEDP, fragrances and / or different types of fillers, surfactants such as anionic synthetic surfactants, detergent builders, polymerized dirt releasers, detergent chelators, stabilizers, fillers, dyes, colorants, dye transfer inhibitors, alkoxylated polycarbonates, defoamers, structural agents, binders, leachants, bleach activators, clay detergents, anti-redeposition agents, polymeric dispersants, brighteners, fabric softeners, fragrances and / or other compounds known in the art.

[0279] The soap bar can be processed in conventional laundry soap bar manufacturing equipment, such as, but not limited to, mixers, pressing machines (e.g., two-stage vacuum pressing machines), extruders, cutters, logo-stamper machines, cooling tunnels, and packaging machines. The invention is not limited to preparing soap bars by any single method. The premix of the invention can be added to the soap at different stages of the process. For example, a premix comprising soap, enzymes, optionally one or more additional enzymes, protease inhibitors, and salts of monovalent cations and organic anions can be prepared, and then the mixture can be extruded. The enzymes and optionally additional enzymes can be added simultaneously, for example, in solid form, as enzyme inhibitors (e.g., protease inhibitors). In addition to the mixing and extrusion steps, the process may further include the following steps: grinding, extrusion, cutting, stamping, cooling, and / or packaging.

[0280] use

[0281] This invention further relates to the use of variations according to the invention in cleaning processes (e.g., laundry or hard surface cleaning, including automated dishwashing and industrial cleaning). The dirt and stains important for cleaning consist of many different substances, and a range of different enzymes, all with different substrate specificities, have been developed for use in both laundry and hard surface cleaning (e.g., dishwashing). These enzymes are believed to provide enzymatic cleaning benefits because they specifically improve stain removal during the cleaning process in which they are applied compared to the same process without enzymes. Cleaning enzymes known in the art include, for example, proteases, amylases, lipases, keratins, cellulases, endoglucanases, xyloglucanases, pectins, pectin lyases, xanthan gums, peroxidases, halogenated peroxidases, catalases, and mannanases.

[0282] In one embodiment, the invention relates to the use of variants of the invention in detergent compositions for cleaning hard surfaces (e.g., dishwashing), or in laundry or for stain removal. In another embodiment, the invention relates to the use of α-amylase variants according to the invention in cleaning processes (e.g., laundry or hard surface cleaning, including but not limited to dishwashing and industrial cleaning). Therefore, in one embodiment, the present invention relates to including the locations corresponding to the positions H1, H2, D3, G4, T5, G7, T8, I9, M10, Q11, Y12, F13, E14, W15, N16, V17, P18, N19, D20, Q22, H23, W24, N25, R26, L27, H28, N29, N30, A31, Q32, N33, L34, K35, N36, A37, G38, I39, T40, A41, I42, W43, I44, P45, P46, A47, W48, K49, G50, T51, S52, Q5 3. N54, D55, V56, G59, A60, Y61, L63, Y64, L66, E68, F69, N70, Q71, K72, G73, T74, V75, R76, T77, K78, T81, K82, A83, E84, L85, E86, R87 , A88, I89, R90, S91, L92, K93, N95, G96, I97, Q98, V99, Y100, V103, H107, K108, G109, G110, A111, D112, F113, T114, E115, R116, V117 , Q118, A119, V120, E121, V122, N123, P124, Q125, N126, R127, N128, Q129, E130, V131, S132, G133, T134, Y135, Q136, I137, E138, A13 9. W140, T141, G142, F143, N144, F145, P146, G147, G149, N150, Q151, S153, S154, F155, K156, W157, R158, W159, Y160, H161, F162, D1 63. G164, T165, D166, W167, D168, Q169, S170, R171, Q172, L173, A174, N175, R176, I177, Y178, K179, F180, R181, G184, K185, A186, W 187, D188, W189, E190, V191, D192, T193, E194, N195, G196, N197, Y198, D199, Y200, M202, Y203, A204, D205, V206, M208, D209, H210,P211, E212, V213, I214, N215, L217, N218, R219, W220, G221, V222, W223, Y224, A225, N226, T227, L228, N229, L230, D231, F233, V238, H240, I241, K242 S244, F245, M246, R247, D248, W249, L250, G251, H252, V253, R254, G255, Q256, T257, G258, K259, N260, L261, F262, A263, V264, Y267, W268, K269, N270 D271, L272, G273, A274, L275, E276, N277, Y278, L279, S280, K281, T282, N283, W284, T285, M286, S287, A288, V291, P292, L293, H294, Y295, N296, L297 Y298, Q299, A300, S301, N302, S303, S304, G305, N306, Y307, M309, R310, N311, L312, L313, N314, G315, T316, V318, Q319, R320, H321, P322, S323, A325 V326, T327, F328, V329, N331, T334, P336, G337, E338, E341, S342, V344, Q345, G346, W347, F348, K349, P350, L351, A352, A354, T355, L357, T358, E360 Q361, G362, Y363, Q365, V366, F367, Y368, G369, D370, Y372, G373, I374, P375, S376, D377, V379, P380, S381, Y382, R383, Q384, I386, D387, P388, L389 L390、K391、A392、Q394、Q395、Y396、A397、Y398、R400、H402、D403、Y404、F4 05、D406、H407、W408、D409、V410、I411、T414、R415、E416、N418、A419、S420、 H421、P422、S424、G425、L426、A427、T428、I429、M430、S431、D432、G435、G4 36、S437、K438、W439、M440、Y441、V442、R444、Q445、K446、A447、G448、V450、Variants with altered positions at one or more of the following locations are intended for use in cleaning processes such as laundry or hard surface cleaning (including dishwashing and industrial cleaning): where these positions correspond to amino acid positions in the amino acid sequence defined in SEQ ID NO:3.

[0283] In specific embodiments, the present invention relates to the use of variants in cleaning processes such as laundry or hard surface cleaning (including dishwashing and industrial cleaning), the variants comprising a) substitution and / or deletion at two, three, or four positions in the parental α-amylase, said positions corresponding to positions R181, G182, D183, and G184 of the mature polypeptide of SEQ ID NO:4; and

[0284] b) Local location H1, H2, D3, G4, T5, G7, T8, I9, M10, Q11, Y12, F13, E14, W15, N16, V1 7, P18, N19, D20, Q22, H23, W24, N25, R26, L27, H28, N29, N30, A31, Q32, N33, L 34, K35, N36, A37, G38, I39, T40, A41, I42, W43, I44, P45, P46, A47, W48, K49 , G50, T51, S52, Q53, N54, D55, V56, G59, A60, Y61, L63, Y64, L66, E68, F69, N7 0, Q71, K72, G73, T74, V75, R76, T77, K78, T81, K82, A83, E84, L85, E86, R87, A88, I89, R90, S91, L92, K93, N95, G96, I97, Q98, V99, Y100, V103, H107, K108 , G109, G110, A111, D112, F113, T114, E115, R116, V117, Q118, A119, V120, E 121, V122, N123, P124, Q125, N126, R127, N128, Q129, E130, V131, S132, G133 , T134, Y135, Q136, I137, E138, A139, W140, T141, G142, F143, N144, F145, P 146, G147, G149, N150, Q151, S153, S154, F155, K156, W157, R158, W159, Y160 , H161, F162, D163, G164, T165, D166, W167, D168, Q169, S170, R171, Q172, L 173, A174, N175, R176, I177, Y178, K179, F180, R181, G184, K185, A186, W187 , D188, W189, E190, V191, D192, T193, E194, N195, G196, N197, Y198, D199, Y 200, M202, Y203, A204, D205, V206, M208, D209, H210, P211, E212, V213, I214 , N215, L217, N218, R219, W220, G221, V222, W223, Y224, A225, N226, T227, L2 28, N229, L230, D231, F233, V238, H240, I241, K242, S244, F245, M246, R247,D248, W249, L250, G251, H252, V253, R254, G255, Q256, T257, G258, K259, N260, L261, F262, A263, V264, Y267, W268, K269, N270, D271, L272, G273 A274, L275, E276, N277, Y278, L279, S280, K281, T282, N283, W284, T285, M286, S287, A288, V291, P292, L293, H294, Y295, N296, L297, Y298, Q299 A300, S301, N302, S303, S304, G305, N306, Y307, M309, R310, N311, L312, L313, N314, G315, T316, V318, Q319, R320, H321, P322, S323, A325, V326 T327, F328, V329, N331, T334, P336, G337, E338, E341, S342, V344, Q345, G346, W347, F348, K349, P350, L351, A352, A354, T355, L357, T358, E360 Q361, G362, Y363, Q365, V366, F367, Y368, G369, D370, Y372, G373, I374, P375, S376, D377, V379, P380, S381, Y382, R383, Q384, I386, D387, P388 L389, L390, K391, A392, Q394, Q395, Y396, A397, Y398, R400, H402, D403, Y404, F405, D406, H407, W408, D409, V410, I411, T414, R415, E416, N418 A419、S420、H421、P422、S424、G425、L426、A427、T428、I429、M430、S431、 D432、G435、G436、S437、K438、W439、M440、Y441、V442、R444、Q445、K446、 A447、G448、V450、W451、H452、D453、M454、T455、G456、N457、S459、G460、 T463、I464、N465、Q466、D467、W469、H471、F473、N475、G476、G477、V481、And substitutions at one or more positions in W482, wherein these positions correspond to amino acid positions in the amino acid sequence as illustrated in SEQ ID NO:3.

[0285] One embodiment of the invention relates to the use of compositions according to the invention in detergent compositions and in detergent applications, the compositions comprising variations of the invention together with one or more surfactants and optionally one or more detergent components selected from the list of water-soluble solvents, detergent builders and co-builders, bleaching systems, polymers, fabric colorants and accessories, and any mixtures thereof.

[0286] Another embodiment is the use of the composition according to the invention in detergent compositions and in detergent applications, the composition comprising variants of the invention together with one or more surfactants, and one or more additional enzymes selected from the group consisting of proteases, lipases, keratins, cellulases, endoglucans, xyloglucans, pectins, pectin lyases, xanthan gums, peroxidases, halogenated peroxidases, catalases, and mannanases or any mixture thereof.

[0287] On the other hand, the present invention relates to a washing method applicable to both household and industrial laundry. Furthermore, the present invention relates to a method for washing textiles (e.g., fabrics, garments, clothes, etc.), wherein the method comprises treating the textiles with a washing solution containing a detergent composition and the α-amylase of the present invention. The washing can be performed using a household or industrial washing machine or manually using a detergent composition containing the glucosylase of the present invention.

[0288] On the other hand, the present invention relates to a method for washing tableware, applicable to both household and industrial tableware washing. As used herein, the term "tableware washing" refers to both manual and automated tableware washing. Furthermore, the present invention relates to a method for washing hard surfaces (e.g., tableware such as knives, forks, and spoons; ceramics such as plates, glasses, and bowls; and pans), wherein the method comprises treating the hard surface with a washing solution containing a detergent composition and an α-amylase variant of the present invention. Hard surfaces can be washed, for example, using a household or industrial dishwasher or manually using a detergent composition containing the α-amylase of the present invention, optionally together with one or more other enzymes selected from the group consisting of: proteases, amylases, lipases, keratins, cellulases, endoglucanases, xyloglucanases, pectins, pectin lyases, xanthan gums, peroxidases, halogenated peroxidases, catalases, mannanases, or any mixtures thereof.

[0289] In another aspect, the present invention relates to a method for removing stains from a surface, the method comprising contacting the surface with a composition comprising, in a detergent composition and in a detergent application, an α-amylase of the present invention together with one or more surfactants and optionally one or more detergent components selected from the list comprising water-soluble solvents, builder agents and co-builder agents, bleaching systems, polymers, fabric toners and accessories, and any mixture thereof. Another aspect is a method for removing stains from a surface, the method comprising contacting the surface with a composition comprising, in a detergent composition and in a detergent application, a variant of the α-amylase of the present invention together with one or more surfactants and one or more additional enzymes selected from the group comprising proteases, lipases, keratins, cellulases, endoglucanases, xyloglucanases, pectins, pectin lyases, xanthan gums, peroxidases, halogenated peroxidases, catalases, and mannanases, and any mixture thereof.

[0290] The invention is further described through the following examples, but these should not be construed as limiting the scope of the invention.

[0291] Example

[0292] Example 1: Generation of a variant according to the present invention

[0293] Variations of the present invention have been generated through site-directed mutagenesis.

[0294] Genomic DNA prepared from an organism containing an amylase gene at the Pel locus was used as a template for generating site-directed mutants.

[0295] A fragment of approximately 3.8 kb was generated using a mutagenic forward primer and a PnMi4490 (CAATCCAAGAGAACCCTGATACGGATG-SEQ ID NO:5) reverse primer. This fragment, along with a PnMi4491 (CGGAACGCCTGGCTGACAACACG-SEQ ID NO:6) forward primer, was used as a large primer to yield a 6 kb insert cassette. To facilitate integration into the Pel locus via double crossover during transformation, the cassette, along with the amylase and cat genes, contained upstream and downstream Pel sequences at its ends. Selection was performed on LB agar containing chloramphenicol, and the mutation was confirmed by DNA sequencing of the amylase gene.

[0296] Generate a variant library with substitutions at different positions selected from the following: H1, H2, D3, G4, T5, G7, T8, I9, M10, Q11, Y12, F13, E14, W15, N16, V17, P18, N19, D20, Q22, H23, W24, N25, R26, L27, H28, N29, N30, A31, Q32, N33, L34, K35, N36, A37, G38, I39, T40, A41, I42, W43, I44, P45, P46, A47, W48, K49, G50, T51, S52, Q53, N54, D55, V56, G59, A60, Y61. L63, Y64, L66, E68, F69, N70, Q71, K72, G73, T74, V75, R76, T77, K78, T81, K8 2. A83, E84, L85, E86, R87, A88, I89, R90, S91, L92, K93, N95, G96, I97, Q98, V99, Y100, V103, H107, K108, G109, G110, A111, D112, F113, T114, E115, R11 6. V117, Q118, A119, V120, E121, V122, N123, P124, Q125, N126, R127, N128, Q 129, E130, V131, S132, G133, T134, Y135, Q136, I137, E138, A139, W140, T14 1. G142, F143, N144, F145, P146, G147, G149, N150, Q151, S153, S154, F155, K156, W157, R158, W159, Y160, H161, F162, D163, G164, T165, D166, W167, D1 68. Q169, S170, R171, Q172, L173, A174, N175, R176, I177, Y178, K179, F180, R181, G184, K185, A186, W187, D188, W189, E190, V191, D192, T193, E194, N1 95, G196, N197, Y198, D199, Y200, M202, Y203, A204, D205, V206, M208, D209, H210, P211, E212, V213, I214, N215, L217, N218, R219, W220, G221, V222, W2 23. Y224, A225, N226, T227, L228, N229, L230, D231, F233, V238, H240, I241,K242, S244, F245, M246, R247, D248, W249, L250, G251, H252, V253, R254, G255, Q256, T257, G258, K259, N260, L261, F262, A263, V264, Y267, W268, K26 9, N270, D271, L272, G273, A274, L275, E276, N277, Y278, L279, S280, K281, T282, N283, W284, T285, M286, S287, A288, V291, P292, L293, H294, Y295, N 296, L297, Y298, Q299, A300, S301, N302, S303, S304, G305, N306, Y307, M309, R310, N311, L312, L313, N314, G315, T316, V318, Q319, R320, H321, P322 S323, A325, V326, T327, F328, V329, N331, T334, P336, G337, E338, E341, S342, V344, Q345, G346, W347, F348, K349, P350, L351, A352, A354, T355, L3 57, T358, E360, Q361, G362, Y363, Q365, V366, F367, Y368, G369, D370, Y372, G373, I374, P375, S376, D377, V379, P380, S381, Y382, R383, Q384, I386 D387, P388, L389, L390, K391, A392, Q394, Q395, Y396, A397, Y398, R400, H402, D403, Y404, F405, D406, H407, W408, D409, V410, I411, T414, R415, E41 6、N418、A419、S420、H421、P422、S424、G425、L426、A427、T428、I429、M430 、S431、D432、G435、G436、S437、K438、W439、M440、Y441、V442、R444、Q445、K 446、A447、G448、V450、W451、H452、D453、M454、T455、G456、N457、S459、G46 0、T463、I464、N465、Q466、D467、W469、H471、F473、N475、G476、G477、V481、And W482; where these positions correspond to the amino acid positions in the amino acid sequence described in SEQ ID NO:3.

[0297] Table 1: Variations according to the present invention

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310] Example 2: Specific activity of variants

[0311] To determine whether the resulting variants, as described and listed in Example 2, possess maintained or even improved activity, these variants were evaluated using the Phadebas assay. The following detergent compositions were prepared.

[0312] Preparation standard X (0.175%):

[0313] A 1:2 molar ratio CaCl2 and MgCl2 stock solution has a water hardness of 6000 dH.

[0314] Weigh 104.9g of CaCl2·2H2O (0.713M) into a 1L flask, add 500ml of type I water and stir thoroughly. Weigh and add 72.5g of MgCl2·6H2O (0.357M), dissolve thoroughly, and bring the final volume to 1000ml with type I water.

[0315] 0.535M NaHCO3 solution

[0316] Dissolve 44.9g of sodium bicarbonate in 100ml of type I water.

[0317] The standard X detergent, containing X ionic surfactant, has a water hardness of 12 (12 dH).

[0318] Weigh 1.75 g of standard X detergent (as described above) and transfer it to a 1 L bottle. Add 800 ml of type I water and mix thoroughly. Add 35 mg of X ionic surfactant and mix thoroughly. To adjust the water hardness to 12 dH, add 2 ml of a 1:2 molar ratio CaCl2 and MgCl2 stock solution (6000 dH) and 6 ml of a 0.535 mol NaHCO3 solution, and mix thoroughly. Finally, bring the volume to 1000 ml and stir the mixture for 10 min.

[0319] Preparation standard A (0.33%):

[0320] A 4:1 molar ratio CaCl2 and MgCl2 stock solution has a water hardness of 6000 dH.

[0321] Weigh 125.8g of CaCl2·2H2O into a 1-liter flask, add 500ml of type I water, and stir thoroughly. Weigh and add 43.8g of MgCl2·6H2O, dissolve completely, and then bring the final volume to 1000ml with type I water.

[0322] 0.535M NaHCO3 solution

[0323] Dissolve 44.9g of sodium bicarbonate in 100ml of type I water.

[0324] Standard A detergent has a water hardness of 15 (15 dH).

[0325] Weigh 3.335 g of standard detergent A and transfer it to a 1-liter bottle. Add 865 ml of type I water and mix thoroughly. Add 7.5 ml of 0.535 M NaHCO3, mix thoroughly, and bring the volume to 1 liter with type I water. To adjust the water hardness to 15 dH, add 2.5 ml of a 4:1 molar ratio CaCl2·2H2O and MgCl2·6H2O stock solution (6000 dH) and stir the mixture for 15 min.

[0326] Substrate: Phadebas tablets (McGor Life Sciences)

[0327] Suspend one tablet in 10 ml of detergent solution.

[0328] Buffer: 100mM MOPS buffer, pH 8

[0329] Experimental Procedure

[0330] Preparation of mother plate:

[0331] Colonies were picked from the transformed plate using a colony picker (KBiosystems) and inoculated into 96-well plates containing TBGly medium for growth. The cultures were incubated at 37°C for 3 days, and the supernatant was recovered from the plate by centrifugation.

[0332] Preparation of substrate plate:

[0333] The substrate solution was prepared by dissolving one phadebas tablet in 10 ml of standard X / standard A detergent, and 180 μl of the solution was dispensed into a 96-well microtiter plate using a multidrop instrument with continuous stirring.

[0334] Dilute the culture supernatant 100-fold with buffer, and add 20 μl of the diluted culture to 180 μl of pre-split substrate plate and mix thoroughly. Incubate the plate at 25 °C for 20 min with shaking (900 rpm). After incubation, allow the plate to settle for 5 min. Transfer 50 μl of supernatant to a 384-well plate and measure the absorbance at 620 nm. Determine the concentration of the expressed enzyme using a specific antibody via ELISA. Specific activity is calculated by taking the ratio of activity to concentration, and all activities identified as having a higher specific activity than the parental α-amylase are included. The improvement factor (IF) is calculated as follows: [specific activity of variant] / [specific activity of parental α-amylase].

[0335] Table 2: Obtained by the above method Specific activity of variants according to the invention As shown below (amino acid substitution refers to SEQ ID NO:3).

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355] Variants that show an IF of at least 1.0 against at least one standard detergent are considered to have improved performance when compared with parental α-amylase (i.e., α-amylase having the amino acid sequence set forth in SEQ ID NO:3).

[0356] Example 3: Stability of variants

[0357] The stability of the generated variants described and listed in Example 1 in standard detergents was further evaluated.

[0358] Substrate: Phadebas tablets (McGor Life Sciences)

[0359] Suspend one tablet in 10 ml of detergent solution.

[0360] Measurement procedure:

[0361] Transfer 20 μL of culture supernatant to two 96-well plates named "Non-Stressed" and "Stressed". Add 80 μL of detergent mixed with EDTA and mix thoroughly. Incubate the non-stressed plate at 4 °C for 16 hours and the stressed plate at 43 °C for 16 hours. Dilute the non-stressed and stressed samples 20-fold in buffer. Add 20 μL of the diluted culture to the pre-split substrate plate and mix thoroughly. Incubate the plate at 25 °C for 20 min with shaking (900 rpm). After incubation, allow the plate to settle for 5 min. Transfer 50 μL of supernatant to a 384-well plate and measure the absorbance at 620 nm.

[0362] Stability is calculated as the percentage of residual activity (%RA) between stressed and unstressed samples (%RA = stress / unstressed * 100). Improvement factor (IF) is calculated as: %RA of variant / %RA of WT.

[0363] Table 3: The stability results obtained are listed below.

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375] Variants having an IF of at least 1.0 are considered to have improved stability compared to the parental α-amylase (i.e., an α-amylase having the amino acid sequence as illustrated in SEQ ID NO:3).

[0376] Example 4: Mean hit of variants

[0377] If possible Figure 1-3 It can be seen that numerous variants have been generated and their specific activity and stability in standard A and standard X detergent compositions have been tested. Specifically, Figure 1 The improvement factor (IF) is shown as a measure of the specific activity of the variant in the standard A detergent composition. Figure 2 The IF, a measure of the specific activity of the variant in the standard X detergent composition, is shown. Figure 3 The IF is shown as a measure of the stability of the variant in the standard A detergent composition. The mean hit value for each modified site has been calculated (see Table 4).

[0378] Table 4: Average hit rate at each position

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387] As can be seen from the table above, for at least one of the conditions under which the variants have been tested (i.e., specific activity in standard A and / or standard X detergent compositions and / or stability in standard A detergent composition), the IF at each test location is at least 1.0.

[0388] Example 5: Specific activity and stability of variants of the present invention

[0389] To determine whether the resulting variants, as described and listed in Example 2, possess maintained or even improved activity, these variants were evaluated using the Phadebas assay. The following detergent compositions were prepared.

[0390] Preparation standard X (0.175%):

[0391] A 1:2 molar ratio CaCl2 and MgCl2 stock solution has a water hardness of 6000 dH.

[0392] Weigh 104.9g of CaCl2·2H2O (0.713M) into a 1L flask, add 500ml of type I water and stir thoroughly. Weigh and add 72.5g of MgCl2·6H2O (0.357M), dissolve thoroughly, and bring the final volume to 1000ml with type I water.

[0393] 0.535M NaHCO3 solution

[0394] Dissolve 44.9g of sodium bicarbonate in 100ml of type I water.

[0395] The standard X detergent, containing X ionic surfactant, has a water hardness of 12 (12 dH).

[0396] Weigh 1.75 g of standard X detergent (as described above) and transfer it to a 1 L bottle. Add 800 ml of type I water and mix thoroughly. Add 35 mg of X ionic surfactant and mix thoroughly. To adjust the water hardness to 12 dH, add 2 ml of a 1:2 molar ratio CaCl2 and MgCl2 stock solution (6000 dH) and 6 ml of a 0.535 mol NaHCO3 solution, and mix thoroughly. Finally, bring the volume to 1000 ml and stir the mixture for 10 min.

[0397] Preparation standard A (0.33%):

[0398] A 4:1 molar ratio CaCl2 and MgCl2 stock solution has a water hardness of 6000 dH.

[0399] Weigh 125.8g of CaCl2·2H2O into a 1-liter flask, add 500ml of type I water, and stir thoroughly. Weigh and add 43.8g of MgCl2·6H2O, dissolve completely, and then bring the final volume to 1000ml with type I water.

[0400] 0.535M NaHCO3 solution

[0401] Dissolve 44.9g of sodium bicarbonate in 100ml of type I water.

[0402] Standard A detergent has a water hardness of 15 (15 dH).

[0403] Weigh 3.335 g of standard detergent A and transfer it to a 1-liter bottle. Add 865 ml of type I water and mix thoroughly. Add 7.5 ml of 0.535 M NaHCO3, mix thoroughly, and bring the volume to 1 liter with type I water. To adjust the water hardness to 15 dH, add 2.5 ml of a 4:1 molar ratio CaCl2·2H2O and MgCl2·6H2O stock solution (6000 dH) and stir the mixture for 15 min.

[0404] Substrate: Phadebas tablets (McGor Life Sciences)

[0405] Suspend one tablet in 10 ml of detergent solution.

[0406] Buffer: 100mM MOPS buffer, pH 8

[0407] Experimental Procedure

[0408] Preparation of mother plate:

[0409] Colonies were picked from the transformed plate using a colony picker (K ​​Biosystems) and inoculated into 96-well plates containing TBGly medium for growth. The cultures were incubated at 37°C for 3 days, and the supernatant was recovered from the plate by centrifugation.

[0410] Preparation of substrate plate:

[0411] The substrate solution was prepared by dissolving one phadebas tablet in 10 ml of standard X / standard A detergent, and 180 μl of the solution was dispensed into a 96-well microtiter plate using a multi-point instrument with continuous stirring.

[0412] Dilute the culture supernatant 100-fold with buffer, and add 20 μl of the diluted culture to 180 μl of pre-split substrate plate and mix thoroughly. Incubate the plate at 25 °C for 20 min with shaking (900 rpm). After incubation, allow the plate to settle for 5 min. Transfer 50 μl of supernatant to a 384-well plate and measure the absorbance at 620 nm. Determine the concentration of the expressed enzyme using a specific antibody via ELISA. Specific activity is calculated by taking the ratio of activity to concentration, and all activities identified as having a higher specific activity than the parental α-amylase are included. The improvement factor (IF) is calculated as follows: [specific activity of variant] / [specific activity of parental α-amylase].

[0413] Stability of variants of the invention

[0414] Substrate: Phadebas tablets (McGor Life Sciences)

[0415] Suspend one tablet in 10 ml of detergent solution.

[0416] Measurement procedure:

[0417] Transfer 20 μL of culture supernatant to two 96-well plates named "Non-Stressed" and "Stressed". Add 80 μL of detergent mixed with EDTA and mix thoroughly. Incubate the non-stressed plate at 4 °C for 16 hours and the stressed plate at 43 °C for 16 hours. Dilute the non-stressed and stressed samples 20-fold in buffer. Add 20 μL of the diluted culture to the pre-split substrate plate and mix thoroughly. Incubate the plate at 25 °C for 20 min with shaking (900 rpm). After incubation, allow the plate to settle for 5 min. Transfer 50 μL of supernatant to a 384-well plate and measure the absorbance at 620 nm.

[0418] Stability is calculated as the percentage of residual activity (%RA) between stressed and unstressed samples (%RA = stress / unstressed * 100). Improvement factor (IF) is calculated as: %RA of variant / %RA of WT.

[0419] The specific activities of the variants of the present invention obtained by the above method are shown in Table 5 below (amino acid substitution refers to SEQ ID NO:3).

[0420] Table 5: Specific variants of the invention.

[0421]

[0422]

[0423] The invention described and claimed herein is not limited to the specific aspects disclosed herein, as these aspects are intended to illustrate several aspects of the invention. Any equivalent aspects are intended to be within the scope of the invention. In fact, various modifications to the invention, other than 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 case of conflict, the disclosure including the definition shall prevail. sequence list <110> Novozymes A / S <120> α-Amylase variants and the polynucleotides that encode them <130> 13069-WO-PCT <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 1458 <212> DNA <213> Bacillus species <400> 1 catcacgatg ggacgaacgg aacgattatg cagtattttg aatggaacgt tccgaatgat 60 ggacaacatt ggaaccgctt acacaacaac gctcaaaatt taaaaaatgc cggaattaca 120 gcaatctgga ttccacctgc gtggaaagga acgagccaaa atgatgtagg ctacggtgcg 180 tatgaccttt atgaccttgg tgaatttaac caaaaaggaa cggtccgtac gaaatatgga 240 acaaaagcag aattagaacg agcgattcgt tcgttaaagg cgaacgggat tcaagtgtat 300 ggcgatgttg ttatgaacca taaaggcgga gctgatttca ccgagcgtgt tcaagcggtt 360 gaagtgaacc cgcaaaaccg aaaccaagaa gtgtctggca cttatcaaat cgaagcatgg 420 acagggttca attttcctgg acgtggcaat caacattctt cgtttaaatg gcgctggtat 480 catttcgatg ggacggattg ggaccagtct cgccaactcg caaatcgtat ttataagttt 540 agaggagacg gaaaagcatg ggactgggaa gttgacactg aaaatgggaa ctatgattac 600 ttaatgtatg cagacgttga catggatcat ccagaagtga ttaacgaact aaaccgttgg 660 ggcgtctggt acgcgaatac ccttaattta gacggcttcc gactggatgc agtgaaacat 720 attaaattta gcttcatgcg tgattggtta gggcatgttc gcgggcaaac gggcaagaat 780 ctttttgccg ttgcagagta ttggaagaat gacctagggg ctttagaaaa ttatttaagc 840 aaaacaaatt ggacgatgag cgcctttgat gttccgcttc attacaacct ttatcaagcg 900 tcaaatagta gcggaaatta cgacatgaga aacttgttaa atggaacact cgttcaacgt 960 catccgagcc atgcggttac gtttgtcgat aaccacgaca cacagcctgg agaagccctc 1020 gaatcgttcg ttcaaggctg gtttaaacca ctagcttatg caacgattct tacgagagag 1080 caaggctacc cacaagtgtt ttacggcgat tattatggca tcccaagtga cggtgttcca 1140 agctaccgtc aacagatcga cccactttta aaagctcgtc aacaatatgc ttatggtaga 1200 cagcacgatt actttgatca ttgggatgta attggctgga cacgtgaagg aaacgcatct 1260 cacccgaact caggacttgc aaccattatg tctgatggtc caggtggatc aaaatggatg 1320 tatgttggcc gtcagaaagc tggcgaagtg tggcatgaca tgactggaaa ccgcagtggc 1380 actgtgacaa ttaatcaaga cggctgggga cacttttttg tcaacggcgg ctctgtctcc 1440 gtatgggtga aacgataa 1458 <210> 2 <211> 514 <212> PRT <213> Bacillus species <220> <221> Mature polypeptide <222> (30)..(514) <400> 2 Met Asn Arg Trp Lys Ala Ala Phe Ser Trp Met Leu Ser Leu Ala Leu ‑25 ‑20 ‑15 Val Phe Thr Leu Phe Tyr Thr Pro Ser Ser Ala Ser Ala His His Asp ‑10 ‑5 ‑1 1 Gly Thr Asn Gly Thr Ile Met Gln Tyr Phe Glu Trp Asn Val Pro Asn 5 10 15 Asp Gly Gln His Trp Asn Arg Leu His Asn Asn Ala Gln Asn Leu Lys 20 25 30 35 Asn Ala Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Trp Lys Gly Thr 40 45 50 Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu Gly 55 60 65 Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Ala 70 75 80 Glu Leu Glu Arg Ala Ile Arg Ser Leu Lys Ala Asn Gly Ile Gln Val 85 90 95 Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp Phe Thr Glu 100 105 110 115 Arg Val Gln Ala Val Glu Val Asn Pro Gln Asn Arg Asn Gln Glu Val 120 125 130 Ser Gly Thr Tyr Gln Ile Glu Ala Trp Thr Gly Phe Asn Phe Pro Gly 135 140 145 Arg Gly Asn Gln His Ser Ser Phe Lys Trp Arg Trp Tyr His Phe Asp 150 155 160 Gly Thr Asp Trp Asp Gln Ser Arg Gln Leu Ala Asn Arg Ile Tyr Lys 165 170 175 Phe Arg Gly Asp Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu Asn 180 185 190 195 Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met Asp His Pro 200 205 210 Glu Val Ile Asn Glu Leu Asn Arg Trp Gly Val Trp Tyr Ala Asn Thr 215 220 225 Leu Asn Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe 230 235 240 Ser Phe Met Arg Asp Trp Leu Gly His Val Arg Gly Gln Thr Gly Lys 245 250 255 Asn Leu Phe Ala Val Ala Glu Tyr Trp Lys Asn Asp Leu Gly Ala Leu 260 265 270 275 Glu Asn Tyr Leu Ser Lys Thr Asn Trp Thr Met Ser Ala Phe Asp Val 280 285 290 Pro Leu His Tyr Asn Leu Tyr Gln Ala Ser Asn Ser Ser Gly Asn Tyr 295 300 305 Asp Met Arg Asn Leu Leu Asn Gly Thr Leu Val Gln Arg His Pro Ser 310 315 320 His Ala Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly Glu Ala 325 330 335 Leu Glu Ser Phe Val Gln Gly Trp Phe Lys Pro Leu Ala Tyr Ala Thr 340 345 350 355 Ile Leu Thr Arg Glu Gln Gly Tyr Pro Gln Val Phe Tyr Gly Asp Tyr 360 365 370 Tyr Gly Ile Pro Ser Asp Gly Val Pro Ser Tyr Arg Gln Gln Ile Asp 375 380 385 Pro Leu Leu Lys Ala Arg Gln Gln Tyr Ala Tyr Gly Arg Gln His Asp 390 395 400 Tyr Phe Asp His Trp Asp Val Ile Gly Trp Thr Arg Glu Gly Asn Ala 405 410 415 Ser His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp Gly Pro Gly 420 425 430 435 Gly Ser Lys Trp Met Tyr Val Gly Arg Gln Lys Ala Gly Glu Val Trp 440 445 450 His Asp Met Thr Gly Asn Arg Ser Gly Thr Val Thr Ile Asn Gln Asp 455 460 465 Gly Trp Gly His Phe Phe Val Asn Gly Gly Ser Val Ser Val Trp Val 470 475 480 Lys Arg 485 <210> 3 <211> 485 <212> PRT <213> Bacillus species <400> 3 His His Asp Gly Thr Asn Gly Thr Ile Met Gln Tyr Phe Glu Trp Asn 1 5 10 15 Val Pro Asn Asp Gly Gln His Trp Asn Arg Leu His Asn Asn Ala Gln 20 25 30 Asn Leu Lys Asn Ala Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Trp[[ID=N]] 35 40 45 Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60 Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly 65 70 75 80 Thr Lys Ala Glu Leu Glu Arg Ala Ile Arg Ser Leu Lys Ala Asn Gly 85 90 95 Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110 Phe Thr Glu Arg Val Gln Ala Val Glu Val Asn Pro Gln Asn Arg Asn 115 120 125 Gln Glu Val Ser Gly Thr Tyr Gln Ile Glu Ala Trp Thr Gly Phe Asn 130 135 140 Phe Pro Gly Arg Gly Asn Gln His Ser Ser Phe Lys Trp Arg Trp Tyr 145 150 155 160 His Phe Asp Gly Thr Asp Trp Asp Gln Ser Arg Gln Leu Ala Asn Arg 165 170 175 Ile Tyr Lys Phe Arg Gly Asp Gly Lys Ala Trp Asp Trp Glu Val Asp 180 185 190 Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met 195 200 205 Asp His Pro Glu Val Ile Asn Glu Leu Asn Arg Trp Gly Val Trp Tyr 210 215 220 Ala Asn Thr Leu Asn Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His 225 230 235 240 Ile Lys Phe Ser Phe Met Arg Asp Trp Leu Gly His Val Arg Gly Gln 245 250 255 Thr Gly Lys Asn Leu Phe Ala Val Ala Glu Tyr Trp Lys Asn Asp Leu 260 265 270 Gly Ala Leu Glu Asn Tyr Leu Ser Lys Thr Asn Trp Thr Met Ser Ala 275 280 285 Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Gln Ala Ser Asn Ser Ser 290 295 300 Gly Asn Tyr Asp Met Arg Asn Leu Leu Asn Gly Thr Leu Val Gln Arg 305 310 315 320 His Pro Ser His Ala Val Thr Phe Val Asp Asn His Asp Thr Gln Pro 325 330 335 Gly Glu Ala Leu Glu Ser Phe Val Gln Gly Trp Phe Lys Pro Leu Ala 340 345 350 Tyr Ala Thr Ile Leu Thr Arg Glu Gln Gly Tyr Pro Gln Val Phe Tyr 355 360 365 Gly Asp Tyr Tyr Gly Ile Pro Ser Asp Gly Val Pro Ser Tyr Arg Gln 370 375 380 Gln Ile Asp Pro Leu Leu Lys Ala Arg Gln Gln Tyr Ala Tyr Gly Arg 385 390 395 400 Gln His Asp Tyr Phe Asp His Trp Asp Val Ile Gly Trp Thr Arg Glu 405 410 415 Gly Asn Ala Ser His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425 430 Gly Pro Gly Gly Ser Lys Trp Met Tyr Val Gly Arg Gln Lys Ala Gly 435 440 445 Glu Val Trp His Asp Met Thr Gly Asn Arg Ser Gly Thr Val Thr Ile 450 455 460 Asn Gln Asp Gly Trp Gly His Phe Phe Val Asn Gly Gly Ser Val Ser 465 470 475 480 Val Trp Val Lys Arg 485 <210> 4 <211> 483 <212> PRT <213> Bacillus species <400> 4 His His Asp Gly Thr Asn Gly Thr Ile Met Gln Tyr Phe Glu Trp Asn 1 5 10 15 Val Pro Asn Asp Gly Gln His Trp Asn Arg Leu His Asn Asn Ala Gln 20 25 30 Asn Leu Lys Asn Ala Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Trp 35 40 45 Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60 Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly 65 70 75 80 Thr Lys Ala Glu Leu Glu Arg Ala Ile Arg Ser Leu Lys Ala Asn Gly 85 90 95 Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110 Phe Thr Glu Arg Val Gln Ala Val Glu Val Asn Pro Gln Asn Arg Asn 115 120 125 Gln Glu Val Ser Gly Thr Tyr Gln Ile Glu Ala Trp Thr Gly Phe Asn 130 135 140 Phe Pro Gly Arg Gly Asn Gln His Ser Ser Phe Lys Trp Arg Trp Tyr 145 150 155 160 His Phe Asp Gly Thr Asp Trp Asp Gln Ser Arg Gln Leu Ala Asn Arg 165 170 175 Ile Tyr Lys Phe Arg Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu 180 185 190 Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met Asp His 195 200 205 Pro Glu Val Ile Asn Glu Leu Asn Arg Trp Gly Val Trp Tyr Ala Asn 210 215 220 Thr Leu Asn Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys 225 230 235 240 Phe Ser Phe Met Arg Asp Trp Leu Gly His Val Arg Gly Gln Thr Gly 245 250 255 Lys Asn Leu Phe Ala Val Ala Glu Tyr Trp Lys Asn Asp Leu Gly Ala 260 265 270 Leu Glu Asn Tyr Leu Ser Lys Thr Asn Trp Thr Met Ser Ala Phe Asp 275 280 285 Val Pro Leu His Tyr Asn Leu Tyr Gln Ala Ser Asn Ser Ser Gly Asn 290 295 300 Tyr Asp Met Arg Asn Leu Leu Asn Gly Thr Leu Val Gln Arg His Pro 305 310 315 320 Ser His Ala Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly Glu 325 330 335 Ala Leu Glu Ser Phe Val Gln Gly Trp Phe Lys Pro Leu Ala Tyr Ala 340 345 350 Thr Ile Leu Thr Arg Glu Gln Gly Tyr Pro Gln Val Phe Tyr Gly Asp 355 360 365 Tyr Tyr Gly Ile Pro Ser Asp Gly Val Pro Ser Tyr Arg Gln Gln Ile 370 375 380 Asp Pro Leu Leu Lys Ala Arg Gln Gln Tyr Ala Tyr Gly Arg Gln His 385 390 395 400 Asp Tyr Phe Asp His Trp Asp Val Ile Gly Trp Thr Arg Glu Gly Asn 405 410 415 Ala Ser His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp Gly Pro 420 425 430 Gly Gly Ser Lys Trp Met Tyr Val Gly Arg Gln Lys Ala Gly Glu Val 435 440 445 Trp His Asp Met Thr Gly Asn Arg Ser Gly Thr Val Thr Ile Asn Gln 450 455 460 Asp Gly Trp Gly His Phe Phe Val Asn Gly Gly Ser Val Ser Val Trp 465 470 475 480 Val Lys Arg <210> 5 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence <400> 5 caatccaaga gaaccctgat acggatg 27 <210> 6 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Artificial sequence <400> 6 cggaacgcct ggctgacaac acg 23

Claims

1. An α-amylase variant, wherein the variant has α-amylase activity, and wherein the variant is substituted at position H1 in SEQ ID NO: 3 with H1V, H1G, H1S, or H1C, wherein the position corresponds to an amino acid position in the amino acid sequence shown in SEQ ID NO: 3, wherein the α-amylase variant has an improvement factor of >1.0 for a measure of specific activity in a Model A or Model X detergent composition compared to the parental α-amylase shown in SEQ ID NO:

3.

2. A polynucleotide encoding the α-amylase variant according to claim 1.

3. A nucleic acid construct comprising the polynucleotide as described in claim 2.

4. An expression vector comprising the polynucleotide according to claim 2.

5. A host cell comprising the polynucleotide of claim 2, the nucleic acid construct of claim 3, or the expression vector of claim 4.

6. A composition comprising the α-amylase variant according to claim 1.

7. The composition according to claim 6, further comprising at least one additional active component.

8. The composition according to claim 7, wherein the additional active ingredient is an enzyme, wherein the enzyme is selected from protease, lipase, cellulase, pectic acid lyase or mannanase.

9. The composition according to any one of claims 6 to 8, wherein the composition is a detergent composition, and the detergent composition is a liquid detergent composition or a powdered detergent composition.

10. The composition according to any one of claims 6 to 8, wherein the composition is a liquid laundry detergent or liquid tableware detergent composition, or a powdered laundry detergent or a powdered tableware detergent composition.

11. The composition of claim 10, wherein the composition is an automatic dishwashing (ADW) liquid detergent composition, or the composition is a soap bar, or the composition is an ADW unit dose detergent composition.

Images