Alpha-amylase variants

Abstract

The present invention relates to alpha-amylase variants, in particular to variants of a parent alpha-amylase. The present invention also relates to polynucleotides encoding these variants, nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

Description

[0001] This invention application is a divisional application of the invention patent application filed on June 29, 2012, with application number 201280032708.4 (international application number PCT / EP2012 / 062748) entitled "α-Amylase Variant".

[0002] References to sequence lists

[0003] This application contains a sequence list in a computer-readable form, which is incorporated herein by reference. Background Technology Invention Field

[0004] The present invention relates to variants of α-amylase, polynucleotides encoding these variants, methods for producing these variants, and methods for using these variants.

[0005] Description of related technologies

[0006] α-Amylase (α-1,4-glucan-4-glucan hydrolase, EC3.2.1.1) constitutes a group of enzymes that catalyze the hydrolysis of starch and other linear and branched 1,4-glycoside oligosaccharides and polysaccharides.

[0007] Alpha-amylase has a long history of industrial use in several known applications such as detergents, baking, brewing, starch liquefaction, and saccharification, for example in the preparation of high-fructose syrups, or as a component in the production of ethanol from starch. These and other applications of alpha-amylase are known and utilize alpha-amylases derived from microorganisms, particularly bacterial alpha-amylases.

[0008] The first bacterial α-amylase to be used was that from Bacillus licheniformis, also known as Termamyl, which has been well characterized and whose crystal structure has been determined. Alkaline amylases (such as AA560) form a specific group of α-amylases already used in detergents.

[0009] Many of these known bacterial amylases have been modified to improve their functionality in specific applications.

[0010] Methods for increasing the thermostability of α-amylases have been extensively studied. Suzuki et al. (1989) disclosed a chimeric α-amylase in which a specific region of Bacillus amyloliquefaciens α-amylase was replaced with a corresponding region of Bacillus licheniformis α-amylase. The purpose of constructing this chimeric α-amylase was to identify the region responsible for thermostability. This region was found to contain amino acid residues 177-186 and 255-270 of Bacillus amyloliquefaciens α-amylase.

[0011] Igarashi et al. (1998) showed that the thermostability of AmyS-type amylases could be increased by deleting two amino acid residues R179-G180 (AmyS number) from the ring (F 178 to A184). However, Shiau et al. (2003) showed that AmyS enzymes with deletions in the same ring had lower corn starch hydrolysis specific activity at high temperatures than their parent enzymes, thus negating one of the main advantages of AmyS amylases.

[0012] For environmental reasons, lowering temperatures during washing, dishwashing, and / or cleaning processes has become increasingly important. However, most enzymes, including amylases, have an optimal temperature above the temperatures typically used in low-temperature washing. α-Amylases are key enzymes used in detergent compositions, and their use for removing starch stains during laundry or dishwashing has become increasingly important. Therefore, it is important to find α-amylase variants that retain their detergent performance, stain removal effect, and / or activity at lower temperatures. However, despite the efficiency of current detergent enzyme compositions, many stains remain difficult to remove completely. These problems are exacerbated by increased washing temperatures (e.g., cold water) and shorter wash cycles. Therefore, there is a need for starch-degrading enzymes that can function at low temperatures while retaining or increasing other desirable properties such as specific activity (starch-degrading activity), stability, and / or detergent performance.

[0013] Therefore, an object of the present invention is to provide an α-amylase variant that can be used in washing, dishwashing, and / or cleaning processes at low temperatures, such as 5°C-35°C. Another object of the present invention is to provide an α-amylase variant that has improved low-temperature washing performance compared to the parental α-amylase or compared to the α-amylase of any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. Invention Summary

[0014] This invention relates to isolated variants of parental α-amylase, said variant comprising alterations at two or more positions corresponding to the G304, W140, W189, D134, E260, F262, W284, W347, W439, W469, G476, and G477 positions of the mature polypeptide of SEQ ID NO:1, wherein each alteration is independently a substitution, deletion, or insertion, and said variant has at least 80% but less than 100% sequence identity with the mature polypeptide of any one of SEQ ID NOs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, and said variant has α-amylase activity.

[0015] The present invention also relates to isolated polynucleotides containing these variants, nucleic acid constructs containing these polynucleotides, vectors and host cells; and methods for producing these variants.

[0016] The present invention also relates to methods for preparing such variants.

[0017] Detailed Description of the Invention

[0018] This invention relates to isolated variants of parental α-amylase, said variant comprising alterations at two or more positions corresponding to the G304, W140, W189, D134, E260, F262, W284, W347, W439, W469, G476, and G477 positions of the mature polypeptide of SEQ ID NO:1, wherein each alteration is independently a substitution, deletion, or insertion, and said variant has at least 80% but less than 100% sequence identity with the mature polypeptide of any one of SEQ ID NOs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, and said variant has α-amylase activity.

[0019] definition

[0020] α-Amylase activity: The term “α-amylase activity” refers to the activity of α-1,4-glucan-4-glucan hydrolases (EC3.2.1.1), which constitute a group of enzymes that catalyze the hydrolysis of starch and other linear and branched 1,4-glycoside oligosaccharides and polysaccharides.

[0021] Variants: The term "variant" refers to a polypeptide with altered (i.e., substitution, insertion, and / or deletion) α-amylase activity at one or more 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 1-3 amino acids adjacent to an amino acid occupying a position.

[0022] Mutant: The term “mutant” refers to a polynucleotide that encodes a variant.

[0023] Wild-type enzymes: The term "wild-type" α-amylase refers to α-amylase expressed by naturally occurring microorganisms (such as bacteria, yeast, or filamentous fungi found in nature).

[0024] Parent or parental α-amylase: The term "parent" or "parental α-amylase" refers to an α-amylase that has been modified to produce the enzyme variant of the present invention. The parent can be a naturally occurring (wild-type) polypeptide or a variant thereof.

[0025] Separated variants: The term “separated variants” means variants that have been artificially modified. In one respect, the variant is at least 1% pure, for example at least 5% pure, at least 10% pure, at least 20% pure, at least 40% pure, at least 60% pure, at least 80% pure, and at least 90% pure, as determined by SDS-PAGE.

[0026] Substantially pure variant: The term "substantially pure variant" means a formulation containing, by weight, up to 10%, up to 8%, up to 6%, up to 5%, up to 4%, up to 3%, up to 2%, up to 1%, and up to 0.5% of other polypeptide substances naturally or recombinantly bound to the polypeptide. Preferably, the variant is at least 92% pure, for example, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, and 100% pure, based on the total polypeptide substance present in the formulation. The variants of the present invention are preferably in a substantially pure form. This can be achieved, for example, by well-known recombinant methods or by classical purification methods.

[0027] Mature polypeptide: The term "mature polypeptide" refers to a polypeptide in its final form after translation and any post-translational modifications such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.

[0028] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" refers to the polynucleotide that encodes a mature polypeptide with α-amylase activity.

[0029] Sequence consistency: The association between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence consistency".

[0030] For the purposes of this invention, the degree of sequence identity between two amino acid sequences was determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol. 48:443-453), as performed in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, TrendsGenet. 16:276-277), preferably version 3.0.0 or later. Optional parameters used were a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The Needle-marked "Longest Consistency" output (obtained using the –nobrief option) was used as the consistency percentage and calculated as follows:

[0031] (identical residues x 100) / (length of alignment - total number of vacancies in alignment)

[0032] For the purposes of this invention, the degree of sequence identity between two deoxyribonucleotide sequences was determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, above), as performed in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, above), preferably version 3.0.0 or later. Optional parameters used were a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EDNAFULL (EMBOSS version of NCBINUC 4.4) substitution matrix. The Needle-labeled “Longest Consistency” output (obtained using the –nobrief option) was used as the consistency percentage and calculated as follows:

[0033] (identical deoxyribonucleotides x 100) / (alignment length - total number of vacancies in the alignment)

[0034] The term "fragment" refers to a polypeptide in which one or more amino acids are removed from the amino and / or carboxyl termini of a mature polypeptide; wherein the fragment has α-amylase activity.

[0035] Subsequence: The term “subsequence” refers to a polynucleotide in which one or more nucleotides are deleted from the 5’ and / or 3’ ends of the coding sequence of a mature polypeptide; wherein the subsequence encodes a fragment having α-amylase activity.

[0036] Allelic variants: The term "allelic variant" refers to any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variations occur naturally through mutation and can lead to polymorphism within a population. Gene mutations can be silent (without change in the encoded polypeptide) or can encode a polypeptide with a modified amino acid sequence. Allelic variants of a polypeptide are polypeptides encoded by allelic variants of a gene.

[0037] Isolated polynucleotides: The term "isolated polynucleotide" refers to a polynucleotide that has been artificially modified. In one respect, isolated polynucleotides are at least 1% pure, for example at least 5% pure, at least 10% pure, at least 20% pure, at least 40% pure, at least 60% pure, at least 80% pure, at least 90% pure, and at least 95% pure, as determined by agarose gel electrophoresis. Polynucleotides can be genomic, cDNA, RNA, semi-synthetic, synthetically derived, or any combination thereof.

[0038] Substantially Pure Polynucleotide: The term "substantially pure polynucleotide" means a polynucleotide formulation that is free of other external or unwanted nucleotides and is in a form suitable for use within a gene-modified polypeptide production system. Thus, a substantially pure polynucleotide contains, by weight, up to 10%, up to 8%, up to 6%, up to 5%, up to 4%, up to 3%, up to 2%, up to 1%, and up to 0.5% of other polynucleotides naturally or recombinantly bound to that polynucleotide. However, substantially pure polynucleotides may contain naturally occurring 5' and 3' untranslated regions, such as promoters and terminators. Preferably, the substantially pure polynucleotide is at least 90% pure by weight, for example, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and at least 99.5%. The polynucleotides of the present invention are preferably in a substantially pure form.

[0039] Coding sequence: The term "coding sequence" refers to a polynucleotide that directly specifies the amino acid sequence of its polypeptide product. The boundaries of a coding sequence are typically determined by an open reading frame (ORF), which usually begins with an ATG start codon or alternative start codons such as GTG and TTG and ends with a stop codon such as TAA, TAG, and TGA. Coding sequences can be DNA, cDNA, synthetic, or recombinant polynucleotides.

[0040] cDNA: The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from mature spliced ​​mRNA molecules obtained from eukaryotic cells. cDNA lacks intron sequences that can be present in the corresponding genomic DNA. The initial primary RNA transcript is a precursor of mRNA, which undergoes a series of steps (including splicing) before it appears as mature spliced ​​mRNA.

[0041] Nucleic acid construct: The term "nucleic acid construct" refers to a single-stranded or double-stranded nucleic acid molecule isolated from a naturally occurring gene or modified to contain nucleic acid segments in a form not normally found in nature or synthetically produced. When the nucleic acid construct contains the control sequence required to express the coding sequence of this invention, the term "nucleic acid construct" is synonymous with the term "expression cassette".

[0042] The term "control sequence" refers to all components necessary for the expression of polynucleotides encoding variants of the present invention. Each control sequence may be native or foreign relative to the polynucleotide encoding the variant, or may be native or foreign to each other. Such control sequences include, but are not limited to, leader sequences, polyadenylated sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At a minimum, control sequences include promoters and transcription and translation termination signals. Control sequences may be fitted with adapters to introduce specific restriction sites that facilitate the connection of the control sequence to the coding region of the polynucleotide encoding the variant.

[0043] Operable ligation: The term “operable ligation” refers to a configuration in which a control sequence is positioned relative to the coding sequence of a polynucleotide so that the control sequence guides the expression of the coding sequence.

[0044] Expression: The term “expression” includes any steps involved in variant generation, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0045] Expression vector: The term “expression vector” refers to a linear or circular DNA molecule that contains a polynucleotide encoding a variant and is operatively linked to additional nucleotides that cause its expression.

[0046] Host cell:

[0047] The term "host cell" refers to any cell type that is readily transformed, transfected, transduced, etc., using nucleic acid constructs or expression vectors containing the polynucleotides of this invention. The term "host cell" also encompasses any parental cell progeny that is not entirely identical to the parental cell due to mutations occurring during replication.

[0048] Starch Removal Processes: The term "starch removal process" refers to any kind of process by which starch is removed (or converted), such as a washing process in which starch is removed from textiles, for example, textile cleaning such as laundry. Starch removal processes can also be hard surface cleaning, such as dishwashing, or it can be a general cleaning process, such as industrial cleaning or institutional cleaning. This term typically also includes other starch removal processes or starch conversion, ethanol production, starch liquefaction, textile desizing, paper and pulp production, brewing, and detergents.

[0049] Improved properties: The term "improved properties" refers to characteristics associated with variants that are improved compared to the parent. Such improved properties include, but are not limited to, thermal activity, thermal stability, pH activity, pH stability, substrate / cofactor specificity, improved surface properties, product specificity, increased stability or solubility in the presence of pretreated biomass, improved stability under storage conditions, and chemical stability.

[0050] Washing performance: In the context of this invention, the term "washing performance" is used as the ability of an enzyme to remove starch or starch-containing stains present on the object to be cleaned, such as during the cleaning of clothing or hard surfaces like dishwashing. Washing performance can be quantified by calculating the so-called intensity value (Int) defined in the AMSA description and in the beaker washing performance test below in the Methods section.

[0051] Improved washing performance: The term "improved washing performance" is defined herein as the washing performance relative to the parental amylase, or relative to the activity of an α-amylase having the same amino acid sequence as the variant but without deletions at one or more designated positions, or relative to the activity of an α-amylase having the amino acid sequence shown in SEQ ID NO:4, where the variant enzyme exhibits altered washing performance, such as increased stain removal. The term "washing performance" includes general cleaning such as hard surface cleaning, as in dishwashing, and also includes washing performance on textiles such as clothing, and also includes industrial and institutional cleaning.

[0052] Low temperature: "Low temperature" is a temperature of 5℃-35℃, preferably 5℃-30℃, more preferably 5℃-25℃, even more preferably 5℃-20℃, most preferably 5℃-15℃, and particularly 5℃-10℃.

[0053] In a preferred embodiment, "low temperature" is a temperature of 10°C-35°C, preferably 10°C-30°C, more preferably 10°C-25°C, most preferably 10°C-20°C, and particularly 10°C-15°C.

[0054] The rules for variant naming are for the purposes of this invention, and the mature polypeptide disclosed in SEQ ID NO:1 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 polypeptides disclosed in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. Based on the comparison results, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol. 48:443-453) was used, preferably in version 3.0.0 or later of the Needle program in the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), to determine the amino acid position number corresponding to any amino acid residue in the mature polypeptide disclosed in SEQ ID NO:1.

[0055] The identification of the corresponding amino acid residues in another α-amylase can be confirmed by comparing multiple polypeptide sequences using "ClustalW" (Larkin et al., 2007, Bioinformatics 23:2947-2948).

[0056] When another enzyme has deviated from the mature polypeptides of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, making it impossible for conventional sequence-based comparison methods to detect their relationship (Lindahl and Elofsson, 2000, J.Mol.Biol. 295:613-615), alternative paired sequence comparison algorithms can be used. Higher sensitivity can be achieved in sequence-based searches using search procedures that utilize probabilistic representations (spectrums) of polypeptide families to retrieve data. For example, the PSI-BLAST procedure generates spectra through an iterative database search process and is able to 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, Journal of Molecular Biology 287:797-815; McGuffin and Jones, 2003, Bioinformatics 19:874-881) utilize information from multiple sources (PSI-BLAST, secondary structure prediction, structural alignment spectra, and solvation potential) as input to a neural network predicting the structural folding of a query sequence. Similarly, the method of Gough et al., 2000, Journal of Molecular Biology 313:903-919 can be used to align structurally unknown sequences with superfamily models existing in the SCOP database. These alignment results can then be used to generate homology models of peptides, and the accuracy of such models can be evaluated using various tools developed for this purpose.

[0057] 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 been structurally aligned, and these alignments are available and downloadable. Various algorithms, such as distance alignment matrices (Holm and Sander, Proteins 33:88-96) or combined extensions (Shindyalov and Bourne, 1998, Protein Engineering 11:739-747), can be used to align two or more protein structures, and implementations of these algorithms can be further used to query structure databases containing structures of interest to discover potential structural homologs (e.g., Holm and Park, 2000, Bioinformatics 16:566-567).

[0058] In describing the α-amylase variants of the present invention, the nomenclature described below is used for ease of reference. Recognized IUPAC single-letter or three-letter amino acid abbreviations are used.

[0059] Replacement For amino acid substitutions, the following nomenclature is used: original amino acid, position, and the amino acid to be substituted. Therefore, the substitution of threonine at position 226 with alanine is named "Thr226Ala" or "T226A".

[0060] Multiple mutations are separated by plus signs ("+"), for example, "Gly205Arg+Ser411Phe" or "G205R+S411F" means that glycine (G) is replaced with arginine (R) at positions 205 and 411, respectively, and serine (S) is replaced with phenylalanine (F).

[0061] Missing For amino acid deletions, the following nomenclature is used: original amino acid, position *. Therefore, glycine deleted at position 195 is named "Gly195*" or "G195*". Multiple deletions are separated by plus signs ("+"), for example, "Gly195*+Ser411*" or "G195*+S411*".

[0062] insert For amino acid insertions, the following nomenclature is used: original amino acid, position, original amino acid, inserted amino acid. Therefore, inserting lysine after glycine at position 195 would be named "Gly195GlyLys" or "G195GK". Insertions of multiple amino acids are named as [original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2; etc.]. For example, inserting lysine and alanine after glycine at position 195 would be named "Gly195GlyLysAla" or "G195GKA".

[0063] In this case, the inserted amino acid residue is numbered according to the position number of the amino acid residue preceding the inserted amino acid residue.

[0064] In the above example, the sequence would therefore be:

[0065] 195 195 195a 195b G GKA

[0066] Variants containing multiple changes are separated by ("+") plus signs, for example, "Arg170Tyr+Gly195Glu" or "R170Y+G195E" represent the substitution of arginine and glycine at positions 170 and 195 with tyrosine and glutamic acid, respectively.

[0067] Different permutationsWhere different substitutions can be introduced at a single position, the different substitutions are separated by commas. For example, "Arg170Tyr,Glu" means that arginine is replaced by tyrosine or glutamic acid at position 170. Therefore, "Tyr167Gly,Ala+Arg170Gly,Ala" indicates the following variants: "Tyr167Gly+Arg170Gly", "Tyr167Gly+Arg170Ala", "Tyr167Ala+Arg170Gly", and "Tyr167Ala+Arg170Ala".

[0068] The parental α-amylase can be a polypeptide with at least 80% sequence identity to the mature polypeptide of SEQ ID NO:1. In one aspect, the parent has at least 80%, for example, at least 85%, at least 87%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO:1, said mature polypeptide having α-amylase activity. In another aspect, the amino acid sequence of the parent is approximately ten amino acids different from that of the mature polypeptide of SEQ ID NO:1, for example, differing by five amino acids, four amino acids, three amino acids, two amino acids, and one amino acid.

[0069] The parent preferably comprises or consists of the amino acid sequence of SEQ ID NO:1. Alternatively, the parent comprises or consists of the mature polypeptide of SEQ ID NO:1.

[0070] In another embodiment, the parent is an allelic variant of the mature polypeptide of SEQ ID NO:1.

[0071] The parental α-amylase can also be a polypeptide with at least 80% sequence identity to the mature polypeptide of SEQ ID NO:2.

[0072] In another aspect, the parent has at least 80%, for example at least 85%, at least 87%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide of SEQ ID NO:2, said mature polypeptide having α-amylase activity. In another aspect, the amino acid sequence of the parent is approximately equal to or less than ten amino acids of the mature polypeptide of SEQ ID NO:2, for example, differing by five amino acids, four amino acids, three amino acids, two amino acids, and one amino acid.

[0073] The parent preferably comprises or consists of the amino acid sequence of SEQ ID NO:2. Alternatively, the parent comprises or consists of the mature polypeptide of SEQ ID NO:2.

[0074] In another embodiment, the parent is an allelic variant of the mature polypeptide of SEQ ID NO:2.

[0075] The parental α-amylase can also be a polypeptide with at least 80% sequence identity to the mature polypeptide of SEQ ID NO:3.

[0076] In another aspect, the parent has at least 80%, for example at least 85%, at least 87%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide of SEQ ID NO:3, said mature polypeptide having α-amylase activity. In another aspect, the amino acid sequence of the parent is approximately equal to or less than ten amino acids of the mature polypeptide of SEQ ID NO:3, for example, differing by five amino acids, four amino acids, three amino acids, two amino acids, and one amino acid.

[0077] The parent preferably comprises or consists of the amino acid sequence of SEQ ID NO:3. Alternatively, the parent comprises or consists of the mature polypeptide of SEQ ID NO:3.

[0078] In another embodiment, the parent is an allelic variant of the mature polypeptide of SEQ ID NO:3.

[0079] The parental α-amylase can also be a polypeptide with at least 80% sequence identity to the mature polypeptide of SEQ ID NO:4.

[0080] In another aspect, the parent has at least 80%, for example at least 85%, at least 87%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide of SEQ ID NO:4, said mature polypeptide having α-amylase activity. In another aspect, the amino acid sequence of the parent is approximately equal to or less than ten amino acids of the mature polypeptide of SEQ ID NO:4, for example differing by five amino acids, four amino acids, three amino acids, two amino acids, and one amino acid.

[0081] The parent preferably comprises or consists of the amino acid sequence of SEQ ID NO:4. Alternatively, the parent comprises or consists of the mature polypeptide of SEQ ID NO:4.

[0082] In another embodiment, the parent is an allelic variant of the mature polypeptide of SEQ ID NO:4.

[0083] The parental α-amylase can also be a polypeptide with at least 80% sequence identity to the mature polypeptide of SEQ ID NO:5.

[0084] In another aspect, the parent has at least 80%, for example at least 85%, at least 87%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide of SEQ ID NO:5, said mature polypeptide having α-amylase activity. In another aspect, the amino acid sequence of the parent is approximately equal to or less than ten amino acids of the mature polypeptide of SEQ ID NO:5, for example, differing by five amino acids, four amino acids, three amino acids, two amino acids, and one amino acid.

[0085] The parent preferably comprises or consists of the amino acid sequence of SEQ ID NO:5. Alternatively, the parent comprises or consists of the mature polypeptide of SEQ ID NO:5.

[0086] In another embodiment, the parent is an allelic variant of the mature polypeptide of SEQ ID NO:5.

[0087] The parental α-amylase can also be a polypeptide with at least 80% sequence identity to the mature polypeptide of SEQ ID NO:6.

[0088] In another aspect, the parent has at least 80%, for example at least 85%, at least 87%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide of SEQ ID NO:6, said mature polypeptide having α-amylase activity. In another aspect, the amino acid sequence of the parent is approximately equal to or less than ten amino acids of the mature polypeptide of SEQ ID NO:6, for example, differing by five amino acids, four amino acids, three amino acids, two amino acids, and one amino acid.

[0089] The parent preferably comprises or consists of the amino acid sequence of SEQ ID NO:6. Alternatively, the parent comprises or consists of the mature polypeptide of SEQ ID NO:6.

[0090] In another embodiment, the parent is an allelic variant of the mature polypeptide of SEQ ID NO:6.

[0091] The parental α-amylase can also be a polypeptide with at least 80% sequence identity to the mature polypeptide of SEQ ID NO:7.

[0092] In another aspect, the parent has at least 80%, for example at least 85%, at least 87%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide of SEQ ID NO:7, said mature polypeptide having α-amylase activity. In another aspect, the amino acid sequence of the parent is approximately equal to or less than ten amino acids of the mature polypeptide of SEQ ID NO:7, for example, differing by five amino acids, four amino acids, three amino acids, two amino acids, and one amino acid.

[0093] The parent preferably comprises or consists of the amino acid sequence of SEQ ID NO:7. Alternatively, the parent comprises or consists of the mature polypeptide of SEQ ID NO:7.

[0094] In another embodiment, the parent is an allelic variant of the mature polypeptide of SEQ ID NO:7.

[0095] The parental α-amylase can also be a polypeptide with at least 80% sequence identity to the mature polypeptide of SEQ ID NO:8.

[0096] In another aspect, the parent has at least 80%, for example at least 85%, at least 87%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide of SEQ ID NO:8, said mature polypeptide having α-amylase activity. In another aspect, the amino acid sequence of the parent is approximately equal to or less than ten amino acids of the mature polypeptide of SEQ ID NO:8, for example, differing by five amino acids, four amino acids, three amino acids, two amino acids, and one amino acid.

[0097] The parent preferably comprises or consists of the amino acid sequence of SEQ ID NO:8. Alternatively, the parent comprises or consists of the mature polypeptide of SEQ ID NO:8.

[0098] In another embodiment, the parent is an allelic variant of the mature polypeptide of SEQ ID NO:8.

[0099] The parental α-amylase can also be a polypeptide with at least 80% sequence identity to the mature polypeptide of SEQ ID NO:9.

[0100] In another aspect, the parent has at least 80%, for example at least 85%, at least 87%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide of SEQ ID NO:9, said mature polypeptide having α-amylase activity. In another aspect, the amino acid sequence of the parent is approximately equal to or less than ten amino acids of the mature polypeptide of SEQ ID NO:9, for example, differing by five amino acids, four amino acids, three amino acids, two amino acids, and one amino acid.

[0101] The parent preferably comprises or consists of the amino acid sequence of SEQ ID NO:9. Alternatively, the parent comprises or consists of the mature polypeptide of SEQ ID NO:9.

[0102] In another embodiment, the parent is an allelic variant of the mature polypeptide of SEQ ID NO:9.

[0103] The parental α-amylase can also be a polypeptide with at least 80% sequence identity to the mature polypeptide of SEQ ID NO:10.

[0104] In another aspect, the parent has at least 80%, for example at least 85%, at least 87%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide of SEQ ID NO:10, said mature polypeptide having α-amylase activity. In another aspect, the amino acid sequence of the parent is approximately equal to or less than ten amino acids of the mature polypeptide of SEQ ID NO:10, for example, differing by five amino acids, four amino acids, three amino acids, two amino acids, and one amino acid.

[0105] The parent preferably comprises or consists of the amino acid sequence of SEQ ID NO:10. Alternatively, the parent comprises or consists of the mature polypeptide of SEQ ID NO:10.

[0106] In another embodiment, the parent is an allelic variant of the mature polypeptide of SEQ ID NO:10.

[0107] The parental α-amylase can also be a polypeptide with at least 80% sequence identity to the mature polypeptide of SEQ ID NO:11.

[0108] In another aspect, the parent has at least 80%, for example at least 85%, at least 87%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide of SEQ ID NO:11, said mature polypeptide having α-amylase activity. In another aspect, the amino acid sequence of the parent is approximately equal to or less than ten amino acids of the mature polypeptide of SEQ ID NO:11, for example, differing by five amino acids, four amino acids, three amino acids, two amino acids, and one amino acid.

[0109] The parent preferably comprises or consists of the amino acid sequence of SEQ ID NO:11. Alternatively, the parent comprises or consists of the mature polypeptide of SEQ ID NO:11.

[0110] In another embodiment, the parent is an allelic variant of the mature polypeptide of SEQ ID NO:11.

[0111] The parental α-amylase can also be a polypeptide with at least 80% sequence identity to the mature polypeptide of SEQ ID NO:12.

[0112] In another aspect, the parent has at least 80%, for example at least 85%, at least 87%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide of SEQ ID NO:12, said mature polypeptide having α-amylase activity. In another aspect, the amino acid sequence of the parent is approximately equal to or less than ten amino acids of the mature polypeptide of SEQ ID NO:12, for example, differing by five amino acids, four amino acids, three amino acids, two amino acids, and one amino acid.

[0113] The parent preferably comprises or consists of the amino acid sequence of SEQ ID NO:12. Alternatively, the parent comprises or consists of a mature polypeptide of SEQ ID NO:12.

[0114] In another embodiment, the parent is an allelic variant of the mature polypeptide of SEQ ID NO:12.

[0115] The amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or fragments thereof, can be used to design nucleic acid probes to identify and clone parental DNA encoding strains from different genera or species, according to methods well known in the art. Specifically, such probes can be used to hybridize with the genome or cDNA of the genus or species of interest according to standard DNA blotting procedures in order to identify and isolate the corresponding genes therein. Such probes can be much shorter than the entire sequence, but should have a length of at least 14, for example at least 25, at least 35, or at least 70 nucleotides. Preferably, the nucleic acid probe has a length of at least 100 nucleotides, such as at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, or at least 900 nucleotides. Both DNA and RNA probes can be used. Typically, these probes are labeled for detecting corresponding genes (e.g., using...). 32 P, 3 H, 35 S, biotin, or avidin). Such probes are covered by this invention.

[0116] DNA from genomic DNA or cDNA libraries prepared from these other organisms that hybridizes to the probes described above and encodes the parent can be screened. Genomic DNA or other DNA from these other organisms can be separated by agarose or polyacrylamide gel electrophoresis or other separation techniques. DNA from these libraries or separated DNA can be transferred to and immobilized on nitrocellulose or other suitable carrier materials for DNA blotting.

[0117] For the purposes of this invention, hybridization refers to the hybridization of a polynucleotide with a labeled nucleotide probe under low to very high stringency conditions, said nucleotide probe corresponding to a polynucleotide or a subsequence of the polynucleotide encoding SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. Molecules hybridized with this probe can be detected using, for example, X-ray film or any other detection method known in the art.

[0118] In one respect, the nucleic acid probe is a polynucleotide or fragment thereof encoding a polypeptide of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.

[0119] For long probes at least 100 nucleotides in length, very low to very high stringency conditions are defined as optimally following standard DNA blotting procedures, pre-hybridizing and hybridizing at 42°C in 5X SSPE, 0.3% SDS, and 200 μg / ml of cleaved and denatured salmon sperm DNA, and for very low and low stringency in 25% formamide, for moderate and moderate-high stringency in 35% formamide, or for high and very high stringency in 50% formamide for 12 to 24 hours. Finally, the vector material is washed three times with 2X SSC and 0.2% SDS at 45°C (very low stringency), 50°C (low stringency), 55°C (moderate stringency), 60°C (moderate-high stringency), 65°C (high stringency), or 70°C (very high stringency), each for 15 minutes.

[0120] For short probes ranging from approximately 15 to approximately 70 nucleotides in length, stringency conditions are defined as optimally following standard DNA blotting procedures using the T values ​​calculated according to the method of Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA) 48:1390). m Pre-hybridization and hybridization were performed at approximately 5°C to approximately 10°C in 0.9M NaCl, 0.09M Tris-HCl pH 7.6, 6mM EDTA, 0.5% NP-40, 1X Denhardt's solution, 1mM sodium pyrophosphate, 1mM sodium dihydrogen phosphate, 0.1mM ATP, and 0.2mg yeast RNA per mL for 12 to 24 hours. Finally, the vector material was subjected to pre-hybridization and hybridization at the calculated T0. m Wash once at approximately 5°C to approximately 10°C in 6X SCC with 0.1% SDS for 15 minutes, and then wash twice with 6X SSC for 15 minutes each time.

[0121] Parents can be obtained from microorganisms of any genus. For the purposes of this invention, as used herein in conjunction with the given sources, the term "obtained from" should mean that the variant encoded by the polynucleotide is produced by that source or by a cell in which a polynucleotide from that source has been inserted. In one aspect, the parent is secreted extracellularly.

[0122] The parent can be a bacterial α-amylase. For example, the parent can be a Gram-positive bacterial polypeptide, such as Bacillus α-amylase, Clostridium α-amylase, Enterococcus α-amylase, Geobacillus α-amylase, Lactobacillus α-amylase, Lactococcus α-amylase, Oceanobacillus α-amylase, Staphylococcus α-amylase, Streptococcus α-amylase, or Streptomyces α-amylase. α-amylase (reptomyces), or Gram-negative bacterial polypeptides such as Campylobacter α-amylase, Escherichia coli α-amylase, Flavobacterium α-amylase, Fusobacterium α-amylase, Helicobacter α-amylase, Ilyobacter α-amylase, Neisseria α-amylase, Pseudomonas α-amylase, Salmonella α-amylase, or Ureaplasma α-amylase.

[0123] In one respect, the parental strains are *Bacillus alkalophilus* α-amylase, *Bacillus amyloliquefaciens* α-amylase, *Bacillus brevis* α-amylase, *Bacillus circulans* α-amylase, *Bacillus clausii* α-amylase, *Bacillus coagulans* α-amylase, *Bacillus firmus* α-amylase, *Bacillus lautus* α-amylase, *Bacillus lentus* α-amylase, *Bacillus licheniformis* α-amylase, *Bacillus megaterium* α-amylase, and *Bacillus pumilus* α-amylase. α-amylase from Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis.

[0124] In another respect, the parent is α-amylase from Streptococcus equi, α-amylase from Streptococcus pyogenes, α-amylase from Streptococcus uberis, or α-amylase from Streptococcus equi subsp. Zooepidemicus.

[0125] On the other hand, the parent strain does not produce α-amylase from Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans.

[0126] The parent can be a fungal α-amylase. For example, the parent can be a yeast α-amylase, such as Candida α-amylase, Kluyveromyces α-amylase, Pichia α-amylase, Saccharomyces α-amylase, Schizosaccharomyces α-amylase, or Yarrowia α-amylase. For example, the parent can be filamentous fungi with α-amylases such as *Acremonium*, *Agaricus*, *Alternaria*, *Aspergillus*, *Aureobasidium*, *Botryospaeria*, *Ceriporiopsis*, and *Chaetomidium*. Amylase, Chrysosporium α-amylase, Claviceps α-amylase, Cochliobolus α-amylase, Coprinopsis α-amylase, Coptotermes α-amylase, Corynascus α-amylase, Cryphonectria α-amylase, Cryptococcus α-amylase, Diplostomum α-amylase α-amylase of *Exidia*, *Filibasidium*, *Fusarium*, *Gibberella*, *Holomastigotoides*, *Humicola*, *Irpex*, *Lentinula*, and *Leptospaeria*. Amylase, α-amylase of *Magnaporthe*, α-amylase of *Melanocarpus*, α-amylase of *Meripilus*, α-amylase of *Mucor*, α-amylase of *Myceliophthora*, α-amylase of *Neocallimastix*, α-amylase of *Neurospora*, α-amylase of *Paecilomyces*, α-amylase of *Penicillium*,α-amylases from the genera *Phanerochaete*, *Piromyces*, *Poitrasia*, *Pseudoplectania*, *Pseudotrichonympha*, *Rhizomucor*, *Schizophyllum*, *Scytalidium*, and *Ta...* α-amylase from the genera *Laromyces*, *Thermoascus*, *Thielavia*, *Tolypocladium*, *Trichoderma*, *Trichophaea*, *Verticillium*, *Volvariella*, or *Xylaria*.

[0127] On the other hand, the parent is α-amylase from Saccharomyces carlsbergensis, α-amylase from Saccharomyces cerevisiae, α-amylase from Saccharomyces diastaticus, α-amylase from Saccharomyces douglasii, α-amylase from Saccharomyces kluyveri, α-amylase from Saccharomyces norbensis, or α-amylase from Saccharomyces oviformis.

[0128] On the other hand, the parental strains include α-amylases from *Acremonium cellulolyticus*, *Aspergillus aculeatus*, *Aspergillus foetidus*, *Aspergillus fumigatus*, *Aspergillus japonicus*, *Aspergillus nidus*, *Aspergillus niger*, *Aspergillus oryzae*, *Chrysosporium inops*, *Chrysosporium keratinophilum*, *Chrysosporium lucknowense*, *Chrysosporium merdarium*, *Chrysosporium pannicola*, and *Chrysosporium queenslandense*. α-Amylase of *Chrysosporium tropicum*, *Chrysosporium zonatum*, *Fusarium bactridioides*, *Fusarium cerealis*, *Fusarium crookwellense*, *Fusarium culmorum*, *Fusarium graminearum*, *Fusarium graminum*, *Fusarium heterosporum*, *Fusarium negundi*, *Fusarium oxysporum*, *Fusarium reticulatum*, and *Fusarium rosenbergii*. Fusarium roseum α-amylase, Fusarium sambucinum α-amylase, Fusarium sarcochroum α-amylase, Fusarium sporotrichioides α-amylase, Fusarium sulphureum α-amylase, Fusarium sulphureum α-amylase, Fusarium sulphureum α-amylaseα-Amylase of *Fusarium trichothecioides*, *Fusarium venenatum*, *Humicola grisea*, *Humicola insolens*, *Humicola lanuginosa*, *Irpex lacteus*, *Mucor miehei*, *Myceliophthora thermophila*, *Neurosporacrassa*, *Penicillium funiculosum*, *Penicillium purpurogenum*, *Phanerochaetechrysosporium*, and *Thielavia*. α-amylase of *Thielavia albomyces*, *Thielavia albopilosa*, *Thielavia australeinsis*, *Thielavia fimeti*, *Thielavia microspora*, *Thielavia ovispora*, *Thielavia peruviana*, *Thielavia setosa*, *Thielavia spededonium*, *Thielavia subthermophila*, *Thielavia terrestris*, *Trichoderma harzianum*, *Trichoderma* α-Amylase of Trichoderma koningii, α-Amylase of Trichoderma longibrachiatum, α-Amylase of Trichoderma reesei, or α-Amylase of Trichoderma viride.

[0129] In another respect, the parent is a Bacillus α-amylase, for example, the α-amylase of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.

[0130] It should be understood that, for the aforementioned species, the present invention includes complete and incomplete states, as well as other taxonomic equivalents, such as asexual forms, regardless of the species name known through them. Those skilled in the art will readily identify the appropriate equivalents.

[0131] Strains of these species are readily accessible to the public at many culture collections, such as the American Type Culture Collection (ATCC), the German Microbial Culture Collection (DSM), the Centralbureau Voor Schimmelcultures (CBS), and the Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

[0132] The aforementioned probes can be used to identify the parent and obtain it from other sources, including microorganisms isolated from nature (e.g., soil, compost, water, etc.) or directly from natural materials (e.g., soil, compost, water, etc.). Techniques for directly isolating microorganisms and DNA from their natural habitat are well known in the art. The polynucleotide encoding the parent can then be derived by similarly screening the genome or cDNA library of another microorganism or a mixed DNA sample. Once the polynucleotide encoding the parent has been detected with the probe, it can be isolated or cloned using techniques known to those skilled in the art (see, for example, Sambrook et al., 1989, above).

[0133] The parent can be a heterozygous polypeptide, in which a portion of one polypeptide is fused to the N-terminus or C-terminus of a portion of another polypeptide.

[0134] The parent can also be a fusion polypeptide or a cleavable fusion polypeptide, wherein one polypeptide is fused to the N-terminus or C-terminus of another polypeptide. Fusion polypeptides are generated by fusing a polynucleotide encoding one polypeptide with a polynucleotide encoding another polypeptide. Techniques for generating fusion polypeptides are known in the art and involve linking the 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 promoter and terminator. Fusion proteins can also be constructed using integrin technology, wherein the fusion is generated post-translationally (Cooper et al., EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266:776-779).

[0135] The fusion peptide may further include a cleavage site between the two peptides. After the fusion protein is secreted, this site is cleaved, releasing both peptides. Examples of cleavage sites include, but are not limited to, Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3:568-576; Svetina et al., 2000, Journal of Biotechnology (J. Biotechnol.) 76:245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63:3488-3493; Ward et al., 1995, Biotech (Biotech The sites disclosed in (Nology) 13:498-503; and Contreras et al., 1991, Biotechnology 9:378-381; Eaton et al., 1986, Biochemistry 25:505-512; Collins-Racie et al., 1995, Biotechnology 13:982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6:240-248; and Stevens, 2003, Drug Discovery World 4:35-48.

[0136] Preparation of variants

[0137] The present invention also relates to a method for obtaining variants having α-amylase activity, the method comprising: (a) introducing a modification at two or more (several) positions into a parental α-amylase, said positions corresponding to positions 140, 181, 189, 134, 195, 206, 243, 260, 262, 284, 304, 347, 439, 469, 476, and 477 of the mature polypeptide of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12, wherein the variant is numbered according to SEQ ID NO:1 and has α-amylase activity; and (b) recovering the variant.

[0138] In one aspect, the present invention relates to a method for obtaining variants having α-amylase activity, the method comprising: (a) introducing a modification at two or more sites into a parental α-amylase, said sites corresponding to W140, R181, W189, D134, N195, V206, Y243, E260, F262, W284, G304, W347, W439, W469, G476, and G477 of the mature polypeptide of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12, wherein the variant is numbered according to SEQ ID NO:1 and has α-amylase activity; and (b) recovering the variant.

[0139] In one embodiment, the introduced change is a substitution.

[0140] In yet another embodiment, the present invention relates to a method for obtaining variants having α-amylase activity, the method comprising: (a) introducing substitutions at two or more sites into a parental α-amylase, said sites corresponding to G304RKEQ, W140YF, W189EGT, D134E, E260ADCQLMFPSWVGHIKNRTY, F262GP, W284DHFYR, W347HFY, W439RG, G476EQRK, G477EQKMR of mature polypeptides of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12, wherein the variants are numbered according to SEQ ID NO:1 and have α-amylase activity; and (b) recovering the variants.

[0141] In a preferred embodiment, the introduced substitutions are two or more of G304R, W140YF, W189EGT, D134E, E260GHIKNRTY, W284DFR, W439RG, G476EK, and G477EKMR. In a more preferred embodiment, the introduced substitutions are G304R, W140YF, E260GHIKNPRTY, and G476EQRK. In one or even more preferred embodiment, the method for obtaining variants having α-amylase activity comprises: (a) introducing substitutions G304R, W140Y, E260G, and G476K into a parental α-amylase in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12, wherein the variants are numbered according to SEQ ID NO:1 and have α-amylase activity; and (b) recovering the variants. Variants can be prepared using any mutagenesis method known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc.

[0142] Site-directed mutagenesis is a technique that produces one or more mutations at one or more defined sites in a polynucleotide encoding a parent.

[0143] Site-directed mutagenesis can be performed in vitro by PCR, which involves using primers containing oligonucleotides with the desired mutation. In vitro site-directed mutagenesis can also be performed by cassette mutagenesis, which involves cleavage by a restriction enzyme at a site in a plasmid containing a polynucleotide encoding the parent and subsequent ligation of the oligonucleotide containing the mutation into the polynucleotide. Typically, the restriction enzymes used to digest the plasmid and the oligonucleotide are the same, allowing the sticky ends of the plasmid and the insert to ligate to each other. See, for example, Scherer and Davis, 1979, Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA) 76:4949-4955; and Barton et al., 1990, Nucleic Acids Res. 18:7349-4966.

[0144] 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.

[0145] Any site-directed mutagenesis method can be used in this invention. Many commercially available kits are available for preparing variants.

[0146] Synthetic gene construction necessitates the in vitro synthesis of designed polynucleotide molecules to encode polypeptides of interest. Gene synthesis can be performed using numerous techniques, such as the multi-microchip-based techniques described by Tian et al. (2004, Nature 432:1050-1054), which involve the synthesis and assembly of oligonucleotides on optically programmable microfluidic chips.

[0147] Single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested using known mutagenesis, recombination, and / or truncation methods, followed by relevant screening procedures, such as those disclosed in Reidhaar-Olson and Sauer, 1988, Science, 241:53-57; Bowie and Sauer, 1989, Proceedings of the National Academy of Sciences, 86:2152-2156; WO 95 / 17413 or WO 95 / 22625.

[0148] 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).

[0149] Mutagenesis / reorganization methods can be combined with high-throughput automated screening methods to detect the activity of cloned mutagenic peptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17:893-896). Mutagenic DNA molecules encoding active peptides can be recovered from 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.

[0150] Semi-synthetic gene construction is accomplished by combining multiple aspects of synthetic gene construction, and / or site-directed mutagenesis, and / or random mutagenesis and / or shuffling.

[0151] Semi-synthetic gene construction is represented by methods that utilize polynucleotide fragments synthesized in combination with PCR technology. Defined regions of the gene can thus be synthesized de novo, while other regions can be amplified using site-specific mutagenic primers, and other regions can be amplified using either error-prone or non-error-prone PCR. Polynucleotide sequence shuttle can then be performed.

[0152] variants

[0153] The present invention also provides variants of the parental α-amylase comprising alterations at two or more positions corresponding to the G304, W140, W189, D134, E260, F262, W284, W347, W439, W469, G476, and G477 positions of the mature polypeptide of SEQ ID NO:1, wherein each alteration is independently a substitution, insertion, or deletion (preferably a substitution), and the variants possess α-amylase activity. Thus, variants with improved low-temperature washing performance compared to the parental α-amylase or compared to the α-amylases of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 are provided.

[0154] In one embodiment, the variant has at least 80%, for example, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the parental α-amylase.

[0155] In another embodiment, the present invention relates to isolated variants of α-amylase, said variant comprising alterations at two or more positions corresponding to the G304, W140, W189, D134, E260, F262, W284, W347, W439, W469, G476, and G477 positions of the mature polypeptide of SEQ ID NO:1, wherein each alteration is independently a substitution, deletion, or insertion, and said variant has at least 80% but less than 100% sequence identity with the mature polypeptide of any one of SEQ ID NOs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, and said variant has α-amylase activity.

[0156] In another embodiment, the variant has at least 85%, at least 87%, at least 90%, at least 95%, such as at least 96%, at least 97%, at least 98%, and at least 99%, but less than 100%, sequence identity with the mature polypeptide of SEQ ID NO:1.

[0157] In another embodiment, the variant has at least 85%, at least 87%, at least 90%, at least 95%, such as at least 96%, at least 97%, at least 98%, and at least 99%, but less than 100%, sequence identity with the mature polypeptide of SEQ ID NO:2.

[0158] In another embodiment, the variant has at least 85%, at least 87%, at least 90%, at least 95%, such as at least 96%, at least 97%, at least 98%, and at least 99%, but less than 100%, sequence identity with the mature polypeptide of SEQ ID NO:3.

[0159] In another embodiment, the variant has at least 85%, at least 87%, at least 90%, at least 95%, such as at least 96%, at least 97%, at least 98%, and at least 99%, but less than 100%, sequence identity with the mature polypeptide of SEQ ID NO:4.

[0160] In another embodiment, the variant has at least 85%, at least 87%, at least 90%, at least 95%, such as at least 96%, at least 97%, at least 98%, and at least 99%, but less than 100%, sequence identity with the mature polypeptide of SEQ ID NO:5.

[0161] In another embodiment, the variant has at least 85%, at least 87%, at least 90%, at least 95%, such as at least 96%, at least 97%, at least 98%, and at least 99%, but less than 100%, sequence identity with the mature polypeptide of SEQ ID NO:6.

[0162] In another embodiment, the variant has at least 85%, at least 87%, at least 90%, at least 95%, such as at least 96%, at least 97%, at least 98%, and at least 99%, but less than 100%, sequence identity with the mature polypeptide of SEQ ID NO:7.

[0163] In another embodiment, the variant has at least 85%, at least 87%, at least 90%, at least 95%, such as at least 96%, at least 97%, at least 98%, and at least 99%, but less than 100%, sequence identity with the mature polypeptide of SEQ ID NO:8.

[0164] In another embodiment, the variant has at least 85%, at least 87%, at least 90%, at least 95%, such as at least 96%, at least 97%, at least 98%, and at least 99%, but less than 100%, sequence identity with the mature polypeptide of SEQ ID NO:9.

[0165] In another embodiment, the variant has at least 85%, at least 87%, at least 90%, at least 95%, such as at least 96%, at least 97%, at least 98%, and at least 99%, but less than 100%, sequence identity with the mature polypeptide of SEQ ID NO:10.

[0166] In another embodiment, the variant has at least 85%, at least 87%, at least 90%, at least 95%, such as at least 96%, at least 97%, at least 98%, and at least 99%, but less than 100%, sequence identity with the mature polypeptide of SEQ ID NO:11.

[0167] In another embodiment, the variant has at least 85%, at least 87%, at least 90%, at least 95%, such as at least 96%, at least 97%, at least 98%, and at least 99%, but less than 100%, sequence identity with the mature polypeptide of SEQ ID NO:12.

[0168] In one aspect, the number of changes in the variants of the invention is 2-20, for example, 2-10 and 2-5, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 changes.

[0169] In one aspect, the variant includes changes at two or more positions corresponding to positions G304, W140, W189, D134, E260, F262, W284, W347, W439, W469, G476, and G477. In another aspect, the variant includes changes at two positions corresponding to any one of positions G304, W140, W189, D134, E260, F262, W284, W347, W439, W469, G476, and G477. In yet another aspect, the variant includes changes at three positions corresponding to any one of positions G304, W140, W189, D134, E260, F262, W284, W347, W439, W469, G476, and G477. In another aspect, the variant includes changes at four positions corresponding to any one of positions G304, W140, W189, D134, E260, F262, W284, W347, W439, W469, G476, and G477. In another aspect, the variant includes changes at five positions corresponding to any one of positions G304, W140, W189, D134, E260, F262, W284, W347, W439, W469, G476, and G477. In yet another aspect, the variant includes changes at six positions corresponding to any one of positions G304, W140, W189, D134, E260, F262, W284, W347, W439, W469, G476, and G477. In another aspect, the variant includes changes at each of the positions corresponding to G304, W140, W189, D134, E260, F262, W284, W347, W439, W469, G476, and G477. These positions correspond to the position of SEQ ID NO:1. Preferably, these changes are substitutions.

[0170] In one embodiment, the variant includes permutations at two, three, or four positions selected from the group consisting of G304, W140, W189, D134, E260, F262, W284, W347, W439, W469, G476, and G477.

[0171] In a preferred embodiment, the variant includes permutations at two, three, or four locations, the locations being selected from the group consisting of G304, W140, E260, and G476.

[0172] In one aspect of the invention, the variant includes two or more permutations selected from the group consisting of G304RKEQ, W140YF, W189EGT, D134E, E260ADCQLMFPSWVGHIKNRTY, F262GP, W284DHFYR, W347HFY, W439RG, G476EQRK, and G477EQKMR.

[0173] Preferably, variations of the invention include substitutions at two, three, or four positions, said substitutions being selected from the group consisting of G304R, W140YF, E260GHIKNPRTY, and G476EQRK. In a more preferred embodiment, the substitutions at two, three, or four positions are selected from the group consisting of G304R, W140Y, E260G, and G476K.

[0174] In one embodiment, the variant further comprises one or more substitutions selected from the group consisting of T51IL, S52Q, N54K, G109A, E194D, N195F, V206Y, Y243F, G109A, G273DV, G337N, K72R, R181H, S303G, and Y100I. In a preferred embodiment, one or more other substitutions are selected from the group consisting of N195F, V206Y, and Y243F. Preferably, the variant comprises two or three of these substitutions. Thus, improved low-temperature washing performance and improved resistance to Ca are provided compared to the parental α-amylase or the α-amylases of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. 2+ A variant of depleted stability.

[0175] In another aspect of the invention, the variant comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:11. Two or more substitutions of the mature polypeptide of NO:12, wherein the substitutions are selected from the group consisting of D134E, E260G, E260H, E260I, E260K, E260N, E260R, E260T, G109A, G273D, G273V, G337N, G476E, G477E, G477M, G477R, K72R, R181H, S303G, W140F, W140Y, W189E, W189G, W189T, W284D and Y100I.

[0176] Variations can also include changes in one or more other locations.

[0177] For example, variants can include changes at positions corresponding to G182*+D183* or D183*+G184*.

[0178] In another aspect, the present invention relates to variants comprising substitutions at positions corresponding to the position of the polypeptide of SEQ ID NO:1, said substitutions being selected from the group consisting of:

[0179] W140Y+N195F+V206Y+Y243F+E260G+G477E,

[0180] W140Y+N195F+V206Y+Y243F+E260T+W284D,

[0181] W140Y+N195F+V206Y+Y243F+W284D,

[0182] G109A+W140Y+N195F+V206Y+Y243F+E260G,

[0183] W140Y+N195F+V206Y+Y243F+E260G,

[0184] N195F+V206Y+Y243F+E260K+W284D,

[0185] D134E+G476E,

[0186] W140Y+N195F+V206Y+Y243F+E260G+G476E,

[0187] W140Y+W189G+N195F+V206Y+Y243F+E260G,

[0188] W140Y+N195F+V206Y+Y243F+E260G+S303G,

[0189] W140Y+W189T+N195F+V206Y+Y243F+E260G,

[0190] W140Y+N195F+V206Y+Y243F+E260G+W284D,

[0191] Y100I+W140Y+N195F+V206Y+Y243F+E260G,

[0192] W140Y+N195F+V206Y+Y243F+E260G+G337N,

[0193] W140Y+N195F+V206Y+Y243F+E260G+W439R

[0194] G109A+W140Y+E194D+N195F+V206Y+Y243F+E260G

[0195] G109A+W140Y+N195F+V206Y+Y243F+E260G+G476E

[0196] T51I+Y100I+G109A+W140Y+N195F+V206Y+Y243F+E260G

[0197] T51I+G109A+W140Y+N195F+V206Y+Y243F+E260G+W439R

[0198] T51I+S52Q+N54K+G109A+W140Y+N195F+V206Y+Y243F+E260G+G476E

[0199] W140Y+N195F+V206Y+Y243F+E260G+G304R+G476K

[0200] W140Y+N195F+V206Y+Y243F+E260G+W284R+G477K

[0201] W140Y+N195F+V206Y+Y243F+E260G+W284F+G477R, and

[0202] N195F+V206Y+Y243F+E260G+W284D.

[0203] In another aspect, the present invention relates to variants comprising substitutions at positions corresponding to the position of the polypeptide of SEQ ID NO:1, the substitutions being selected from the group consisting of:

[0204] W140Y+N195F+V206Y+Y243F+E260G+G477E,

[0205] W140Y+N195F+V206Y+Y243F+E260T+W284D,

[0206] W140Y+N195F+V206Y+Y243F+W284D,

[0207] G109A+W140Y+N195F+V206Y+Y243F+E260G,

[0208] W140Y+N195F+V206Y+Y243F+E260G,

[0209] N195F+V206Y+Y243F+E260K+W284D,

[0210] D134E+G476E,

[0211] W140Y+N195F+V206Y+Y243F+E260G+G476E,

[0212] W140Y+W189G+N195F+V206Y+Y243F+E260G,

[0213] W140Y+N195F+V206Y+Y243F+E260G+S303G,

[0214] W140Y+W189T+N195F+V206Y+Y243F+E260G,

[0215] W140Y+N195F+V206Y+Y243F+E260G+W284D,

[0216] Y100I+W140Y+N195F+V206Y+Y243F+E260G,

[0217] W140Y+N195F+V206Y+Y243F+E260G+G337N,

[0218] W140Y+N195F+V206Y+Y243F+E260G+W439R

[0219] G109A+W140Y+E194D+N195F+V206Y+Y243F+E260G

[0220] G109A+W140Y+N195F+V206Y+Y243F+E260G+G476E

[0221] T51I+Y100I+G109A+W140Y+N195F+V206Y+Y243F+E260G

[0222] T51I+G109A+W140Y+N195F+V206Y+Y243F+E260G+W439R

[0223] T51I+S52Q+N54K+G109A+W140Y+N195F+V206Y+Y243F+E260G+G476E

[0224] W140Y+N195F+V206Y+Y243F+E260G+G304R+G476K

[0225] W140Y+N195F+V206Y+Y243F+E260G+W284R+G477K

[0226] W140Y+N195F+V206Y+Y243F+E260G+W284F+G477R, and

[0227] N195F+V206Y+Y243F+E260G+W284D.

[0228] In yet another aspect, the present invention relates to variants comprising changes within a position corresponding to the position of the polypeptide of SEQ ID NO:1, said changes being selected from the group consisting of:

[0229] D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+G477E,

[0230] D183*+G184*+W140Y+N195F+V206Y+Y243F+E260T+W284D,

[0231] D183*+G184*+W140Y+N195F+V206Y+Y243F+W284D,

[0232] D183*+G184*+G109A+W140Y+N195F+V206Y+Y243F+E260G,

[0233] D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G,

[0234] D183*+G184*+N195F+V206Y+Y243F+E260K+W284D,

[0235] D183*+G184*+D134E+G476E,

[0236] D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+G476E,

[0237] D183*+G184*+W140Y+W189G+N195F+V206Y+Y243F+E260G,

[0238] D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+S303G,

[0239] D183*+G184*+W140Y+W189T+N195F+V206Y+Y243F+E260G,

[0240] D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+W284D,

[0241] D183*+G184*+Y100I+W140Y+N195F+V206Y+Y243F+E260G,

[0242] D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+G337N,

[0243] D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+W439R

[0244] D183*+G184*+G109A+W140Y+E194D+N195F+V206Y+Y243F+E260G

[0245] D183*+G184*+G109A+W140Y+N195F+V206Y+Y243F+E260G+G476E

[0246] D183*+G184*+T51I+Y100I+G109A+W140Y+N195F+V206Y+Y243F+E260G

[0247] D183*+G184*+T51I+G109A+W140Y+N195F+V206Y+Y243F+E260G+W439R

[0248] D183*+G184*+T51I+S52Q+N54K+G109A+W140Y+N195F+V206Y+Y243F+E260G+G476E

[0249] D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+G304R+G476K

[0250] D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+W284R+G477K

[0251] D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+W284F+G477R, and

[0252] D183*+G184*+N195F+V206Y+Y243F+E260G+W284D.

[0253] In another aspect, the present invention relates to variants comprising changes within a position corresponding to the position of the polypeptide of SEQ ID NO:1, said changes being selected from the group consisting of:

[0254] D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+G477E,

[0255] D183*+G184*+W140Y+N195F+V206Y+Y243F+E260T+W284D,

[0256] D183*+G184*+W140Y+N195F+V206Y+Y243F+W284D,

[0257] D183*+G184*+G109A+W140Y+N195F+V206Y+Y243F+E260G,

[0258] D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G,

[0259] D183*+G184*+N195F+V206Y+Y243F+E260K+W284D,

[0260] D183*+G184*+D134E+G476E,

[0261] D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+G476E,

[0262] D183*+G184*+W140Y+W189G+N195F+V206Y+Y243F+E260G,

[0263] D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+S303G,

[0264] D183*+G184*+W140Y+W189T+N195F+V206Y+Y243F+E260G,

[0265] D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+W284D,

[0266] D183*+G184*+Y100I+W140Y+N195F+V206Y+Y243F+E260G,

[0267] D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+G337N,

[0268] D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+W439R

[0269] D183*+G184*+G109A+W140Y+E194D+N195F+V206Y+Y243F+E260G

[0270] D183*+G184*+G109A+W140Y+N195F+V206Y+Y243F+E260G+G476E

[0271] D183*+G184*+T51I+Y100I+G109A+W140Y+N195F+V206Y+Y243F+E260G

[0272] D183*+G184*+T51I+G109A+W140Y+N195F+V206Y+Y243F+E260G+W439R

[0273] D183*+G184*+T51I+S52Q+N54K+G109A+W140Y+N195F+V206Y+Y243F+E260G+G476E

[0274] D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+G304R+G476K

[0275] D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+W284R+G477K

[0276] D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+W284F+G477R, and

[0277] D183*+G184*+N195F+V206Y+Y243F+E260G+W284D.

[0278] Essential amino acids in the parent can be identified using methods 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 resulting mutant molecule is tested for α-amylase activity to identify amino acid residues crucial to the molecule's activity. See also Hilton et al., 1996, Journal of Biochemistry 271:4699-4708. The active site of α-amylase or other biological interactions can also be determined by physical analysis of the structure, such as by techniques like nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, along with the proposed contact site amino acid mutation. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, Journal of Molecular Biology 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identity of essential amino acids can also be inferred from the analysis of similarity to the peptide, wherein the peptide is related to the parent.

[0279] Polynucleotides

[0280] The present invention also relates to isolated polynucleotides encoding any variant of the invention.

[0281] Nucleic acid constructs

[0282] The present invention also relates to nucleic acid constructs comprising a polynucleotide encoding a variant of the invention operatively linked to one or more control sequences, wherein the control sequences direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

[0283] Polynucleotides can be manipulated in a variety of ways to provide variant expression. Depending on the expression vector, manipulating the polynucleotide before insertion into the vector may be desirable or necessary.

[0284] Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art. The control sequence can be a promoter sequence recognized by the host cell for the expression of the polynucleotide. The promoter sequence contains a transcriptional control sequence that mediates variant expression. The promoter can be any nucleic acid sequence exhibiting transcriptional activity in the host cell, including mutant, truncated, and heterozygous promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to those of the host cell.

[0285] Examples of suitable promoters guiding the transcription of the nucleic acid constructs of this invention in bacterial host cells include promoters derived from the Bacillus amyloliquefaciens α-amylase gene (amyQ), Bacillus licheniformis α-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus thermophilus maltose amylase gene (amyM), Bacillus subtilis fructan sucrase gene (sacB), Bacillus subtilis xylA and xylB genes, Escherichia coli lac operon, Streptomyces agarase gene (dagA), and prokaryotic β-lactamase gene (Villa-Kamaroff et al., 1978, Proceedings of the National Academy of Sciences 75:3727-3731), and the tac promoter (DeBoer et al., 1983, Proceedings of the National Academy of Sciences 80:21-25).

[0286] Other promoters are described in Gilbert et al., 1980, Scientific American, 242:74-94, “Useful proteins from recombinant bacteria”, and by Sambrook et al. (1989, above).

[0287] Examples of suitable promoters for guiding the transcription of the nucleic acid constructs of this invention in filamentous fungal host cells include Aspergillus nidulans acetamase, Aspergillus niger neutral α-amylase, Aspergillus niger acid-stable α-amylase, Aspergillus niger or Aspergillus awamori glucosylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum trypsin-like protease (WO 96 / 00787), Fusarium venenatum amylase (WO 00 / 56900), Fusarium venenatum Daria (WO 00 / 56900), Fusarium venenatum Quinn (WO 00 / 56900), and Rhizomucor The promoters obtained from the genes of *Trichoderma reesei* lipase, *Rhizopus reesei* aspartic protease, *Trichoderma reesei* β-glucosidase, *Trichoderma reesei* cellobiose hydrolase I, *Trichoderma reesei* cellobiose hydrolase II, *Trichoderma reesei* endoglucanase I, *Trichoderma reesei* endoglucanase II, *Trichoderma reesei* endoglucanase III, *Trichoderma reesei* endoglucanase IV, *Trichoderma reesei* endoglucanase V, *Trichoderma reesei* xylanase I, *Trichoderma reesei* xylanase II, and *Trichoderma reesei* β-xylosidase, as well as NA2-tp i promoters (modified promoters comprising a gene encoding neutral α-amylase in Aspergillus, wherein the untranslated leader sequence has been replaced by an untranslated leader sequence from a gene encoding triose phosphate isomerase in Aspergillus; non-restrictive examples include modified promoters comprising a gene encoding neutral α-amylase in Aspergillus niger, wherein the untranslated leader sequence has been replaced by an untranslated leader sequence from a gene encoding triose phosphate isomerase in Aspergillus nidulans or Aspergillus oryzae); and their mutant, truncated, and heterozygous promoters.

[0288] In yeast hosts, useful promoters were obtained from the genes of *Saccharomyces cerevisiae* enolase (ENO-1), *Saccharomyces cerevisiae* galactokinase (GAL1), *Saccharomyces cerevisiae* alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2 / GAP), *Saccharomyces cerevisiae* triose phosphate isomerase (TPI), *Saccharomyces cerevisiae* metallothionein (CUP1), and *Saccharomyces cerevisiae* 3-phosphate glycerate kinase.

[0289] Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8:423-488.

[0290] The control sequence can also be a suitable transcription terminator sequence that is recognized by the host cell to terminate transcription. The terminator sequence is operatively linked to the 3' end of the multinucleotide encoding the variant. Any terminator that is functional in the host cell can be used.

[0291] Preferred terminators for use in filamentous fungal host cells were obtained from the genes of Aspergillus nidulans antaminobenzoate synthase, Aspergillus niger glucosidase, Aspergillus niger α-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0292] Preferred terminators for use in yeast host cells were obtained from the genes of Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase.

[0293] Other useful terminators for yeast host cells are described above by Romanos et al., 1992.

[0294] The control sequence can also be a suitable leader sequence, which is the untranslated region of an mRNA important for translation in the host cell. This leader sequence is operatively linked to the 5' end of a multinucleotide encoding the variant. Any leader sequence that is functional in the host cell can be used.

[0295] Preferred leader sequences for use in filamentous fungal host cells were obtained from the genes of Aspergillus oryzae TAKA amylase and Aspergillus nidulans triphosphate isomerase.

[0296] Suitable leader sequences for yeast host cells were obtained from the genes of Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0297] The control sequence can also be a polyadenylated sequence, a sequence operatively linked to the 3' end of a sequence encoding a variant and recognized by the host cell during transcription as a signal to add polyadenylated residues to the transcribed mRNA. Any polyadenylated sequence that is functional in the host cell can be used.

[0298] Preferred polyadenylated sequences for use in filamentous fungal host cells were obtained from the genes of Aspergillus nidulans antaminobenzoate synthase, Aspergillus niger glucosidase, Aspergillus niger α-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0299] Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Molecular Cellular Biology, 15:5983-5990.

[0300] The control sequence can also be a signal peptide coding region encoding a signal peptide, wherein the signal peptide is linked to the N-terminus of the variant and directs the variant's entry into the cell via a secretory pathway. The 5' end of the polynucleotide coding sequence may intrinsically contain a signal peptide coding region naturally linked within the translation reading frame to a segment encoding the coding region of the variant. Alternatively, the 5' end of the coding sequence may contain a foreign signal peptide coding region relative to the coding sequence. A foreign signal peptide coding region may be required where the coding sequence does not naturally contain one. Alternatively, a foreign signal peptide coding region may simply replace the native signal peptide coding region to enhance the variant's secretion. However, any signal peptide coding region that directs the expressed variant into the host cell via a secretory pathway can be used.

[0301] Effective signal peptide coding sequences for bacterial host cells are obtained from the genes of *Bacillus NCIB 11837* for maltose amylase, *Bacillus licheniformis* for subtilis protease, *Bacillus licheniformis* for β-lactamase, *Bacillus thermophilus* for α-amylase, *Bacillus thermophilus* for neutral proteases (nprT, nprS, nprM), and *Bacillus subtilis* for prsA. Other signal sequences are described by Simonen and Palva, 1993, *Microbiological Reviews* 57:109-137.

[0302] The effective signal peptide coding sequences for filamentous fungal host cells are obtained from the genes of Aspergillus niger neutral amylase, Aspergillus niger glucosylase, Aspergillus oryzae TAKA amylase, Aspergillus oryzae cellulase, Aspergillus oryzae endoglucanase V, Aspergillus pubescens lipase, and Rhizopus oryzae aspartic protease.

[0303] Useful signal peptides for yeast host cells were obtained from the genes of Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences were described by Romanos et al. (1992, above).

[0304] The control sequence can also be a propeptide coding region encoding the propeptide located at the N-terminus of the variant. The resulting polypeptide is called a zymogen or polypeptide precursor (or, in some cases, a zymogen). Polypeptides are usually inactive and can be converted into active polypeptides through catalytic or autocatalytic cleavage of the propeptide. Propeptide coding regions can be obtained from the genes of *Bacillus subtilis* alkaline protease (aprE), *Bacillus subtilis* neutral protease (nprT), *Heterospora thermophila* laccase (WO 95 / 33836), *Rhizopus oryzae* aspartic protease, and *Saccharomyces cerevisiae* α-factor.

[0305] In cases where both the signal peptide and the propeptide region are present at the N-terminus of the variant, the propeptide region is positioned immediately adjacent to the N-terminus of the variant, and the signal peptide region is positioned immediately adjacent to the N-terminus of the propeptide region.

[0306] It may also be desirable to add regulatory sequences that allow the regulation of variant expression relative to the growth of the host cell. Examples of regulatory systems are those that cause gene expression to be turned on or off in response to chemical or physical stimuli, including the presence of regulatory compounds. Regulatory systems in prokaryotes include the lac, tac, and trp gene-operating systems. In yeast, the ADH2 or GAL1 system can be used. In filamentous fungi, the *Aspergillus niger* glucosylamylase promoter, the *Aspergillus oryzae* TAKA α-amylase promoter, and the *Aspergillus oryzae* glucosylamylase promoter can be used. Other examples of regulatory sequences are those that allow gene amplification. In eukaryotic systems, these regulatory sequences include dihydrofolate reductase genes amplified in the presence of methotrexate and metallothionein genes amplified due to heavy metals. In these cases, the polynucleotide encoding the variant will be operatively linked to the regulatory sequence.

[0307] expression carrier

[0308] The present invention also relates to recombinant expression vectors comprising the polynucleotide of the present invention, a promoter, and transcription and translation termination signals. Various nucleotides and control sequences can be linked together to produce a recombinant expression vector, which may contain one or more suitable restriction sites to allow insertion or substitution of the polynucleotide encoding the variant at such sites. Alternatively, the polynucleotide can be expressed by inserting the polynucleotide or a nucleic acid construct containing the polynucleotide into a suitable vector for expression. In producing the expression vector, the coding sequence is positioned in the vector such that the coding sequence is operatively linked to a suitable control sequence for expression.

[0309] Recombinant expression vectors can be any vector (e.g., plasmids or viruses) that can suitably undergo recombinant DNA procedures and can induce polynucleotide expression. Typically, the choice of vector will depend on its compatibility with the host cell into which the vector will be introduced. Vectors can be linear or closed circular plasmids.

[0310] The vector can be a self-replicating vector, that is, a vector that exists as an extrachromosomal entity whose replication does not depend on chromosome replication, such as a plasmid, extrachromosomal element, mini-chromosome, or artificial chromosome. The vector can contain any means to ensure self-replication. Alternatively, the vector can be one that, when introduced into a host cell, integrates into the genome and replicates along with the chromosome already integrated into it. Furthermore, a single vector or plasmid, or two or more vectors or plasmids containing the total DNA of the genome to be introduced into the host cell, or transposons, can be used.

[0311] The vector preferably contains one or more selection markers that allow for the selection of transformed cells, transfected cells, transduced cells, etc. A selection marker is a gene whose product provides resistance to biocides or viruses, heavy metals, prototrophic to auxotrophic traits, etc.

[0312] Examples of bacterial selection markers include the dal gene from Bacillus subtilis or Bacillus licheniformis, or markers that confer antibiotic resistance such as ampicillin, chloramphenicol, kanamycin, or tetracycline. Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selection markers for filamentous fungal host cells include, but are not limited to, amdS (acetamipase), argB (ornithine carbamoyltransferase), bar (phosphinicotinic acid acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (ornithine-5'-phosphate decarboxylase), sC (adenosyl sulfate transtransferase), and trpC (o-aminobenzoate synthase), and their equivalents. Preferred markers for Aspergillus cells are the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae, and the bar gene of Streptomyces hygroscopicus.

[0313] The vector preferably contains elements that allow the vector to integrate into the host cell genome or to replicate autonomously in the cell independently of the genome.

[0314] To integrate into the host cell genome, the vector can rely on a multinucleotide sequence encoding a variant or any other vector element for integration into the genome via homologous or non-homologous recombination. Alternatively, the vector can contain additional nucleotide sequences that guide integration into the host cell genome via homologous recombination at a precise location on the chromosome. To increase the likelihood of integration at a precise location, the integrative element should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, that are highly homologous to the corresponding target sequence to enhance the probability of homologous recombination. The integrative element can be any sequence homologous to the target sequence in the host cell genome. Furthermore, the integrative element can be a non-coding or coding nucleotide sequence. On the other hand, the vector can integrate into the host cell genome via non-homologous recombination.

[0315] For autonomous replication, the vector may also contain an origin of replication, which enables the vector to replicate autonomously within the host cell discussed in this discussion. The origin of replication can be any plasmid replicon that mediates autonomous replication and functions within the cell. The terms "origin of replication" or "plasmid replicon" refer to the nucleotide sequence that enables a plasmid or vector to replicate in vivo.

[0316] Examples of bacterial origins of replication (OCRs) are the plasmids pBR322, pUC19, pACYC177, and pACYC184, which allow replication in *E. coli*, and pUB110, pE194, and pTA1060, which allow replication in *Bacillus*. The starting point for replication.

[0317] Examples of replication origins used in yeast host cells include the 2-micron replication origin, ARS1, ARS4, a combination of ARS1 and CEN3, and a combination of ARS4 and CEN6.

[0318] Examples of useful origins of replication in filamentous fungal cells are AMA1 and ANS1 (Gems et al., 1991, Gene 98:61-67; Cullen et al., 1987, Nucleic Acids Res. 15:9163-9175; WO 00 / 24883). The AMA1 gene can be isolated and plasmids or vectors containing the gene can be constructed according to the methods disclosed in WO 00 / 24883.

[0319] One or more copies of the polynucleotide of the present invention can be inserted into a host cell to increase the generation of variants. An increase in the copy number of the polynucleotide can be achieved by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene along with the polynucleotide, wherein cells containing an amplified copy of the selectable marker gene and thus an additional copy of the polynucleotide can be selected by culturing the cells in the presence of a suitable selectant.

[0320] The procedures used to connect the above-mentioned elements to construct the recombinant expression vector of the present invention are well known to those skilled in the art (see, for example, Sambrook et al., 1989, above) to obtain substantially pure variants.

[0321] host cells

[0322] This invention also relates to recombinant host cells containing inventive polynucleotides operatively linked to one or more control sequences that direct the generation of variants of the invention. A construct or vector containing the polynucleotide is introduced into the host cell, thereby maintaining the construct or vector as a chromosomal integrase or as a self-replicating extrachromosomal vector as described earlier. The term "host cell" encompasses any parental cell progeny that is not entirely identical to the parental cell due to mutations occurring during replication. The selection of the host cell will depend largely on the genes encoding the variant and their origin.

[0323] The host cell can be any cell that is useful in the recombinant production of variants, such as prokaryotes or eukaryotes.

[0324] 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*, *Selenobacter*, *Neisseria*, *Pseudomonas*, *Salmonella*, and *Ureaplasma*.

[0325] The bacterial host cell can be any Bacillus cell, including but not limited to Bacillus alkalinophilus cells, Bacillus amyloliquefaciens cells, Bacillus brevis cells, Bacillus circulans cells, Bacillus clausti cells, Bacillus coagulans cells, Bacillus sturdier cells, Bacillus brilliance cells, Bacillus retardans cells, Bacillus licheniformis cells, Bacillus megaterium cells, Bacillus brevis cells, Bacillus thermophilus cells, Bacillus subtilis cells, and Bacillus thuringiensis cells.

[0326] The bacterial host cell can also be any streptococcal cell, including but not limited to Streptococcus equi cells, Streptococcus pyogenes cells, Streptococcus mammae cells, and Streptococcus equi veterinary subspecies cells.

[0327] The bacterial host cell can also be any Streptomyces cell, including but not limited to non-color-producing Streptomyces cells, Streptomyces avermitilis cells, sky-blue Streptomyces cells, gray Streptomyces cells, and light blue-purple Streptomyces cells.

[0328] It can be achieved, for example, through protoplast transformation (see, e.g., Chang and Cohen, 1979, Modern Molecular Biology, 168:111-115), by using competent cells (see, e.g., Young and Spizizen, 1961, Journal of Bacteriology, 81:823-829, or Dubnau and Davidoff-Abelson, 19...). DNA can be introduced into Bacillus cells via, for example, protoplast transformation (see, for example, Hanahan, 1983, J.Mol.Biol. 166:557-580) or electroporation (see, for example, Dower et al., 1988, Biotechniques 6:742-751) or conjugation (see, for example, Koehler and Thorne, J.Bacteriol. 169:5271-5278). DNA can also be introduced into Escherichia coli cells via, for example, protoplast transformation (see, for example, Hanahan, 1983, J.Mol.Biol. 166:557-580) or electroporation (see, for example, Dower et al., 1988, Nucleic Acids Res. 16:6127-6145). DNA can be introduced into Streptomyces cells, for example, through protoplast transformation and electroporation (see, e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49:399-405), through conjugation (see, e.g., Mazodier et al., 1989, Journal of Bacteriology 171:3583-3585), or through transduction (see, e.g., Burke et al., 2001, Proceedings of the National Academy of Sciences 98:6289-6294). DNA can be introduced into Pseudomonas cells, for example, by electroporation (see, e.g., Choi et al., 2006, J. Microbiol. Methods 64:391-397) or by binding (see, e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71:51-57).DNA can be introduced into streptococcal cells, for example, via natural competent cells (see, e.g., Perry and Kuramitsu, Infect. Immun. 32:1295-1297), via protoplast transformation (see, e.g., Catt and Jollick, 1991, Microbios 68:189-207), via electroporation (see, e.g., Buckley et al., 1999, Applied and Environmental Microbiology 65:3800-3804), or via conjugation (see, e.g., Clewell, 1981, Microbiol. Rev. 45:409-436). However, any method known in the art for introducing DNA into host cells can be used.

[0329] The host cell can also be a eukaryotic cell, such as a mammalian, insect, plant, or fungal cell.

[0330] The host cell can be a fungal cell. As used herein, “fungus” includes Ascomycota, Basidiomycota, Chytridiomycota, Zygomycota, Oomycota, and all mitotic fungi (as defined by Hawksworth et al., in Ainsworth and Bisby's Dictionary of The Fungi, 8th ed., 1995, CAB International, University Press, Cambridge, UK).

[0331] The host cell for fungi can be a yeast cell. As used herein, "yeast" includes ascospore-producing yeasts (Endomycetales), basidiospore-producing yeasts, and yeasts belonging to the class Fungi Imperfecti (Blastomycetes). Since yeast classification may change in the future, for the purposes of this invention, yeast should be defined as described in *Biology and Activities of Yeast* (Skinner, FA; Passmore, SM; and Davenport, RR, eds., Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0332] Yeast host cells can be cells of the genera *Candida*, *Hansenula*, *Kluyveromyces*, *Pichia pastoris*, *Saccharomyces*, *Saccharomyces*, or *Ascomyces*, such as *Kluyveromyces lactis*, *Saccharomyces cerevisiae*, *Saccharomyces cerevisiae*, *Saccharomyces sacchariformis*, *Saccharomyces douglasii*, *Kluyveromyces kluyveromyces*, *Nordiya*, *Ovo*, or *Lipolysis*.

[0333] The host cell for fungi can be a filamentous fungal cell. "Filamentous fungi" includes all filamentous forms of the subphylum Eumycota and Oomycota (as defined above by Hawkesworth et al., 1995). Filamentous fungi are typically characterized by a mycelial wall composed of chitosan, cellulose, glucan, mannan, and other complex polysaccharides. Vegetative growth is characterized by hyphal elongation, and carbon metabolism is obligate aerobic. In contrast, yeasts, such as Saccharomyces cerevisiae, exhibit vegetative growth through budding of single-celled cells, and their carbon metabolism can be fermentative.

[0334] The host cells for filamentous fungi can be cells of the genera *Cladosporium*, *Aspergillus*, *Aureobasidium*, *Bjerkandera*, *Pseudomonas*, *Aureobasidium*, *Coprinus*, *Coriolus*, *Cryptococcus*, *Ustilago maydis*, *Fusarium*, *Pyrophyllus*, *Gastroderma*, *Mucor*, *Hydrophyllus*, *Neurospora*, *Penicillium*, *Penicillium*, *Phlebia*, *Phyllostachys*, *Schizophyllum*, *Cynophyllus*, *Thermophilus*, *Ascomycetes*, *Cladosporium*, *Cyclophorus*, *Cladophora*, *Trametes*, or *Trichoderma*.

[0335] For example, the host cells for filamentous fungi can be Aspergillus amblymorii cells, Aspergillus scurvy cells, Aspergillus fumigatus cells, Aspergillus japonicus cells, Aspergillus nidus cells, Aspergillus oryzae cells, Bjerkandera adusta cells, Ceriporiopsis aneirina cells, Ceriporiopsis caregiea cells, Ceriporiopsis gilvescens cells, Ceriporiopsis pannocinta cells, Ceriporiopsis rivulosa cells, Ceriporiopsis subrufa cells, Ceriporiopsis subvermispora cells, Chrysosporium inops cells, Chrysosporium keratophilum cells, Chrysosporium lucida cells, Chrysosporium coprolifera cells, Chrysosporium pubescens cells, Chrysosporium pubescens cells, Chrysosporium lucida ... zonatum cells, *Coprinus gracilistylus* cells, *Coriolus hirsutus* cells, *Fusarium cerealis* cells, *Fusarium graminearum* cells, *Fusarium xanthophytum* cells, *Fusarium graminearum* cells, *Fusarium graminearum* cells, *Fusarium heterosporum* cells, *Fusarium hyacinthum* cells, *Fusarium oxysporum* cells, *Fusarium reticulata* cells, *Fusarium rosenbergii* cells, *Fusarium oleraceus* cells, *Fusarium peltata* cells, *Fusarium venenatum* cells, *Potentilla rotundifolia* cells, *Potentilla pubescens* cells, *Mucor* cells, *Heatophilic pyrophyllosis* cells, *Neurospora crassa* cells, *Penicillium purpureum* cells, *Phlebia radiata* cells, *Pleurotus eryngii* cells. eryngii) cells, terrestrial fusiform cells, Trametes villosa cells, Trametes versicolor cells, Trichoderma harzianum cells, Trichoderma koningii cells, Trichoderma longibranch cells, Trichoderma reesei cells, or Trichoderma viride cells.

[0336] Fungal cells can be transformed through a process involving protoplast formation, protoplast transformation, and cell wall regeneration in a manner known per se. Suitable procedures for transforming Aspergillus and Trichoderma host cells are described in EP 238023 and Yelton et al., 1984, Proceedings of the National Academy of Sciences 81:1470-1474. Suitable methods for transforming Fusarium species are described in Malardier et al., 1989, Gene 78:147-156 and WO 96 / 00787. Yeast can be transformed using the procedures described in the following literature: Becker and Guarente, cited in Abelson, JN and Simon, MI eds., Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Vol. 194, pp. 182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proceedings of the National Academy of Sciences 75:1920.

[0337] Method of generation

[0338] The present invention also relates to a method for generating a variant, the method comprising: (a) culturing a host cell of the present invention under conditions suitable for expressing the variant; and (b) recovering the variant.

[0339] Host cells are cultured in a suitable nutrient medium for generating variants using methods known in the art. For example, cells can be cultured by shake flask culture or by small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid-state fermentation) in a laboratory or industrial fermenter, said culture being carried out in a suitable medium and under conditions that allow for peptide expression and / or isolation. Culture is performed in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts using procedures known in the art. 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 a variant is secreted into the nutrient medium, the variant can be recovered directly from the medium. If no variant is secreted, it can be recovered from cell lysates.

[0340] Variances can be detected using methods known in the art that are specific to the variant. These detection methods may include 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 variants.

[0341] The variant can be recovered using methods known in the art. For example, the variant can be recovered from nutrient media using routine procedures, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

[0342] Variants can be purified by a variety of methods known in the art, including but not limited to chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic chromatography, focusing chromatography, and size exclusion chromatography), electrophoresis (e.g., preparative isoelectric focusing), differential dissolution (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, for example, Protein Purification, edited by J.-C. Janson and Lars Ryden, VCH Publishing, New York, 1989) to obtain substantially pure variants.

[0343] In terms of alternatives, variants are not recycled; instead, the present invention’s host cells expressing variants are used as the source of variants.

[0344] Composition

[0345] The present invention also relates to compositions comprising variants of the invention. Preferably, the composition is enriched with such variants. The term "enrichment" means that the α-amylase activity of the composition has been increased, for example, having an enrichment factor of 1.1.

[0346] The composition may contain variants as the main enzymatic component, such as a single-component composition. Alternatively, the composition may contain a variety of enzymatic activities, such as aminopeptidase, amylase, glycosylase, carboxypeptidase, catalase, cellulase, chitinase, keratinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, α-galactosidase, β-galactosidase, glucosylamylase, α-glucosidase, β-glucosidase, halogenated peroxidase, invertase, laccase, lipase, mannosidase, oxidase, pectin lyase, peptidylglutaminase, peroxidase, phytase, polyphenol oxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase. Other enzymes can be produced, for example, by microorganisms belonging to the following genera: *Aspergillus*, such as *Aspergillus echinosporum*, *Aspergillus avocado*, *Aspergillus sulphureus*, *Aspergillus fumigatus*, *Aspergillus japonicus*, *Aspergillus niger*, or *Aspergillus oryzae*; *Fusarium*, such as *Fusarium cerealis*, *Fusarium graminearum*, *Fusarium graminearum*, *Fusarium graminearum*, *Fusarium toruloseum*, *Fusarium venenatum*; *Pyrophyllus*, such as *Pyrophyllus salivarius* or *Pyrophyllus pubescens*; or *Trichoderma*, such as *Trichoderma harzianum*, *Trichoderma konjac*, *Trichoderma longifolium*, *Trichoderma reesei*, or *Trichoderma viride*.

[0347] The composition can be prepared according to methods known in the art, and it can be in the form of a liquid or dry composition. For example, the composition can be in the form of particles or microparticles. Variants can be stabilized according to methods known in the art.

[0348] According to the present invention, the above-mentioned α-amylase variants can typically be components in cleaning compositions such as detergent compositions, for example, laundry detergent compositions or dishwashing detergent compositions. Liquid laundry detergent compositions are particularly preferred.

[0349] Such cleaning compositions contain cleaning / detergent additives, preferably mixtures of components. Typically, the cleaning additives will be present in the composition in an amount from 0.001 to 99.9 wt%, more typically from 0.01 to 80 wt%.

[0350] In another preferred aspect, the composition comprises one or more surfactants, which may be nonionic (including semi-polar) and / or anionic and / or cationic and / or zwitterionic and / or amphoteric and / or semi-polar nonionic and / or mixtures thereof. The surfactants are typically present in the composition at levels ranging from about 0.1% to 60% by weight, or from 0.5% to 50% by weight, or from 1% to 40% by weight.

[0351] use

[0352] The present invention also relates to methods using α-amylase variants. The α-amylase variants of the present invention can be used in detergent compositions, low-temperature laundry, dishwashing, and / or cleaning processes.

[0353] Example

[0354] pNP-G7 assay for determining α-amylase activity

[0355] α-Amylase activity can be determined using a G7-pNP substrate. G7-pNP, abbreviated as 4,6-ethylidene (G7)-p-nitrophenyl (G1)-α,D-maltoheptaglycoside, is a block oligosaccharide cleavable by endoamylases such as α-amylase. After cleavage, the α-glucosidase contained in the kit further digests and hydrolyzes the substrate to release free PNP molecules, which are yellow and therefore can be measured spectrophotometrically at λ = 405 nm (400-420 nm). The kit containing G7-pNP substrate and α-glucosidase is manufactured by Roche / Hitachi (catalog number 11876473). Reagents:

[0356] The G7-pNP substrate from this kit contains 22 mM 4,6-ethylidene-G7-pNP and 52.4 mM Hepes (2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid), pH 7.0.

[0357] The α-glucosidase reagent contains 52.4 mM Hepes, 87 mM NaCl, 12.6 mM MgCl2, 0.075 mM CaCl2, and >4 kU / L of α-glucosidase.

[0358] A substrate working solution is prepared by mixing 1 mL of α-glucosidase reagent with 0.2 mL of G7-pNP substrate. This substrate working solution should be prepared immediately before use.

[0359] Dilution buffer: 50 mM MOPS, 0.05% (w / v) Triton X100 (polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether (C 14 H 22 O(C2H4O) n (n=9-10), 1mM CaCl2, pH 8.0. Procedure:

[0360] Dilute the amylase sample to be analyzed in dilution buffer to ensure the pH of the diluted sample is 7. The assay is performed by transferring 20 μl of the diluted enzyme sample to a 96-well microtiter plate and adding 80 μl of substrate working solution. Mix the solutions and pre-incubate at room temperature for 1 min, then measure the absorbance at OD 405 nm every 20 seconds over a 5-minute timeframe.

[0361] The slope (absorbance per minute) of the time-dependent absorption curve is proportional to the specific activity (activity / mg enzyme) of the α-amylase in question under a given series of conditions. The amylase sample should be diluted to a level where the slope is below 0.4 absorbance units / minute.

[0362] Automated Mechanical Stress Measurement Method (AMSA) for Clothing

[0363] To assess the washing performance in clothing, washing experiments were conducted using the Automated Mechanical Stress Assay (AMSA). AMSA allows for the testing of the washing performance of large volumes of small-volume enzyme-detergent solutions. An AMSA plate has numerous slots for the test solution and a lid that firmly presses against all slot openings to the clothing sample (the textile to be washed). During the washing cycle, the plate, test solution, textile, and lid are vigorously shaken to bring the test solution into contact with the textile and apply mechanical stress using a regular, periodic oscillation method. For further description, see WO 02 / 42740, particularly the paragraph “Specific Method Examples” on pages 23-24.

[0364] Overall description of washing performance

[0365] A test solution was prepared comprising water (10°dH), 0.8 g / L detergent (e.g., 5.1 g / L European liquid detergent as described below), and the enzyme of the present invention (e.g., at concentrations of 0, 0.8, and / or 1.2 mg enzyme protein / L). Fabrics with starch stains (e.g., CS-28 from the Test Material Center BV (Vlardingen, Netherlands, Email 120, 3133KT)) were added and washed at 20°C for 20 minutes. After thorough rinsing under running tap water and drying in the dark, the light intensity or reflectance of the stained fabric was subsequently measured as a measure of washing performance. A test with 0 mg enzyme protein / L was used as a blank to obtain the δ attenuation value. Preferably, mechanical action was applied during the washing step, for example, by shaking, rotating, or agitating the washing solution together with the fabric. The AMSA washing performance test was conducted under the experimental conditions detailed below.

[0366] Table 1: AMSA Experimental Conditions

[0367]

[0368] Amylase dilution buffer: Dilutes amylase in ultrapure water (MilliQ water) with a low concentration of calcium (0.1 mM) to stabilize the amylase during storage and 0.01% Triton X-100 to reduce the risk of enzyme protein adsorption to containers and pipettes.

[0369] By adding CaCl2, MgCl2, and NaHCO3 (Ca) to the test system 2+ :Mg 2+ :HCO3 - =3:1:4.5), adjust the water hardness to 10°dH. After washing, rinse the textiles in tap water and then dry.

[0370] Washability is measured as the color brightness of washed textiles. Brightness can also be described as the intensity of light reflected from a sample when illuminated by white light. When a sample is soiled, the intensity of the reflected light is lower than that of a clean sample. Therefore, the intensity of reflected light can be used to measure washability.

[0371] Color measurement using a professional flatbed scanner (Kodak iQsmart, Kodak, Midtager 29, DK-2605) (Denmark), the scanner is used to capture images of washed textiles.

[0372] To extract the light intensity value from the scanned image, the 24-bit pixel values ​​from the image are converted into red, green, and blue (RGB) values. The intensity value (Int) is calculated by summing these RGB values ​​as a vector and then multiplying by the length of the resulting vector.

[0373]

[0374] textile :

[0375] Textile sample CS-28 (rice starch on cotton) was obtained from the Test Materials Centre BV (mailbox 120, 3133 KT, Vlaardingen, Netherlands). Table 3 shows the results of AMSA clothing tests for different variants. In this result, the index is 100. The performance results of the parental α-amylase were assigned a value of 100 and the results of the variants were compared with this value.

[0376] AMSA washing performance

[0377] The washing performance of the variant and the corresponding parental α-amylase was tested using the AMSA assay method as described in the Methods section. Results are given as (variant performance - blank performance) divided by (parental performance - blank performance) multiplied by 100, where the blank performance was obtained by washing under the same conditions but in the absence of α-amylase. Finally, the mean of the relative performances at two concentrations, 0.8 and 1.2 mg / L, was calculated. The results are presented in Table 3.

[0378] Terg-O-tometer (TOM) Washing Test

[0379] Tergo-To-Meter (TOM) is a medium-scale model washing system that can be used to simultaneously test 12 different washing conditions. TOM is essentially a large, temperature-controlled water bath containing up to 12 open metal beakers submerged within it. Each beaker constitutes a small top-loader type washing machine, and during the experiment, each contains a solution of a specific detergent / enzyme system and stained and unstained fabrics to test its performance. Mechanical stress is achieved by rotating stirring arms that agitate the liquid inside each beaker. Because the TOM beakers are lidless, samples can be extracted during TOM experiments, and online information during the washing process can be analyzed.

[0380] The TOM model washing system is primarily used for medium-scale testing of detergents and enzymes under US or LA / AP washing conditions. In TOM experiments, several factors can be varied, such as the ballast-to-dirt ratio and the fabric-to-wash ratio. Therefore, TOM provides a link between small-scale experiments (such as AMSA and microwashing) and more time-consuming full-scale experiments in top-loading washing machines.

[0381] equipment A water bath with 12 steel beakers and 1 rotating arm / beaker, with a capacity of 500 or 1200 mL detergent solution. The temperature range is from 5°C to 80°C. The water bath should be filled with deionized water. The rotation speed can be set up to 70 to 120 rpm.

[0382] TOM washing performance

[0383] The water hardness is adjusted to the strength described below by adding CaCl2, MgCl2, and NaHCO3. The washing solution is prepared in a tank with the desired amount of detergent, temperature, and water hardness as described below. The detergent is dissolved during magnetic stirring for 10 minutes. (The washing solution is used within 30 to 60 minutes after preparation).

[0384] In the Terg-O-Tometer, set the temperature and rotation speed (rpm) in the water bath according to the following settings. While adjusting the temperature according to the settings (tolerance is + / - 0.5°C), add the washing solution to the TOM beaker in the amounts described below.

[0385] Stir the mixture in the beaker at 120 rpm. Add two portions of rice starch sample (CS-28) and the dirt ballast to each beaker and wash according to the times described below. Rinse the sample in cold tap water for 5 minutes. Allow the sample to air dry overnight in the dark.

[0386] textile Textile sample CS-28 (rice starch on cotton) was obtained from the Fraldingen Test Materials Centre BV (email 120, 3133KT) in the Netherlands.

[0387] Foul Ballast : Dirt ballast, obtained from the Fraser Dingen Test Materials Centre BV (Phone 120, 3133KT), Netherlands, on cotton / polyester rice starch (EMPA 162). Bistro gravy (063KC), Frij chocolate milkshake, Heinz spaghetti (113KC), and Herseys double chocolate were obtained from Warwick Equest Ltd, UK (Durhamshire, DH86BN, Consett, Consett Business Park, Unit 55).

[0388] The results of the TOM washing test for different variants are shown in Table 3. In this result, the index is 100. The value 100 was assigned to the performance results of the parental α-amylase (SEQ ID NO:7) and the results of the variants were compared with this value.

[0389] Table 2: Experimental Conditions

[0390]

[0391] The detergent and test materials are as follows:

[0392]

[0393] Washability was measured as the color brightness of the washed textiles, expressed as attenuation values. Attenuation measurements were performed using a Macbeth 7000 Color Eye spectrophotometer. Measurements were taken for each dried sample. Due to the risk of interference from the background, the sample was placed on top of four layers of fabric during attenuation measurements. Attenuation was measured at 460 nm. UV filters were not included. The average attenuation of the samples was calculated.

[0394] Example 1

[0395] Detergency performance of α-amylase in liquid detergents in Europe (EU) (Example 1A) and North America (Example 1B) (US)

[0396] As described above, the washing performance of the tested variant and the corresponding parent α-amylase (SEQ ID NO:7) was tested. The results are given as (variant performance - blank performance) divided by (parental performance - blank performance) multiplied by 100, where the blank is the performance obtained by washing under the same conditions but in the absence of α-amylase.

[0397] Example 1A : European Heavy Stain Liquid Laundry Detergent Composition

[0398]

[0399]

[0400] 1 Random graft copolymers are polyvinyl acetate-grafted polyethylene oxide copolymers having a polyethylene oxide backbone and multiple polyvinyl acetate side chains. The molecular weight of the polyethylene oxide backbone is approximately 6000, the weight ratio of polyethylene oxide to polyvinyl acetate is approximately 40 to 60, and there is no more than one grafting point per 50 ethylene oxide units.

[0401] Example 1B: North American Heavy Stain Liquid Laundry Detergent Composition

[0402] Sodium alkylethoxylate 17.29 Sodium alkylbenzenesulfonate 7.73 Branched alkyl sulfates 3.3 <![CDATA[C 12-18 Alkyl 1,5-9 ethoxylates 1.31 <![CDATA[C 12 Dimethylamine oxide 1.03 Citric acid 0.67 <![CDATA[C 12-18 Fatty acids 1.52 Sodium borate (Borax) 2.53 <![CDATA[Ethoxylated polyethyleneimine 2 > 1.44 Phosphonic chelating agents 0.34 Disodium dihydroxybenzene-3,5-disulfonic acid salt hydrate 0.19 Brightener 0.29 <![CDATA[Amphoteric alkoxylated oil cleaning polymer 3 > 1.93 Small quantities: dyes, fragrances, enzymes, enzyme stabilizers, solvents, structural agents, pH adjusters balance

[0403] 2 Polyethyleneimine with 20 ethoxylated groups / -NH (MW=600).

[0404] 3 The amphoteric alkoxylated oleo-cleaning polymer is a polyethyleneimine (MW=600) having 24 ethoxylated groups / -NH and 16 propoxylated groups / -NH.

[0405] Table 3: Washing Performance

[0406]

[0407]

[0408] The washing performance test results clearly demonstrate that, at the test temperature, the performance of the variants is improved relative to their corresponding parent molecules (SEQ ID NO:7).

[0409] Example 2

[0410] Full-scale washing machine performance evaluation of amylase variants in liquid detergents

[0411] I. Preparation of Detergent Test Compositions

[0412] In this experiment, four test compositions were prepared based on liquid detergent Example 1A. The detergent base was prepared from Example 1A, which was enzyme-free and adjusted to pH 8.2.

[0413] Prepare the following four detergent formulations:

[0414] Table 4

[0415]

[0416] *Added as an active enzyme protein

[0417] The enzyme is supplied by Novosibirsk A / S of Denmark as 'Natalase 200L'.

[0418] 2 Variant 1 is the amylase variant of the present invention, *Bacillus amylase* sp722 SEQ ID NO:1, having the following two deletions: D183*+G184* and containing substitutions for W140Y, N195F, V206Y, Y243F, E260G, and W284D. It is also referred to as SP722+D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+W284D.

[0419] 3 Variant 2 is the amylase variant of the present invention of wild-type Bacillus amylase sp722 SEQ ID NO:1, which has the following two deletions: D183*+G184* and includes substitutions W140Y, N195F, V206Y, Y243F, and W284D. It is also referred to as SP722+D183*+G184*+W140Y+N195F+V206Y+Y243F+W284D.

[0420] II. Test Fabrics

[0421] Three amylase-sensitive stains: CS-28 rice starch, PCS-28 rice starch, and CS-29 tapioca starch (5cm x 5cm) (supplied by the Netherlands Test Materials Centre) were applied to 20cm x 20cm white knitted cotton fabric (supplied by Warwick Equest Durham, UK). Two amylase-sensitive stains: BBQ sauce and Frijj chocolate milkshake (2.5cm in diameter) were applied to 20cm x 20cm white knitted cotton fabric (supplied by Warwick Equest Durham, UK). Eight replicates (two replicates across four different machines) were used for each test preparation.

[0422] Table 5:

[0423]

[0424]

[0425] III. Test Washing Program

[0426] This method involves using a Western European Hotpoint washing machine, model Aquarius WF541. Amylase-sensitive stains were washed with the test reagent as described above, along with a mixed soiling and cleaning ballast load as described above. The washing machine, containing 13L of water with a Clarke hardness of 10° and the test fabric and ballast load, was used at 15°C for a quick cotton wash cycle of 1 hour and 15 minutes. After washing, the test fabric was hung to dry indoors. The washing process was repeated for three additional wash cycles.

[0427] The Stain Removal Index (SRI) was then measured (e.g., by comparing the L*a*b* value of the unwashed product with the L*a*b* value of the washed product) to quantify the stain removal performance of the detergent composition.

[0428] The performance index is also measured by calculating ((performance of instance C or instance D - performance of comparison instance A) divided by (performance of comparison instance B - performance of comparison instance A) multiplied by 100).

[0429] IV. Comparison of Samples

[0430] Table 6 : Average SRI of 5 types of stains (8 replicates of each stain)

[0431]

[0432] Table 7 : Performance index of CS-29 cassava starch (8 replicates)

[0433]

[0434]

[0435] By comparing samples washed with the composition of Example A (containing nil enzyme) with samples washed with Examples B (containing Natalase), C, and D (containing variants 1 and 2, respectively), it is evident that the stain removal performance is improved by the addition of amylase. Based on Example A (nil enzyme) as a reference, by comparing samples washed with the composition of Example B (containing Natalase) with samples washed with Examples C and D (containing variants 1 and 2, respectively), it is evident that variants 1 and 2 of the present invention both achieve better stain removal performance than... Significantly higher level of stain removal.

[0436] Example 3

[0437] Full-scale washing machine performance evaluation of amylase variants in liquid detergents

[0438] I. Preparation of Detergent Test Compositions

[0439] In this experiment, four test compositions were prepared based on the liquid detergent example 1B above. The detergent base was prepared from example 1B, which was enzyme-free and adjusted to pH 8.2.

[0440] The following four formulations were prepared:

[0441] Table 8 :

[0442]

[0443] *Added as an active enzyme protein

[0444] The 'Natalase 200L' is supplied by Novosim A / S of Denmark.

[0445] 4 Variant 3 is the amylase variant of the present invention of wild-type Bacillus amylase sp722 SEQ ID NO:1, which has the following two deletions: D183*+G184* and includes substitutions of W140Y, N195F, V206Y, Y243F, E260G, and G477E. It is also referred to as SP722+D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+G477E

[0446] 5Variant 4 is a variant of the amylase of the present invention, *Bacillus amylase* sp722 SEQ ID NO:1, having the following two deletions: D183*+G184* and including substitutions of G109A, W140Y, N195F, V206Y, Y243F, and E260G. It is also referred to as SP722+D183*+G184*+G109A+W140Y+N195F+V206Y+Y243F+E260G.

[0447] II. Test Fabrics

[0448] Three amylase-sensitive stains: PCS-28 rice starch, PS-28 rice starch, and CS-26 corn starch (5cm x 5cm) (supplied by the Netherlands Test Materials Centre) were applied to 20cm x 20cm white knitted cotton fabric (supplied by Warwick Equest Durham, UK). Two amylase-sensitive stains: BBQ sauce and chocolate pudding baby food (2.5cm in diameter) were applied to 20cm x 20cm white knitted cotton fabric (supplied by Warwick Equest Durham, UK). Eight replicates (two replicates across four different machines) were used for each test preparation.

[0449] Table 9 :

[0450]

[0451] III. Test Washing Program

[0452] This method involves using a Kenmore washing machine, model 600 series, from North America. Amylase-sensitive stains are washed with the test formulation described above, along with a mixed soiling and cleaning ballast load as described above.

[0453] A washing machine containing 0.78 g / L of test reagent, 6° Clarke water hardness, the test fabric, and ballast was used to wash the fabric at 15°C with a 12-minute super wash and a single rinse. After washing, the test fabric was hung to dry indoors.

[0454] Repeat the washing process for another 3 washing cycles.

[0455] The stain removal index (e.g., by comparing the L*a*b* value of the unwashed product with the L*a*b* value of the washed product) was then measured to quantify the stain removal performance of the detergent composition.

[0456] The performance index is also measured by calculating ((performance of instance C or instance D - performance of comparison instance A) divided by (performance of comparison instance B - performance of comparison instance A) multiplied by 100).

[0457] IV. Comparison of Samples

[0458] Table 10 : Average SRI of 5 types of stains (8 replicates of each stain)

[0459]

[0460]

[0461] Table 11 : Performance index of CS-26 corn starch (8 replicates)

[0462] Comparison Example A -- Comparison Example B 100 According to Example C of the present invention 221 Example D of the present invention 238

[0463] By comparing samples washed with the composition of Example A (containing nil enzyme) with samples washed with Examples B (containing Natalase), C, and D (containing variants 3 and 4, respectively), it is evident that the stain removal performance is improved by the addition of amylase. Based on Example A (nil enzyme) as a reference, by comparing samples washed with the composition of Example B (containing Natalase) with samples washed with Examples C and D (containing variants 3 and 4, respectively), it is evident that variants 3 and 4 of the present invention both achieve better stain removal performance than... Significantly higher level of stain removal.

[0464] Example 4

[0465] Full-scale washing machine performance evaluation of amylase variants in liquid detergents

[0466] I. Preparation of Detergent Test Compositions

[0467] In this experiment, four test compositions were prepared based on the following liquid detergent formulation 4A. A detergent base was prepared from formulation 4A, which was enzyme-free and adjusted to pH 8.2.

[0468] Table 12; Formulation 4A Heavy-duty liquid laundry detergent composition

[0469]

[0470]

[0471] 1 Random graft copolymers are polyvinyl acetate-grafted polyethylene oxide copolymers having a polyethylene oxide backbone and multiple polyvinyl acetate side chains. The molecular weight of the polyethylene oxide backbone is approximately 6000, the weight ratio of polyethylene oxide to polyvinyl acetate is approximately 40 to 60, and there is no more than one grafting point per 50 ethylene oxide units.

[0472] The following seven detergent formulations were prepared:

[0473] Table 13 :

[0474]

[0475] *Added as an active enzyme protein

[0476] The enzyme is supplied by Novosibirsk A / S of Denmark as 'Natalase 200L'.

[0477] 2 Variant 5 is the amylase variant of the present invention of wild-type Bacillus amylase sp722 SEQ ID NO:1, which has the following two deletions: D183*+G184* and includes substitutions of G109A, W140Y, N195F, V206Y, Y243F, E260G, and G476E. It is also referred to as SP722+D183*+G184*+G109A+W140Y+N195F+V206Y+Y243F+E260G+G476E.

[0478] 3 Variant 6 is the amylase variant of the present invention of wild-type Bacillus amylase sp722 SEQ ID NO:1, which has the following two deletions: D183*+G184* and includes substitutions of T51I, G109A, W140Y, N195F, V206Y, Y243F, E260G, and W439R. It is also referred to as SP722+D183*+G184*+T51I+G109A+W140Y+N195F+V206Y+Y243F+E260G+W439R.

[0479] 4 Variant 7 is the amylase variant of the present invention of wild-type Bacillus amylase sp722 SEQ ID NO:1, which has the following two deletions: D183*+G184* and includes substitutions of T51I, S52Q, N54K, G109A, W140Y, N195F, V206Y, Y243F, E260G, and G476E. It is also referred to as SP722+D183*+G184*+T51I+S52Q+N54K+G109A+W140Y+N195F+V206Y+Y243F+E260G+G476E.

[0480] 5Variant 8 is the amylase variant of the present invention of wild-type Bacillus amylase sp722 SEQ ID NO:1, which has the following two deletions: D183*+G184* and includes substitutions of W140Y, N195F, V206Y, Y243F, E260G, G304R, and G476K. It is also referred to as SP722+D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+G304R+G476K.

[0481] 6 Variant 9 is the amylase variant of the present invention of wild-type Bacillus amylase sp722 SEQ ID NO:1, which has the following two deletions: D183*+G184* and includes substitutions of W140Y, N195F, V206Y, Y243F, E260G, W284R, and G477K. It is also referred to as SP722+D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+W284R+G477K.

[0482] II. Test Fabrics

[0483] Three amylase-sensitive stains: PCS-28 rice starch, CS-128 cooked rice starch, and CS-26 corn starch (5cm x 5cm) (supplied by the Netherlands Test Materials Centre) were applied to 20cm x 20cm white knitted cotton fabric (supplied by Warwick Equest, Durham, UK). Two amylase-sensitive stains: Chilli Con Carne and Heinz spaghetti (2.5cm in diameter) were applied to 20cm x 20cm white knitted cotton fabric (supplied by Warwick Equest, Durham, UK). Eight replicates (two replicates across four different machines) were used for each test preparation.

[0484] Table 14

[0485]

[0486] III. Test Washing Program

[0487] This method involves using a Western European Hotpoint washing machine, model Aquarius WF541. Amylase-sensitive stains were washed with the test formulation described above, along with a mixed dirt and cleaning ballast load as described above.

[0488] A washing machine containing 6 g / L of the test reagent, 13 L of water at a Clark hardness of 10°, and the test fabric and ballast were used to wash the fabric in a rapid cotton wash cycle at 15°C for 1 hour and 15 minutes. After washing, the test fabric was hung to dry indoors.

[0489] Repeat the washing process for another 3 washing cycles.

[0490] The Stain Removal Index (SRI) was then measured (e.g., by comparing the L*a*b* value of the unwashed product with the L*a*b* value of the washed product) to quantify the stain removal performance of the detergent composition.

[0491] The performance index is also measured by calculating ((performance of instance C, D, E, F or G - performance of comparison instance A) divided by (performance of comparison instance B - performance of comparison instance A) multiplied by 100).

[0492] IV. Comparison of Samples

[0493] Table 15 : Average SRI of 5 types of stains (8 replicates of each stain)

[0494]

[0495] Table 16 : Performance index of Mexican hot meat sauce (8 replicates)

[0496] Comparison Example A -- Comparison Example B 100 According to Example C of the present invention 169 Example D of the present invention 145 Example E of the present invention 181 Example F of the present invention 178 According to Example G of the present invention 188

[0497] By comparing samples washed with the composition of Example A (containing nil enzyme) with samples washed with Examples B (containing Natalase), C, D, E, F and G (containing variants 5, 6, 7, 8 and 9, respectively), it is evident that the stain removal performance is improved by the addition of amylase.

[0498] Based on Example A (nil enzyme) as a reference, by comparing samples washed with the composition of Example B (containing Natalase) with samples washed with Examples C, D, E, F, and G (containing variants 5, 6, 7, 8, and 9, respectively), it is evident that variants 5, 6, 7, 8, and 9 of the present invention can achieve higher performance than... Significantly higher level of stain removal.

[0499] Example 5

[0500] Full-scale washing machine performance evaluation of amylase variants in liquid detergents

[0501] I. Preparation of Detergent Test Compositions

[0502] In this experiment, four test compositions were prepared based on liquid detergent formulation 5A. A detergent base was prepared from formulation 5A, which was enzyme-free and adjusted to pH 8.2.

[0503] Table 17 : Formulation of 5A Heavy Stain Liquid Laundry Detergent Composition

[0504] Sodium alkylethoxylate 14.81 Sodium alkylbenzenesulfonate 3.53 Branched alkyl sulfates 2.44 <![CDATA[C 12-18 Alkyl-1,5-9-ethoxylates 0.88 <![CDATA[C 12 Dimethylamine oxide 0.56 Citric acid 2.05 <![CDATA[C 12-18 Fatty acids 1.48 <![CDATA[ethoxylated polyethyleneimine 2 > 1.51 Phosphonic chelating agents 0.53 Brightener 0.19 <![CDATA[Amphoteric alkoxylated polymers 3 > 1.25 Small quantities: dyes, fragrances, enzymes, enzyme stabilizers, solvents, structural agents, pH adjusters Balance

[0505] 2 Polyethyleneimine with 20 ethoxylated groups / -NH (MW=600).

[0506] 3 Amphoteric alkoxylated polymers are polyethyleneimine (MW 600) prepared from polymers derived from polymers containing 24 ethoxylated groups / -NH and 16 propoxylated groups / -NH.

[0507] Table 18

[0508]

[0509] The following six formulations were prepared:

[0510] *Added as an active enzyme protein

[0511] The 'Natalase 200L' is supplied by Novosim A / S of Denmark.

[0512] 2 Variant 7 is the amylase variant of the present invention of wild-type Bacillus amylase sp722 SEQ ID NO:1, which has the following two deletions: D183*+G184* and includes substitutions of T51I, S52Q, N54K, G109A, W140Y, N195F, V206Y, Y243F, E260G, and G476E. It is also referred to as SP722+D183*+G184*+T51I+S52Q+N54K+G109A+W140Y+N195F+V206Y+Y243F+E260G+G476E.

[0513] 3Variant 8 is the amylase variant of the present invention of wild-type Bacillus amylase sp722 SEQ ID NO:1, which has the following two deletions: D183*+G184* and includes substitutions of W140Y, N195F, V206Y, Y243F, E260G, G304R, and G476K. It is also referred to as SP722+D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+G304R+G476K.

[0514] 4 Variant 9 is the amylase variant of the present invention of wild-type Bacillus amylase sp722 SEQ ID NO:1, which has the following two deletions: D183*+G184* and includes substitutions of W140Y, N195F, V206Y, Y243F, E260G, W284R, and G477K. It is also referred to as SP722+D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+W284R+G477K.

[0515] 5 Variant 10 is an amylase variant of the present invention of wild-type Bacillus amylase sp722 SEQ ID NO:1, which has the following two deletions: D183*+G184* and includes substitutions of W140Y, N195F, V206Y, Y243F, E260G, W284F, and G477R. It is also referred to as SP722+D183*+G184*+W140Y+N195F+V206Y+Y243F+E260G+W284F+G477R.

[0516] II. Test Fabrics

[0517] Three amylase-sensitive stains: PCS-28 rice starch, PS-28 rice starch, and CS-29 tapioca starch (5cm x 5cm) (supplied by the Netherlands Test Materials Centre) were applied to 20cm x 20cm white knitted cotton fabric (supplied by Warwick Equest, Durham, UK). One amylase-sensitive stain: Heinz spaghetti (2.5cm in diameter) was applied to 20cm x 20cm white knitted cotton fabric (supplied by Warwick Equest, Durham, UK).

[0518] An amylase-sensitive stain: gravy (2.5 cm in diameter) was applied to a 25 cm x 24 cm white knitted cotton fabric (supplied by Accurate Product Development, Fairfield, Ohio, USA). Eight replicates (two duplicates, four different machines) were used for each test preparation.

[0519] Table 19

[0520]

[0521]

[0522] III. Test Washing Program

[0523] This method involves using a Kenmore washing machine, model 600 series, from North America. Amylase-sensitive stains are washed with the test formulation described above, along with a mixed soiling and cleaning ballast load as described above.

[0524] A washing machine containing 0.78 g / L of test reagent, 6° Clarke water hardness, the test fabric, and ballast was used to wash the fabric at 15°C with a 12-minute super wash and a single rinse. After washing, the test fabric was hung to dry indoors.

[0525] Repeat the washing process for another 3 washing cycles.

[0526] The stain removal index (e.g., by comparing the L*a*b* value of the unwashed product with the L*a*b* value of the washed product) was then measured to quantify the stain removal performance of the detergent composition.

[0527] The performance index is also measured by calculating ((performance of instance C, D, E, or F - performance of comparison instance A) divided by (performance of comparison instance B - performance of comparison instance A) multiplied by 100).

[0528] IV. Comparison of Samples

[0529] Table 20 : Average SRI of 5 types of stains (8 replicates of each stain)

[0530]

[0531] Table 21 : Heinz Spaghetti (8 replicates) Performance Index

[0532] Comparison Example A -- Comparison Example B 100 According to Example C of the present invention 266 Example D of the present invention 256 Example E of the present invention 284 Example F according to the present invention 416

[0533] By comparing samples washed with the composition of Example A (containing nil enzyme) with samples washed with Examples B (containing Natalase), C, D, E, and F (containing variants 7, 8, 9, and 10, respectively), it is evident that the stain removal performance is improved by the addition of amylase.

[0534] Based on comparative example A (nil enzyme) as a reference, by comparing samples washed with the composition of example B (containing Natalase) with samples washed with examples C, D, E, and F (containing variants 7, 8, 9, and 10, respectively), it is evident that variants 7, 8, 9, and 10 of the present invention can achieve higher performance than... Significantly higher level of stain removal.

[0535] The invention described and claimed herein is not limited in scope to the specific aspects disclosed herein, as these aspects are intended to illustrate various aspects of the invention. Any equivalent aspects are contemplated within the scope of the invention. In fact, various modifications to the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description. Such modifications are also contemplated to fall within the scope of the appended claims. In the event of conflict, this disclosure, including its definitions, shall prevail. sequence list <110> Novozymes A / S <120> α-Amylase variants <130> 12183-WO-PCD <160> 12 <170> PatentIn version 3.5 <210> 1 <211> 485 <212> PRT <213> Bacillus species <400> 1 His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His 1 5 10 15 Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ser 20 25 30 Asn Leu Arg Asn Arg 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 Arg Ser Gln Leu Glu Ser Ala Ile His Ala Leu Lys Asn Asn Gly 85 90 95 Val Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110 Ala Thr Glu Asn Val Leu Ala Val Glu Val Asn Pro Asn Asn Arg Asn 115 120 125 Gln Glu Ile Ser Gly Asp Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp 130 135 140 Phe Pro Gly Arg Gly Asn Thr Tyr Ser Asp Phe Lys Trp Arg Trp Tyr 145 150 155 160 His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Gln Phe Gln Asn Arg 165 170 175 Ile Tyr Lys Phe Arg Gly Asp Gly Lys Ala Trp Asp Trp Glu Val Asp 180 185 190 Ser Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met 195 200 205 Asp His Pro Glu Val Val Asn Glu Leu Arg Arg Trp Gly Glu Trp Tyr 210 215 220 Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His 225 230 235 240 Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Ala 245 250 255 Thr Gly Lys Glu Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu 260 265 270 Gly Ala Leu Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val 275 280 285 Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly 290 295 300 Gly Asn Tyr Asp Met Ala Lys Leu Leu Asn Gly Thr Val Val Gln Lys 305 310 315 320 His Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325 330 335 Gly Glu Ser Leu Glu Ser Phe Val Gln Glu Trp Phe Lys Pro Leu Ala 340 345 350 Tyr Ala Leu Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360 365 Gly Asp Tyr Tyr Gly Ile Pro Thr His Ser Val Pro Ala Met Lys Ala 370 375 380 Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Asn Phe Ala Tyr Gly Thr 385 390 395 400 Gln His Asp Tyr Phe Asp His His Asn Ile Ile Gly Trp Thr Arg Glu 405 410 415 Gly Asn Thr Thr His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425 430 Gly Pro Gly Gly Glu Lys Trp Met Tyr Val Gly Gln Asn Lys Ala Gly 435 440 445 Gln Val Trp His Asp Ile Thr Gly Asn Lys Pro Gly Thr Val Thr Ile 450 455 460 Asn Ala Asp Gly Trp Ala Asn Phe Ser Val Asn Gly Gly Ser Val Ser 465 470 475 480 Ile Trp Val Lys Arg 485 <210> 2 <211> 485 <212> PRT <213> Bacillus sp. <400> 2 His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr 1 5 10 15 Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Asn Ser Asp Ala Ser 20 25 30 Asn Leu Lys Ser Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp 35 40 45 Lys Gly Ala 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 Arg Ser Gln Leu Gln Ala Ala Val Thr Ser Leu Lys Asn Asn Gly 85 90 95 Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110 Ala Thr Glu Met Val Arg Ala Val Glu Val Asn Pro Asn Asn Arg Asn 115 120 125 Gln Glu Val Thr Gly Glu Tyr Thr Ile Glu Ala Trp Thr Arg Phe Asp 130 135 140 Phe Pro Gly Arg Gly Asn Thr His Ser Ser Phe Lys Trp Arg Trp Tyr 145 150 155 160 His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Arg Leu Asn Asn Arg 165 170 175 Ile Tyr Lys Phe Arg Gly His 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 Ile Asp Met 195 200 205 Asp His Pro Glu Val Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr 210 215 220 Thr Asn Thr Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His 225 230 235 240 Ile Lys Tyr Ser Phe Thr Arg Asp Trp Ile Asn His Val Arg Ser Ala 245 250 255 Thr Gly Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu 260 265 270 Gly Ala Ile Glu Asn Tyr Leu Gln Lys Thr Asn Trp Asn His Ser Val 275 280 285 Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Lys Ser Gly 290 295 300 Gly Asn Tyr Asp Met Arg Asn Ile Phe Asn Gly Thr Val Val Gln Arg 305 310 315 320 His Pro Ser His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325 330 335 Glu Glu Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala 340 345 350 Tyr Ala Leu Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360 365 Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Arg Ser 370 375 380 Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Lys 385 390 395 400 Gln Asn Asp Tyr Leu Asp His His Asn Ile Ile Gly Trp Thr Arg Glu 405 410 415 Gly Asn Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425 430 Gly Ala Gly Gly Ser Lys Trp Met Phe Val Gly Arg Asn Lys Ala Gly 435 440 445 Gln Val Trp Ser Asp Ile Thr Gly Asn Arg Thr Gly Thr Val Thr Ile 450 455 460 Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser 465 470 475 480 Ile Trp Val Asn Lys 485 <210> 3 <211> 485 <212> PRT <213> Bacillus sp. <400> 3 His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr 1 5 10 15 Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Ser Asp Ala Ser 20 25 30 Asn Leu Lys Asp Lys Gly Ile Ser Ala Val Trp Ile Pro Pro Ala Trp 35 40 45 Lys Gly Ala 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 Ile Arg Thr Lys Tyr Gly 65 70 75 80 Thr Arg Asn Gln Leu Gln Ala Ala Val Asn Ala Leu Lys Ser Asn Gly 85 90 95 Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110 Ala Thr Glu Met Val Arg Ala Val Glu Val Asn Pro Asn Asn Arg Asn 115 120 125 Gln Glu Val Ser Gly Glu Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp 130 135 140 Phe Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys Trp Arg Trp Tyr 145 150 155 160 His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Lys Leu Asn Asn Arg 165 170 175 Ile Tyr Lys Phe Arg Gly Asp Gly Lys Gly Trp Asp Trp Glu Val Asp 180 185 190 Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met 195 200 205 Asp His Pro Glu Val Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr 210 215 220 Thr Asn Thr Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His 225 230 235 240 Ile Lys Tyr Ser Phe Thr Arg Asp Trp Ile Asn His Val Arg Ser Ala 245 250 255 Thr Gly Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu 260 265 270 Gly Ala Ile Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val 275 280 285 Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Lys Ser Gly 290 295 300 Gly Asn Tyr Asp Met Arg Gln Ile Phe Asn Gly Thr Val Val Gln Arg 305 310 315 320 His Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325 330 335 Glu Glu Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala 340 345 350 Tyr Ala Leu Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360 365 Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser 370 375 380 Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Arg 385 390 395 400 Gln Asn Asp Tyr Leu Asp His His Asn Ile Ile Gly Trp Thr Arg Glu 405 410 415 Gly Asn Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425 430 Gly Ala Gly Gly Asn Lys Trp Met Phe Val Gly Arg Asn Lys Ala Gly 435 440 445 Gln Val Trp Thr Asp Ile Thr Gly Asn Arg Ala Gly Thr Val Thr Ile 450 455 460 Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser 465 470 475 480 Ile Trp Val Asn Lys 485 <210> 4 <211> 485 <212> PRT <213> Bacillus sp. <400> 4 His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr 1 5 10 15 Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ala 20 25 30 Asn Leu Lys Ser Lys Gly Ile Thr Ala Val 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 Arg Asn Gln Leu Gln Ala Ala Val Thr Ser Leu Lys Asn Asn Gly 85 90 95 Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110 Gly Thr Glu Ile Val Asn Ala Val Glu Val Asn Arg Ser Asn Arg Asn 115 120 125 Gln Glu Thr Ser Gly Glu Tyr Ala Ile Glu Ala Trp Thr Lys Phe Asp 130 135 140 Phe Pro Gly Arg Gly Asn Asn 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 Gln Asn Lys 165 170 175 Ile Tyr Lys Phe Arg Gly Thr 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 His Glu Leu Arg Asn Trp Gly Val Trp Tyr 210 215 220 Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His 225 230 235 240 Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Thr 245 250 255 Thr Gly Lys Pro Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu 260 265 270 Gly Ala Ile Glu Asn Tyr Leu Asn Lys Thr Ser Trp Asn His Ser Val 275 280 285 Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly 290 295 300 Gly Tyr Tyr Asp Met Arg Asn Ile Leu Asn Gly Ser Val Val Gln Lys 305 310 315 320 His Pro Thr His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325 330 335 Gly Glu Ala Leu Glu Ser Phe Val Gln Gln Trp Phe Lys Pro Leu Ala 340 345 350 Tyr Ala Leu Val Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360 365 Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser 370 375 380 Lys Ile Asp Pro Leu Leu Gln Ala Arg Gln Thr Phe Ala Tyr Gly Thr 385 390 395 400 Gln His Asp Tyr Phe Asp His His Asp Ile Ile Gly Trp Thr Arg Glu 405 410 415 Gly Asn Ser Ser His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425 430 Gly Pro Gly Gly Asn Lys Trp Met Tyr Val Gly Lys Asn Lys Ala Gly 435 440 445 Gln Val Trp Arg Asp Ile Thr Gly Asn Arg Thr Gly Thr Val Thr Ile 450 455 460 Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser 465 470 475 480 Val Trp Val Lys Gln 485 <210> 5 <211> 485 <212> PRT <213> Bacillus sp. <400> 5 His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His 1 5 10 15 Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ala 20 25 30 Asn Leu Lys Ser Lys Gly Ile Thr Ala Val 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 Arg Ser Gln Leu Gln Gly Ala Val Thr Ser Leu Lys Asn Asn Gly 85 90 95 Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110 Gly Thr Glu Met Val Asn Ala Val Glu Val Asn Arg Ser Asn Arg Asn 115 120 125 Gln Glu Ile Ser Gly Glu Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp 130 135 140 Phe Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys Trp Arg Trp Tyr 145 150 155 160 His Phe Asp Gly Thr Asp Trp Asp Gln Ser Arg Gln Leu Gln Asn Lys 165 170 175 Ile Tyr Lys Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp 180 185 190 Ile Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met 195 200 205 Asp His Pro Glu Val Ile Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr 210 215 220 Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His 225 230 235 240 Ile Lys Tyr Ser Tyr Thr Arg Asp Trp Leu Thr His Val Arg Asn Thr 245 250 255 Thr Gly Lys Pro Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu 260 265 270 Ala Ala Ile Glu Asn Tyr Leu Asn Lys Thr Ser Trp Asn His Ser Val 275 280 285 Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly 290 295 300 Gly Tyr Phe Asp Met Arg Asn Ile Leu Asn Gly Ser Val Val Gln Lys 305 310 315 320 His Pro Ile His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325 330 335 Gly Glu Ala Leu Glu Ser Phe Val Gln Ser Trp Phe Lys Pro Leu Ala 340 345 350 Tyr Ala Leu Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360 365 Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ser Met Lys Ser 370 375 380 Lys Ile Asp Pro Leu Leu Gln Ala Arg Gln Thr Tyr Ala Tyr Gly Thr 385 390 395 400 Gln His Asp Tyr Phe Asp His His Asp Ile Ile Gly Trp Thr Arg Glu 405 410 415 Gly Asp Ser Ser His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425 430 Gly Pro Gly Gly Asn Lys Trp Met Tyr Val Gly Lys His Lys Ala Gly 435 440 445 Gln Val Trp Arg Asp Ile Thr Gly Asn Arg Ser Gly Thr Val Thr Ile 450 455 460 Asn Ala Asp Gly Trp Gly Asn Phe Thr Val Asn Gly Gly Ala Val Ser 465 470 475 480 Val Trp Val Lys Gln 485 <210> 6 <211> 485 <212> PRT <213> Bacillus sp. <400> 6 His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr 1 5 10 15 Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Ser Asp Ala Ser 20 25 30 Asn Leu Lys Asp Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp 35 40 45 Lys Gly Ala 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 Arg Asn Gln Leu Gln Ala Ala Val Thr Ala Leu Lys Ser Asn Gly 85 90 95 Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110 Ala Thr Glu Trp Val Arg Ala Val Glu Val Asn Pro Ser Asn Arg Asn 115 120 125 Gln Glu Val Ser Gly Asp Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp 130 135 140 Phe Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys Trp Arg Trp Tyr 145 150 155 160 His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Gln Leu Gln Asn Arg 165 170 175 Ile Tyr Lys Phe Arg Gly Asp Gly Lys Gly Trp Asp Trp Glu Val Asp 180 185 190 Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met 195 200 205 Asp His Pro Glu Val Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr 210 215 220 Thr Asn Thr Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His 225 230 235 240 Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Thr 245 250 255 Thr Gly Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Ile 260 265 270 Gly Ala Ile Glu Asn Tyr Leu Ser Lys Thr Asn Trp Asn His Ser Val 275 280 285 Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Arg Ser Gly 290 295 300 Gly Asn Tyr Asp Met Arg Gln Ile Phe Asn Gly Thr Val Val Gln Arg 305 310 315 320 His Pro Thr His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325 330 335 Glu Glu Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala 340 345 350 Tyr Ala Leu Thr Leu Thr Arg Asp Gln Gly Tyr Pro Ser Val Phe Tyr 355 360 365 Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser 370 375 380 Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Lys 385 390 395 400 Gln Asn Asp Tyr Leu Asp His His Asn Met Ile Gly Trp Thr Arg Glu 405 410 415 Gly Asn Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425 430 Gly Pro Gly Gly Asn Lys Trp Met Tyr Val Gly Arg Asn Lys Ala Gly 435 440 445 Gln Val Trp Arg Asp Ile Thr Gly Asn Arg Ser Gly Thr Val Thr Ile 450 455 460 Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser 465 470 475 480 Ile Trp Val Asn Asn 485 <210> 7 <211> 483 <212> PRT <213> Artificial Sequence <220> <223> Variant of SEQ ID NO: 1 <400> 7 His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His 1 5 10 15 Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ser 20 25 30 Asn Leu Arg Asn Arg 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 Arg Ser Gln Leu Glu Ser Ala Ile His Ala Leu Lys Asn Asn Gly 85 90 95 Val Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110 Ala Thr Glu Asn Val Leu Ala Val Glu Val Asn Pro Asn Asn Arg Asn 115 120 125 Gln Glu Ile Ser Gly Asp Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp 130 135 140 Phe Pro Gly Arg Gly Asn Thr Tyr Ser Asp Phe Lys Trp Arg Trp Tyr 145 150 155 160 His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Gln Phe Gln Asn Arg 165 170 175 Ile Tyr Lys Phe Arg Gly Lys Ala Trp Asp Trp Glu Val Asp Ser 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 Val Asn Glu Leu Arg Arg Trp Gly Glu Trp Tyr Thr Asn 210 215 220 Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His Ile Lys 225 230 235 240 Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Ala Thr Gly 245 250 255 Lys Glu Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu Gly Ala 260 265 270 Leu Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val Phe Asp 275 280 285 Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly Gly Asn 290 295 300 Tyr Asp Met Ala Lys Leu Leu Asn Gly Thr Val Val Gln Lys His Pro 305 310 315 320 Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro Gly Glu 325 330 335 Ser Leu Glu Ser Phe Val Gln Glu Trp Phe Lys Pro Leu Ala Tyr Ala 340 345 350 Leu Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr Gly Asp 355 360 365 Tyr Tyr Gly Ile Pro Thr His Ser Val Pro Ala Met Lys Ala Lys Ile 370 375 380 Asp Pro Ile Leu Glu Ala Arg Gln Asn Phe Ala Tyr Gly Thr Gln His 385 390 395 400 Asp Tyr Phe Asp His His Asn Ile Ile Gly Trp Thr Arg Glu Gly Asn 405 410 415 Thr Thr His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp Gly Pro 420 425 430 Gly Gly Glu Lys Trp Met Tyr Val Gly Gln Asn Lys Ala Gly Gln Val 435 440 445 Trp His Asp Ile Thr Gly Asn Lys Pro Gly Thr Val Thr Ile Asn Ala 450 455 460 Asp Gly Trp Ala Asn Phe Ser Val Asn Gly Gly Ser Val Ser Ile Trp 465 470 475 480 Val Lys Arg <210> 8 <211> 483 <212> PRT <213> Artificial Sequence <220> <223> Variant of SEQ ID NO: 2 <400> 8 His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr 1 5 10 15 Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Asn Ser Asp Ala Ser 20 25 30 Asn Leu Lys Ser Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp 35 40 45 Lys Gly Ala 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 Arg Ser Gln Leu Gln Ala Ala Val Thr Ser Leu Lys Asn Asn Gly 85 90 95 Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110 Ala Thr Glu Met Val Arg Ala Val Glu Val Asn Pro Asn Asn Arg Asn 115 120 125 Gln Glu Val Thr Gly Glu Tyr Thr Ile Glu Ala Trp Thr Arg Phe Asp 130 135 140 Phe Pro Gly Arg Gly Asn Thr His Ser Ser Phe Lys Trp Arg Trp Tyr 145 150 155 160 His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Arg Leu Asn 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 Ile Asp Met Asp His 195 200 205 Pro Glu Val Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr Thr Asn 210 215 220 Thr Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His Ile Lys 225 230 235 240 Tyr Ser Phe Thr Arg Asp Trp Ile Asn His Val Arg Ser Ala Thr Gly 245 250 255 Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu Gly Ala 260 265 270 Ile Glu Asn Tyr Leu Gln Lys Thr Asn Trp Asn His Ser Val Phe Asp 275 280 285 Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Lys Ser Gly Gly Asn 290 295 300 Tyr Asp Met Arg Asn Ile Phe Asn Gly Thr Val Val Gln Arg His Pro 305 310 315 320 Ser His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro Glu Glu 325 330 335 Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala Tyr Ala 340 345 350 Leu Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr Gly Asp 355 360 365 Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Arg Ser Lys Ile 370 375 380 Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Lys Gln Asn 385 390 395 400 Asp Tyr Leu Asp His His Asn Ile Ile Gly Trp Thr Arg Glu Gly Asn 405 410 415 Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp Gly Ala 420 425 430 Gly Gly Ser Lys Trp Met Phe Val Gly Arg Asn Lys Ala Gly Gln Val 435 440 445 Trp Ser Asp Ile Thr Gly Asn Arg Thr Gly Thr Val Thr Ile Asn Ala 450 455 460 Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser Ile Trp 465 470 475 480 Val Asn Lys <210> 9 <211> 483 <212> PRT <213> Artificial Sequence <220> <223> Sequence of SEQ ID NO: 3 <400> 9 His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr 1 5 10 15 Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Ser Asp Ala Ser 20 25 30 Asn Leu Lys Asp Lys Gly Ile Ser Ala Val Trp Ile Pro Pro Ala Trp 35 40 45 Lys Gly Ala 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 Ile Arg Thr Lys Tyr Gly 65 70 75 80 Thr Arg Asn Gln Leu Gln Ala Ala Val Asn Ala Leu Lys Ser Asn Gly 85 90 95 Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110 Ala Thr Glu Met Val Arg Ala Val Glu Val Asn Pro Asn Asn Arg Asn 115 120 125 Gln Glu Val Ser Gly Glu Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp 130 135 140 Phe Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys Trp Arg Trp Tyr 145 150 155 160 His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Lys Leu Asn Asn Arg 165 170 175 Ile Tyr Lys Phe Arg Gly Lys Gly Trp Asp Trp Glu Val Asp Thr Glu 180 185 190 Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met Asp His 195 200 205 Pro Glu Val Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr Thr Asn 210 215 220 Thr Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His Ile Lys 225 230 235 240 Tyr Ser Phe Thr Arg Asp Trp Ile Asn His Val Arg Ser Ala Thr Gly 245 250 255 Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu Gly Ala 260 265 270 Ile Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val Phe Asp 275 280 285 Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Lys Ser Gly Gly Asn 290 295 300 Tyr Asp Met Arg Gln Ile Phe Asn Gly Thr Val Val Gln Arg His Pro 305 310 315 320 Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro Glu Glu 325 330 335 Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala Tyr Ala 340 345 350 Leu Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr Gly Asp 355 360 365 Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser Lys Ile 370 375 380 Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Arg Gln Asn 385 390 395 400 Asp Tyr Leu Asp His His Asn Ile Ile Gly Trp Thr Arg Glu Gly Asn 405 410 415 Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp Gly Ala 420 425 430 Gly Gly Asn Lys Trp Met Phe Val Gly Arg Asn Lys Ala Gly Gln Val 435 440 445 Trp Thr Asp Ile Thr Gly Asn Arg Ala Gly Thr Val Thr Ile Asn Ala 450 455 460 Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser Ile Trp 465 470 475 480 Val Asn Lys <210> 10 <211> 483 <212> PRT <213> Artificial Sequence <220> <223> Variant of SEQ ID NO: 4 <400> 10 His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr 1 5 10 15 Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ala 20 25 30 Asn Leu Lys Ser Lys Gly Ile Thr Ala Val 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 Arg Asn Gln Leu Gln Ala Ala Val Thr Ser Leu Lys Asn Asn Gly 85 90 95 Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110 Gly Thr Glu Ile Val Asn Ala Val Glu Val Asn Arg Ser Asn Arg Asn 115 120 125 Gln Glu Thr Ser Gly Glu Tyr Ala Ile Glu Ala Trp Thr Lys Phe Asp 130 135 140 Phe Pro Gly Arg Gly Asn Asn 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 Gln Asn Lys 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 His Glu Leu Arg Asn Trp Gly Val Trp Tyr Thr Asn 210 215 220 Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His Ile Lys 225 230 235 240 Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Thr Thr Gly 245 250 255 Lys Pro Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu Gly Ala 260 265 270 Ile Glu Asn Tyr Leu Asn Lys Thr Ser Trp Asn His Ser Val Phe Asp 275 280 285 Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly Gly Tyr 290 295 300 Tyr Asp Met Arg Asn Ile Leu Asn Gly Ser Val Val Gln Lys His Pro 305 310 315 320 Thr His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro Gly Glu 325 330 335 Ala Leu Glu Ser Phe Val Gln Gln Trp Phe Lys Pro Leu Ala Tyr Ala 340 345 350 Leu Val Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr Gly Asp 355 360 365 Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser Lys Ile 370 375 380 Asp Pro Leu Leu Gln Ala Arg Gln Thr Phe Ala Tyr Gly Thr Gln His 385 390 395 400 Asp Tyr Phe Asp His His Asp Ile Ile Gly Trp Thr Arg Glu Gly Asn 405 410 415 Ser Ser His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp Gly Pro 420 425 430 Gly Gly Asn Lys Trp Met Tyr Val Gly Lys Asn Lys Ala Gly Gln Val 435 440 445 Trp Arg Asp Ile Thr Gly Asn Arg Thr Gly Thr Val Thr Ile Asn Ala 450 455 460 Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser Val Trp 465 470 475 480 Val Lys Gln <210> 11 <211> 483 <212> PRT <213> Artificial sequence <220> <223> Variant of SEQ ID NO: 5 <400> 11 His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His 1 5 10 15 Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ala 20 25 30 Asn Leu Lys Ser Lys Gly Ile Thr Ala Val 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 Arg Ser Gln Leu Gln Gly Ala Val Thr Ser Leu Lys Asn Asn Gly 85 90 95 Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110 Gly Thr Glu Met Val Asn Ala Val Glu Val Asn Arg Ser Asn Arg Asn 115 120 125 Gln Glu Ile Ser Gly Glu Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp 130 135 140 Phe Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys Trp Arg Trp Tyr 145 150 155 160 His Phe Asp Gly Thr Asp Trp Asp Gln Ser Arg Gln Leu Gln Asn Lys 165 170 175 Ile Tyr Lys Phe Arg Gly Lys Ala Trp Asp Trp Glu Val Asp Ile Glu 180 185 190 Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met Asp His 195 200 205 Pro Glu Val Ile Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr Thr Asn 210 215 220 Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His Ile Lys 225 230 235 240 Tyr Ser Tyr Thr Arg Asp Trp Leu Thr His Val Arg Asn Thr Thr Gly 245 250 255 Lys Pro Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu Ala Ala 260 265 270 Ile Glu Asn Tyr Leu Asn Lys Thr Ser Trp Asn His Ser Val Phe Asp 275 280 285 Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly Gly Tyr 290 295 300 Phe Asp Met Arg Asn Ile Leu Asn Gly Ser Val Val Gln Lys His Pro 305 310 315 320 Ile His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro Gly Glu 325 330 335 Ala Leu Glu Ser Phe Val Gln Ser Trp Phe Lys Pro Leu Ala Tyr Ala 340 345 350 Leu Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr Gly Asp 355 360 365 Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ser Met Lys Ser Lys Ile 370 375 380 Asp Pro Leu Leu Gln Ala Arg Gln Thr Tyr Ala Tyr Gly Thr Gln His 385 390 395 400 Asp Tyr Phe Asp His His Asp Ile Ile Gly Trp Thr Arg Glu Gly Asp 405 410 415 Ser Ser His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp Gly Pro 420 425 430 Gly Gly Asn Lys Trp Met Tyr Val Gly Lys His Lys Ala Gly Gln Val 435 440 445 Trp Arg Asp Ile Thr Gly Asn Arg Ser Gly Thr Val Thr Ile Asn Ala 450 455 460 Asp Gly Trp Gly Asn Phe Thr Val Asn Gly Gly Ala Val Ser Val Trp 465 470 475 480 Val Lys Gln <210> 12 <211> 483 <212> PRT <213> Artificial Sequence <220> <223> Variant of SEQ ID NO: 6 <400> 12 His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr 1 5 10 15 Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Ser Asp Ala Ser 20 25 30 Asn Leu Lys Asp Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp 35 40 45 Lys Gly Ala 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 Arg Asn Gln Leu Gln Ala Ala Val Thr Ala Leu Lys Ser Asn Gly 85 90 95 Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110 Ala Thr Glu Trp Val Arg Ala Val Glu Val Asn Pro Ser Asn Arg Asn 115 120 125 Gln Glu Val Ser Gly Asp Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp 130 135 140 Phe Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys Trp Arg Trp Tyr 145 150 155 160 His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Gln Leu Gln Asn Arg 165 170 175 Ile Tyr Lys Phe Arg Gly Lys Gly Trp Asp Trp Glu Val Asp Thr Glu 180 185 190 Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met Asp His 195 200 205 Pro Glu Val Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr Thr Asn 210 215 220 Thr Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His Ile Lys 225 230 235 240 Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Thr Thr Gly 245 250 255 Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Ile Gly Ala 260 265 270 Ile Glu Asn Tyr Leu Ser Lys Thr Asn Trp Asn His Ser Val Phe Asp 275 280 285 Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Arg Ser Gly Gly Asn 290 295 300 Tyr Asp Met Arg Gln Ile Phe Asn Gly Thr Val Val Gln Arg His Pro 305 310 315 320 Thr His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro Glu Glu 325 330 335 Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala Tyr Ala 340 345 350 Leu Thr Leu Thr Arg Asp Gln Gly Tyr Pro Ser Val Phe Tyr Gly Asp 355 360 365 Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser Lys Ile 370 375 380 Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Lys Gln Asn 385 390 395 400 Asp Tyr Leu Asp His His Asn Met Ile Gly Trp Thr Arg Glu Gly Asn 405 410 415 Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp Gly Pro 420 425 430 Gly Gly Asn Lys Trp Met Tyr Val Gly Arg Asn Lys Ala Gly Gln Val 435 440 445 Trp Arg Asp Ile Thr Gly Asn Arg Ser Gly Thr Val Thr Ile Asn Ala 450 455 460 Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser Ile Trp 465 470 475 480 Val Asn Asn

Claims

1. An isolated variant of a parental α-amylase, wherein the variant has α-amylase activity, wherein the following substitutions corresponding to the substitutions numbered using SEQ ID NO: 1 are performed: W140Y+N195F+V206Y+Y243F+E260G, and additional substitutions selected from the following: G109A+G476E, G304R+G476E, and G304R+G476K, wherein the variant has improved washing performance compared to the parental α-amylase of SEQ ID NO:

7.

2. The separated variant according to claim 1, wherein the substitution W140Y+N195F+V206Y+Y243F+E260G+G304R+G476K is performed.

3. Encoding isolated polynucleotides of the isolated variants as described in claim 1 or 2.

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

5. An expression vector comprising the polynucleotide as described in claim 3.

6. A host cell containing the polynucleotide as described in claim 3.

7. A method for generating a variant of a parental α-amylase, the method comprising culturing a host cell as described in claim 6 under conditions suitable for expressing the variant; and recovering the variant.

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