Oxidatively stable alpha-amylase variants

Abstract

The present invention relates to oxidatively stable alpha-amylase variants. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

Description

[0001] References to sequence lists

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

[0003] Invention Field

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

[0005] Related fields description

[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-glycosidic oligosaccharides and polysaccharides.

[0007] Alpha-amylase is a key enzyme used in detergent compositions and its use has become increasingly important for removing starch stains during laundry or dishwashing.

[0008] Some detergents, particularly dishwashing detergents, including bleaching systems, bleach activators, and bleach catalysts, are unstable to α-amylases due to the oxidation of these molecules. Therefore, it is important to identify α-amylase variants that are stable in detergents containing multiple bleaching agents and possess high washing performance, stain removal, and / or activity.

[0009] It is known in the art that α-amylases resistant to bleaching agents and oxidation can be stabilized by replacing methionine at position 197 (using amylase from Bacillus licheniformis for numbering) with, for example, leucine. This has been disclosed, for example, in WO 199418314. However, these prior art oxidically stabilized α-amylases have the disadvantage that their activity is reduced.

[0010] Therefore, an object of the present invention is to provide α-amylase variants that exhibit a high level of stability in detergents (particularly dishwashing detergents and other detergents including bleaching agents or bleaching systems) compared to their parent α-amylases, while simultaneously possessing improved washing performance. A further object is to provide α-amylase variants that exhibit high performance (particularly high washing performance, especially high dishwashing performance).

[0011] The present invention provides α-amylase variants that have improved stability and improved activity compared to their parents. Invention Overview

[0013] The present invention relates to an α-amylase variant of a parental α-amylase, wherein the variant comprises a substitution at one or more sites providing oxidative stability of the variant, wherein the improvement factor of the variant is >1.0 when compared with its parental α-amylase, and wherein the variant has α-amylase activity.

[0014] The present invention also relates to compositions comprising variants according to the present invention.

[0015] In addition, the present invention relates to polynucleotides encoding these variants, nucleic acid constructs containing these polynucleotides, vectors and host cells, and methods for producing these variants.

[0016] Variant Naming Rules

[0017] For the purposes of this invention, the corresponding amino acid residues in another α-amylase are determined using the amino acid sequence as described in SEQ ID NO: 3. The amino acid sequence of the other α-amylase is compared with the amino acid sequence described in SEQ ID NO: 3, and based on this comparison, the Niederman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) implemented in the Nieder program of the EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (preferably version 5.0.0 or later) is used to determine the amino acid position number corresponding to any amino acid residue in the amino acid sequence described in SEQ ID NO: 3. The parameters used are an open space penalty of 10, an open space extension penalty of 0.5, and an EBLOSUM62 (the EMBOSS version of BLOSUM62) replacement matrix.

[0018] The identification of corresponding amino acid residues in another α-amylase can be determined by using several computer programs that compare multiple peptide sequences with their corresponding default parameters. These computer programs include, but are not limited to, MUSCLE (by log-expected multiple sequence comparisons; version 3.5 or later; Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT (version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research 30: 3059-3066; Kato et al., 2005, Nucleic Acids Research 33: 511-518; Kato, 2007, Bioinformatics 23: 372-374; Kato et al., 2009, Methods in Molecular Biology). Biology 537:39-64; Kato Kazuto, 2010, Bioinformatics 26:1899-1900) and EMBOSS EMMA using ClustalW (1.83 or later; Thompson et al., 1994, Nucleic Acid Research 22:4673-4680).

[0019] When other enzymes deviate from the amino acid sequence described in SEQ ID NO: 3, making it impossible for traditional sequence-based comparison methods to detect their relationships (Lindahl and Elofsson, 2000, J. Mol. Biol. 295: 613-615), alternative pairwise sequence comparison algorithms can be used. Greater sensitivity in sequence-based searches can be achieved using search programs that utilize probabilistic representations (characteristic curves) of peptide families to search a database. For example, the PSI-BLAST program generates multiple spectra through an iterative database search process and is capable of detecting distant homologs (Atschul et al., 1997, Nucleic Acid Research 25: 3389-3402). Even greater sensitivity can be achieved if the peptide family or superfamily has one or more representatives in a protein structure database. Procedures such as GenTHREADER (Jones, 1999, J.Mol.Biol. 287: 797-815; McGuffin and Jones, 2003, Bioinformatics 19: 874-881) utilize information from various sources (PSI-BLAST, secondary structure prediction, structural alignment spectra, and solvation potential) as input to neural networks that predict the structural folding of query sequences. Similarly, the method of Gough et al., 2000, J.Mol.Biol. 313: 903-919 can be used to align sequences of unknown structures with superfamily models existing in the SCOP database. These alignments can then be used to generate homology models of peptides, and the accuracy of such models can be evaluated using various tools developed for this purpose.

[0020] For proteins with known structures, several tools and resources are available for retrieving and generating structure alignments. For example, the SCOP superfamily of proteins has already been structurally aligned, and those alignments are accessible and downloadable. Various algorithms, such as distance alignment matrices (Holm and Sander, 1998, 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 also be used to query structure databases with structures of interest to discover possible structural homologs (e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).

[0021] In the description of variations of the invention, the following nomenclature is provided for ease of reference. The recognized IUPAC single-letter or three-letter amino acid abbreviations are used.

[0022] replace. For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Therefore, the substitution of threonine for alanine at position 226 would be named "Thr226Ala" or "T226A". Multiple mutations are separated by plus signs ("+"), for example, "Gly205Arg+Ser411Phe" or "G205R+S411F", representing the substitution of glycine (G) for arginine (R) at positions 205 and 411, respectively, and the substitution of serine (S) for phenylalanine (F).

[0023] Missing For amino acid deletions, use the following nomenclature: original amino acid, position, *. Therefore, a deletion of glycine at position 195 is named “Gly195*” or “G195*”. Multiple deletions are separated by a plus sign (“+”), for example, “Gly195*+Ser411*” or “G195*+S411*”.

[0024] Multiple modifications Variants containing multiple modifications are separated by a plus sign ("+"), such as "Arg170Tyr+Gly195Glu" or "R170Y+G195E", which represent that arginine and glycine at positions 170 and 195 are replaced by tyrosine and glutamic acid, respectively.

[0025] Different modifiers When different modifications can be introduced at a position, these different changes are separated by commas. For example, "Arg170Tyr,Glu" means that arginine at position 170 is replaced by tyrosine or glutamic acid. Therefore, "Tyr167Gly,Ala+Arg170Gly,Ala" represents the following variant:

[0026] “Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and “Tyr167Ala+Arg170Ala”.

[0027] Detailed Description of the Invention

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

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

[0030] SEQ ID NO: 2 is the full-length amino acid sequence of the α-amylase (AAI10), including the signal peptide. The signal peptide is represented by amino acid numbers -29 to -1, and therefore, the mature portion of the α-amylase is represented by amino acid numbers 1 to 485.

[0031] SEQ ID NO: 3 is the mature amino acid sequence of this α-amylase (AAI10).

[0032] SEQ ID NO: 4 is the mature amino acid sequence of the following α-amylase (AAI10), which contains the deletion of amino acids at positions 182 and 183 corresponding to SEQ ID NO: 3.

[0033] SEQ ID NO: 5 is the mature amino acid sequence of α-amylase (SP707).

[0034] SEQ ID NO: 6 is the mature amino acid sequence of α-amylase (AA560).

[0035] SEQ ID NO: 7 is the mature amino acid sequence of α-amylase (K36).

[0036] SEQ ID NO: 8 is the mature amino acid sequence of the protease (Savinase).

[0037] SEQ ID NO: 9 is the mature amino acid sequence of α-amylase (hybrid polypeptide).

[0038] SEQ ID NO: 10 is the mature amino acid sequence of α-amylase (K38).

[0039] SEQ ID NO: 11 is the mature amino acid sequence of α-amylase (KSM-AP1378).

[0040] SEQ ID NO: 12 is the mature amino acid sequence of α-amylase (SP.7-7).

[0041] SEQ ID NO: 13 is the mature amino acid sequence of α-amylase (AAI6).

[0042] Variations of the present invention

[0043] In one aspect, the present invention relates to variants of parental polypeptides having α-amylase activity. Therefore, in a specific aspect, the present invention relates to α-amylase variants of parental α-amylase, wherein the variant comprises substitutions at one or more positions providing oxidative stability of the variant, wherein the improvement factor (as a measure of washing performance) of the variant is >1.0 compared to its parental α-amylase, and wherein the variant has α-amylase activity.

[0044] The inventors of this invention have discovered that variants containing amino acid substitutions at one or more positions providing oxidative stability do not impair the washing performance of the variant, i.e., see the results of Example 3.

[0045] As used herein, the term "α-amylase variant" refers to a polypeptide having α-amylase activity and containing modifications (i.e., substitution, insertion, and / or deletion) at one or more (e.g., several) positions. Substitution means that an amino acid occupying a position is replaced by a different amino acid; deletion means that an amino acid occupying a position is removed; and insertion means that an amino acid is added adjacent to or immediately adjacent to an amino acid occupying a position, all as defined above. Variants of the present invention have at least 20%, for example at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the amino acid sequence set forth in SEQ ID NOs: 3, 4 or the mature amino acid sequence of SEQ ID NO: 2, possessing α-amylase activity.

[0046] As used herein, the term "α-amylase activity" refers to the activity of α-1,4-glucan-4-glucan hydrolase (EC3.2.1.1), which constitutes a group of enzymes that catalyze the hydrolysis of starch and other linear and branched 1,4-glycosidic oligosaccharides and polysaccharides. The terms "α-amylase" and "amylase" are used interchangeably and constitute the same meaning and purpose within the scope of this invention. For the purposes of this invention, α-amylase activity is determined according to the procedure described in the examples. In one embodiment, these variants of the invention have at least 20%, for example at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the amino acid sequence set forth in SEQ ID NOs: 3 or 4, or the mature amino acid sequence set forth in SEQ ID NO: 2, of α-amylase activity.

[0047] As used herein, the term "parental α-amylase" refers to an α-amylase that has been modified to produce an α-amylase variant. For example, an α-amylase having any of the amino acid sequences set forth in SEQ ID NOs: 3 and 4 can be a parent for these variants of the invention. Any amino acid sequence having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 97%, such as at least 99%, sequence identical to any of SEQ ID NOs: 3 and 4 can also be a parental α-amylase for these variants of the invention.

[0048] The parent polypeptide can be obtained from any genus of microorganisms. For the purposes of this invention, the term "obtained from" as used herein in conjunction with a given source shall mean that the parent encoded by the polynucleotide is produced by that source or by a strain in which a polynucleotide from that source has been inserted.

[0049] In some aspects of the invention, the parental α-amylase is an α-amylase of a species of Bacillus, such as the α-amylase of SEQ ID NO: 2, whose mature polypeptide is SEQ ID NO: 3 or 4.

[0050] In some aspects of the invention, the parental α-amylase polypeptide is encoded by the nucleic acid sequence described in SEQ ID NO: 1.

[0051] It should be understood that, for the aforementioned species, this invention covers perfect and imperfect states, and other taxonomic equivalents, such as anamorphs, regardless of their known species names. Those skilled in the art will readily identify the appropriate equivalents.

[0052] Strains of this species are readily available to the public at many culture collections, such as the American Type Culture Collection (ATCC), the German Microbial Culture Collection (DSMZ), the Centralebureau Voor Schimmelcultures (CBS), and the Northern Research Center (NRRL) of the Agricultural Research Culture Collection (ARC).

[0053] The aforementioned probes can be used to identify parental α-amylases and to obtain the parent 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 habitats are well known in the art. The polynucleotide encoding the parental polypeptide can then be obtained by similarly screening genomic DNA or cDNA libraries or mixed DNA samples from another microorganism. Once the polynucleotide encoding the parental polypeptide has been detected with the probe, the polynucleotide can be isolated or cloned using techniques known to those skilled in the art (see, for example, Sambrook et al., 1989, above).

[0054] In one embodiment, the parental α-amylase comprises or is composed of the amino acid sequence set forth in SEQ ID NO: 3 or 4. In another embodiment, the parental α-amylase comprises or is composed of the full-length amino acid sequence set forth in SEQ ID NO: 2. Specifically, the parental α-amylase may comprise or be composed of amino acids 1 to 485 of SEQ ID NO: 2.

[0055] In another embodiment, the parental α-amylase is a fragment containing at least 475 amino acid residues (e.g., at least 480 and at least 485 amino acid residues) as described in SEQ ID NO: 3 or 4.

[0056] In another embodiment, the parental α-amylase is an allelic variant of the amino acid sequence set forth in SEQ ID NO: 3 or 4. Accordingly, the parental α-amylase may be contained in the amino acid sequence set forth in SEQ ID NO: 13.

[0057] As used herein, the term "sequence identity" refers to the correlation between two amino acid sequences or two nucleotide sequences. For the purposes of this invention, the sequence identity between two amino acid sequences is determined using the Niedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol. 48:443-453) implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (preferably version 5.0.0 or later). The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 substitution matrix (the EMBOSS version of BLOSUM62). The Needle output marked "Longest Identity" (obtained using the -nobrief option) is used as the percentage identity and is calculated as follows:

[0058] (identical residues x 100) / (length of alignment - total number of vacancies in alignment).

[0059] For the purposes of this invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, see above) is used to determine sequence identity between two deoxynucleotide sequences. This algorithm is implemented by the Needle program in the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, see above) (preferably version 5.0.0 or later). The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The Needle output labeled "Longest Identity" (obtained using the -nobrief option) is used as the percentage identity and is calculated as follows:

[0060] (identical deoxyribonucleotides x 100) / (alignment length - total number of vacancies in the alignment)

[0061] As used herein, the term "oxidative stability" refers to the property of a protein (such as the α-amylase variant according to the invention) that is not oxidized, i.e., degraded or rendered inactive, due to, for example, the active oxidizing component of a detergent composition containing these oxidizing components. As used herein, the term "oxidizing component" refers to a bleaching system as defined elsewhere herein. Therefore, variants of the invention that possess oxidative stability remain active even in the presence of a bleaching system.

[0062] As used herein, the term "washing performance" refers to the ability of enzymes to remove starch or starch-containing stains present on an object to be cleaned during, for example, cleaning of clothing or hard surfaces (such as dishwashing). The term "washing performance" includes general cleaning such as hard surface cleaning, as in dishwashing, but also includes washing performance on textiles such as clothing, and also includes industrial and institutional cleaning. This washing performance can be quantified by calculating a so-called intensity value, and the result can be displayed as an "improvement factor" (IF). Washing performance can be determined as described in the examples herein.

[0063] As used herein, the term "intensity value" refers to a washing performance that can be measured as luminance, expressed as the intensity of light reflected from a sample when illuminated by white light. When a sample is contaminated, the intensity of the reflected light is lower than that of a clean sample. Therefore, the intensity of the reflected light can be used to measure washing performance, with higher intensity values ​​associated with higher washing performance.

[0064] Color measurements were performed using a professional flatbed scanner (Kodak iQsmart, Kodak), which captures images of the washed textiles.

[0065] To extract light intensity values ​​from a scanned image, the 24-bit pixel values ​​from the image are converted into red, green, and blue (RGB) values. The intensity value (Int) can be calculated by summing the RGB values ​​as vectors and then considering the length of the resulting vector.

[0066]

[0067] As used herein, the term "does not impair the washing performance of the variant" refers to the characteristics of the variant according to the invention, wherein the modifications made in the variant have no negative or any effect on its washing performance.

[0068] In one embodiment, the oxidative stability is determined by an automated mechanical stress test (AMSA), wherein the variant is tested at 55°C for 20 minutes, and wherein the detergent used in the AMSA comprises a bleaching system as described in Example 3.

[0069] As used herein, the term "Automated Mechanical Stress Measurement (AMSA)" refers to a specific assay used to evaluate the washing performance of enzymes (e.g., α-amylase). AMSA testing allows for the examination of the washing performance of large quantities of small-volume enzyme detergent solutions. An AMSA board has numerous slits and caps for the test solution, with the caps forcefully pressing a small piece of textile sample to be washed against all the slits. During the washing period, the board, test solution, textile, and caps vibrate violently, thereby bringing the test solution into contact with the textile and applying mechanical stress in a regular, periodic oscillating manner. Washing performance can be determined under specific conditions, see Example 3. Furthermore, those skilled in the art know how to perform general AMSA to evaluate the washing performance of variations.

[0070] As used herein, the terms "detergent" or "detergent solution" refer to compositions that are considered suitable for use in detergents, such as laundry detergents. The terms "detergent" or "detergent solution" are used interchangeably herein and have the same meaning and purpose unless the context clearly indicates otherwise.

[0071] As used herein, the term "detergent used in the AMSA" refers to the specific detergent used in the AMSA being performed. That is, the detergent used may be the standard Z detergent as described in Example 3.

[0072] As used herein, the term "bleaching system" refers to inorganic and organic bleaching agents suitable as cleaning agents. Inorganic bleaching agents include hydroperoxide salts, such as perborates, percarbonates, superphosphates, persulfates, and persilicates. Inorganic hydroperoxide salts are typically alkali metal salts. Inorganic hydroperoxide salts may be included as crystalline solids without additional protection. Alternatively, the salt may be coated.

[0073] Alkali metal percarbonates, particularly sodium percarbonate, are preferred hydroperoxide compounds for use herein. Most preferably, the percarbonate is incorporated into the product in a coating form, which provides internal stability. Suitable coating materials providing internal stability comprise a mixture of water-soluble alkali metal sulfates and carbonates. Such coatings and coating methods have previously been described in GB 1,466,799. The weight ratio of the mixed salt coating material to the percarbonate is in the range of 1:200 to 1:4, more preferably from 1:99 to 1:9, and most preferably from 1:49 to 1:19. Preferably, the mixed salt is sodium sulfate and sodium carbonate having the general formula Na₂SO₄.n.Na₂CO₃, where n is from 0.1 to 3, preferably from 0.3 to 1.0, and most preferably from 0.2 to 0.5.

[0074] Other suitable coating materials that provide internal stability to the product include sodium silicate, wherein the SiO2:Na2O ratio is from 1.8:1 to 3.0:1, preferably from 1.8:1 to 2.4:1, and / or sodium metasilicate, preferably applied at a SiO2 level of from 2% to 10% (typically from 3% to 5%) by weight of the inorganic hydroperoxide salt. Magnesium silicate may also be included in the coating. Coatings comprising silicates and borates or boric acid or other inorganic substances are also suitable.

[0075] Other coatings, including waxes, oils, and fatty soaps, may also be advantageously used in this invention.

[0076] Potassium persulfate is another inorganic hydrogen peroxide salt that is useful herein. Typical organic bleaching agents are organic peroxy acids, including diacid and tetraacyl peroxides, especially disperoxydodecanoic acid, disperoxytetradecanoic acid, and disperoxyhexadecanoic acid. Benzoyl peroxide is a preferred organic peroxy acid herein. Mono- and dipernonanoic acid, mono- and distearate acid, and N-phthalamide peroxyhexanoic acid are also applicable herein. The diacid peroxide, especially the dibenzoyl peroxide, should preferably be present in particulate form, the particles having a weight-average diameter of from about 0.1 to about 100 micrometers, preferably from about 0.5 to about 30 micrometers, more preferably from about 1 to about 10 micrometers. Preferably, at least about 25%, more preferably at least about 50%, even more preferably at least about 75%, and most preferably at least about 90% of the particles are less than 10 micrometers, preferably less than 6 micrometers. It has been found that diacid peroxides within the aforementioned particle size range provide better stain removal, especially from plastic tableware, compared to larger diacid peroxide particles, while minimizing unwanted deposits and film formation during use in automatic dishwashing machines. This preferred diacid peroxide particle size thus allows the formulator to achieve good stain removal with a low level of diacid peroxide, which reduces deposits and film formation. Conversely, as the diacid peroxide particle size increases, more diacid peroxide is required to achieve good stain removal, which increases the amount of deposits encountered on surfaces during dishwashing. Other typical organic bleaches include peroxides, specifically alkyl peroxides and aryl peroxides. Preferred representatives are (a) peroxybenzoic acid and its cyclic-substituted derivatives, such as alkyl peroxybenzoic acid, but also peroxy-[α]-naphthoic acid and magnesium monoperphthalate, (b) aliphatic or substituted aliphatic peroxy acids, such as peroxylauric acid, peroxystearic acid, [ε]-phthalimide peroxyhexanoic acid [phthaloyliminoperoxyhexanoic acid (PAP)], o-carboxybenzoamide peroxyhexanoic acid, N-nonenyl acyl peroxyacid and N-nonenyl acylamino succinate, and (c) aliphatic and aryliphatic peroxydicarboxylic acids, such as 1,12-disperoxycarboxylic acid, 1,9-disperoxyazelaic acid, disperoxysediic acid, disperoxymethylene acid, disperoxyphthalic acid, 2-decyl-1,4-diacid, and N,N-terephthaloyldi(6-aminoperpropionic acid).

[0077] In a specific embodiment, the bleaching system is added at the following concentrations: at least 5% by weight, for example at least 8% by weight, for example at least 10% by weight, or for example at least 15% by weight.

[0078] As used herein, the term "weight %" refers to a component, such as in a bleaching system, that is present as a percentage based on weight rather than volume. This term is well known to those skilled in the art, who are also capable of calculating such weight % based on knowledge of the composition of a solution (such as a detergent solution).

[0079] In one embodiment, the bleaching system is sodium percarbonate.

[0080] In one embodiment, the improvement factor of the variant is >1.5, wherein the IF has been determined in AMSA, wherein the condition in AMSA is a 20-minute washing cycle at 55°C, and wherein the variant is tested in the presence of a bleaching system (such as sodium percarbonate at a concentration of 8% by weight).

[0081] In one embodiment, the parental α-amylase has the amino acid sequence described in SEQ ID NO: 3, or has an amino acid sequence that is at least 80%, for example at least 85%, for example at least 90%, for example at least 95%, for example at least 98%, identical to the amino acid sequence described in SEQ ID NO: 3.

[0082] In one embodiment, the parental α-amylase has the amino acid sequence set forth in SEQ ID NO: 4, or has an amino acid sequence that is at least 80%, for example at least 85%, for example at least 90%, for example at least 95%, for example at least 98%, identical to the amino acid sequence set forth in SEQ ID NO: 4.

[0083] In one embodiment, the parental α-amylase has the amino acid sequence set forth in SEQ ID NO: 13, or has an amino acid sequence that is at least 80%, for example at least 85%, for example at least 90%, for example at least 95%, for example at least 98%, identical to the amino acid sequence set forth in SEQ ID NO: 13.

[0084] In one embodiment, the parental α-amylase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3.

[0085] In one embodiment, the parental α-amylase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4.

[0086] In one embodiment, the parental α-amylase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 13.

[0087] In another embodiment, the parental α-amylase is a fragment of the polypeptide of SEQ ID NO: 3, wherein the fragment has α-amylase activity.

[0088] In another embodiment, the parental α-amylase is a fragment of the polypeptide of SEQ ID NO: 4, wherein the fragment has α-amylase activity.

[0089] In another embodiment, the parental α-amylase is a fragment of the polypeptide of SEQ ID NO: 13, wherein the fragment has α-amylase activity.

[0090] As used herein, a "fragment" refers to a polypeptide lacking one or more (e.g., several) amino acids at the amino and / or carboxyl terminus of a mature polypeptide; wherein the fragment has α-amylase activity. In one embodiment, a fragment comprises at least 480 amino acid residues, at least 481 amino acid residues, or at least 482 amino acid residues.

[0091] In one embodiment, the variant has at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98%, but less than 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 3.

[0092] In one embodiment, the variant has at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98%, but less than 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 4.

[0093] In one embodiment, the variant has at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98%, but less than 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 13.

[0094] As used herein, the term "sequence consistency" means any definition already described herein. Sequence consistency can be determined as described elsewhere in this document.

[0095] In one embodiment, the variant contains a substitution at position 202, wherein the position corresponds to the amino acid position of the amino acid sequence set forth in SEQ ID NO: 3.

[0096] Unbound by any theory, any amino acid substitution, except for the naturally occurring amino acid at the M202 position corresponding to SEQ ID NO: 3, will provide an oxidically stable variant with an IF (as a measure of washing performance) of at least 1.0.

[0097] Therefore, in one embodiment, the substitution at position 202 is selected from any of the following: M202A, M202R, M202N, M202D, M202C, M202E, M202Q, M202G, M202H, M202I, M202L, M202K, M202F, M202P, M202S, M202T, M202W, M202Y, and M202V, preferably M202L, M202I, M202T, M202F, and M202S, wherein the position corresponds to the position in the amino acid sequence described in SEQ ID NO: 3.

[0098] In one embodiment, the variant includes the deletion of two or more positions corresponding to positions R181, G182, D183, and G184 of the amino acid sequence described in SEQ ID NO: 3.

[0099] As used herein, the term "deletion" refers to the removal of an amino acid from a polypeptide (e.g., an enzyme). Such removal, i.e., deletion, of one or more amino acids can be performed by site-directed mutagenesis or any other method known in the art and by those skilled in the art.

[0100] The variants described in this invention (which contain deletions at two or more positions corresponding to positions R181, G182, D183, and G184 of SEQ ID NO: 3) provide stability to these variants and promote improved washing performance of these variants. It is well known that this specific portion of α-amylase provides stability to α-amylase variants, as described, for example, in WO1996 / 023873. The term "stability" as used in this context refers to the stability of the α-amylase variant during washing. This stability can be determined by measuring the activity of the variant after a washing cycle of 60 min in a detergent solution, for example, at 40°C-60°C. The condition is that variants containing deletions at two or more positions corresponding to positions R181, G182, D183, and G184 of SEQ ID NO: 3 and containing substitutions in one or more amino acids that provide oxidative stability to the variant are within the scope of this invention.

[0101] Therefore, in one embodiment, the variant contains deletions at positions corresponding to R181+G182; R181+D183; R181+G184; G182+D183; G182+G184; or D183+G184, where these positions correspond to those positions in the amino acid sequence set forth in SEQ ID NO: 3.

[0102] These variations may further include one or more additional modifications, such as substitutions, in one or more (e.g., several) other locations.

[0103] Amino acid changes can be of a minor nature, i.e., conserved amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions typically of 1 to 30 amino acids; small amino-terminal or carboxyl-terminal elongations, such as amino-terminal methionine residues; small linker peptides of up to 20-25 residues; or small elongated portions that facilitate purification by altering net charge or another function, such as multihistidine bundles, antigenic epitopes, or binding domains.

[0104] Examples of conserved substitutions are found in the following group: basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that do not typically alter specific activity are known in the art and are described, for example, by H. Neurath and RL Hill, 1979, in *The Proteins*, Academic Press, New York. Common substitutes are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0105] Alternatively, amino acid alterations have the property of changing the physicochemical properties of peptides. For example, amino acid alterations can improve the thermal stability of peptides, change substrate specificity, change the optimal pH, etc.

[0106] Essential amino acids in peptides can be identified using procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, a single alanine mutation is introduced at each residue in the molecule, and the α-amylase activity of the resulting mutant molecule is tested to identify amino acid residues critical to the molecule's activity. See also Hilton et al., 1996, Journal of Biochemistry 271: 4699-4708. Alternatively, mutations in amino acids at hypothetical contact sites can be combined with physical analysis of the structure, such as by techniques like NMR, crystallography, electron diffraction, or photoaffinity labeling, to determine the enzyme's active site or other biological interactions. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, 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 comparisons with related peptides.

[0107] Therefore, in one embodiment, the variant contains 1 to 40 substitutions, such as 1 to 30, such as 1 to 20, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 substitutions.

[0108] In specific embodiments, the number of substitutions is 1 to 20, for example, 1 to 10 and 1 to 5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions.

[0109] In one embodiment, the variant further includes additional modifications.

[0110] These variants can consist of 430 to 490 amino acids, such as 440 to 485, 450 to 485, and 460 to 483 amino acids.

[0111] In one embodiment, the variant comprises or consists of the following modifications: G182*+D183*+M202L, wherein the encoding is as described in SEQ ID NO: 3.

[0112] In a specific embodiment, the variant includes or consists of the following modifications: G182*+D183*+N195F+M202L, wherein the encoding is as described in SEQ ID NO: 3.

[0113] In one aspect, the present invention relates to a variant of a parental α-amylase, wherein the parental α-amylase comprises or is composed of an amino acid sequence having at least 80%, for example at least 85%, for example at least 90%, for example at least 95%, or for example at least 98%, sequence identity with the amino acid sequence set forth in SEQ ID NO: 3, and wherein the variant is composed of a substitution at the position corresponding to position M202 in SEQ ID NO: 3.

[0114] In a specific aspect, the present invention relates to a variant of a parental α-amylase consisting of the amino acid sequence set forth in SEQ ID NO: 3, wherein the variant is composed of a substitution at the position corresponding to position M202 in SEQ ID NO: 3.

[0115] In another aspect, the present invention relates to a variant of a parental α-amylase comprising the amino acid sequence set forth in SEQ ID NO: 3, wherein the variant comprises the modification G182*+D183*+M202L, wherein the encoding is as described in SEQ ID NO: 3.

[0116] In another aspect, the present invention relates to a variant of a parental α-amylase comprising the amino acid sequence set forth in SEQ ID NO: 3, wherein the variant comprises the modification G182*+D183*+N195F+M202L, wherein the encoding is as described in SEQ ID NO: 3.

[0117] Polynucleotides

[0118] The present invention also relates to polynucleotides encoding variants of the invention. Therefore, in particular, the present invention relates to polynucleotides encoding variants that contain substitutions at one or more sites providing oxidative stability to the variant.

[0119] As used herein, the term "polynucleotide-encoded" refers to a polynucleotide that encodes a mature polypeptide having α-amylase activity. In one aspect, the polypeptide-encoding sequence is the nucleotide sequence set forth in SEQ ID NO: 1.

[0120] In one embodiment, the polynucleotide encoding the variant according to the invention has at least 80%, for example at least 85%, for example at least 90%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, but less than 100% sequence identity with the polynucleotide of SEQ ID NO: 1.

[0121] In one embodiment, the polynucleotide encodes a variant containing substitutions and / or deletions at two, three, or four positions corresponding to the amino acid sequence described in SEQ ID NO: 3, namely positions R181, G182, D183, and G184, and a substitution at position M202 corresponding to the amino acid sequence described in SEQ ID NO: 3.

[0122] Nucleic acid constructs

[0123] The present invention also relates to nucleic acid constructs comprising a polynucleotide encoding a variant of the invention, operably linked to one or more control sequences, the one or more control sequences guiding the expression of the encoding sequence in a suitable host cell under conditions compatible with the control sequences. Therefore, in particular, the present invention relates to nucleic acid constructs comprising a polynucleotide encoding a variant containing substitutions at one or more sites providing oxidative stability to the variant, wherein the polynucleotide is operably linked to one or more control sequences.

[0124] As used herein, the term "nucleic acid construct" refers to a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene, or modified in a manner not normally found in nature to contain segments of nucleic acid, or is synthetic, and that the nucleic acid molecule includes one or more control sequences.

[0125] As used herein, the term "operably linked" refers to a configuration in which a control sequence is positioned relative to the coding sequence of a polynucleotide so that the control sequence directs the expression of the coding sequence.

[0126] The polynucleotide can be manipulated in a variety of ways to provide expression of the variant. Depending on the expression vector, manipulation of the polynucleotide before its insertion into the vector may be desired or necessary. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

[0127] The control sequence can be a promoter, which is a polynucleotide recognized by the host cell for the expression of that polynucleotide. The promoter contains a transcriptional control sequence that mediates the expression of that variant. The promoter can be any polynucleotide that exhibits transcriptional activity in the host cell, including variants, truncated and heterozygous promoters, and can be acquired by a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to that of the host cell.

[0128] In one embodiment, the nucleic acid construct comprises a polynucleotide encoding a variant having substitutions and / or deletions at two, three, or four positions corresponding to the amino acid sequence described in SEQ ID NO: 3, at positions R181, G182, D183, and G184, and a substitution at position M202 corresponding to the amino acid sequence described in SEQ ID NO: 3, wherein the polynucleotide is operatively linked to one or more control sequences.

[0129] Expression carrier

[0130] The present invention also relates to recombinant expression vectors comprising polynucleotides encoding variants of the invention, promoters, and transcription and translation termination signals. Therefore, the present invention relates to expression vectors, optionally recombinant expression vectors, comprising polynucleotides encoding variants, the variants including substitutions at one or more sites providing oxidative stability of the variant, promoters, and transcription and translation termination signals.

[0131] As used herein, the term "expression vector" refers to a linear or circular DNA molecule that comprises polynucleotides encoding a variant and is operatively linked to a control sequence that provides for its expression.

[0132] In one embodiment, the expression vector comprises a polynucleotide encoding a variant having substitutions and / or deletions at two, three, or four positions corresponding to the amino acid sequence described in SEQ ID NO: 3, at positions R181, G182, D183, and G184, and at position M202 corresponding to the amino acid sequence described in SEQ ID NO: 3, and wherein the expression vector further comprises a promoter and transcription and translation termination signals.

[0133] 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 the insertion or substitution of a polynucleotide encoding the variant at such sites. Alternatively, the polynucleotide can be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into a suitable vector for expression. In generating the expression vector, the coding sequence is located within the vector, thus making the coding sequence operatively linked to the suitable control sequence for expression.

[0134] Recombinant expression vectors can be any vector (e.g., plasmids or viruses) that facilitates recombinant DNA procedures and induces the expression of polynucleotides. The choice of vector will typically depend on its compatibility with the host cell to which it will be introduced.

[0135] The vector can be a self-replicating vector, that is, a vector that exists as an extrachromosomal entity and replicates independently of chromosome replication, such as a plasmid, extrachromosomal element, microchromosome, or artificial chromosome. The vector can contain any means necessary to ensure self-replication. Alternatively, the vector can be one that integrates into the genome when introduced into a host cell and replicates along with the chromosome in which it is integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids (which together contain the total DNA of the genome to be introduced into the host cell), or transposons can be used.

[0136] The vector preferably contains one or more selective markers that allow for convenient selection of cells such as transformed cells, transfected cells, and transduced cells. These selective markers are genes whose products provide resistance to biocides or viruses, resistance to heavy metals, prototrophy to auxotrophs, etc.

[0137] Those skilled in the art will know which expression vector is most suitable for the specific expression system. Therefore, the present invention is not limited to any specific expression vector, but any expression vector containing polynucleotide variants according to the present invention is considered part of the present invention.

[0138] host cells

[0139] The present invention also relates to recombinant host cells comprising polynucleotides encoding variants of the invention, operably linked to one or more control sequences that direct the generation of variants of the invention. Therefore, the present invention relates to host cells, optionally recombinant host cells, comprising polynucleotides encoding variants comprising substitutions at one or more sites providing oxidative stability of the variant, and operably linked to one or more control sequences that direct the generation of the variant.

[0140] As used herein, the term "host cell" refers to any cell type that is susceptible to transformation, transfection, transduction, etc., using nucleic acid constructs or expression vectors including the polynucleotides of the present invention. The term "host cell" also encompasses any offspring of the parent cell that is not identical to its parent cell due to mutations occurring during replication.

[0141] In one embodiment, the host cell contains a polynucleotide encoding a variant having substitutions and / or deletions at two, three, or four positions corresponding to the amino acid sequence described in SEQ ID NO: 3, at positions R181, G182, D183, and G184, and a substitution at position M202 corresponding to the amino acid sequence described in SEQ ID NO: 3. The polynucleotide is operatively linked to one or more control sequences that direct the generation of the variant.

[0142] A construct or vector containing multiple nucleotides is introduced into a 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 offspring of a parent cell that differs from the parent cell due to mutations occurring during replication. The selection of the host cell will depend largely on the gene encoding the variant and its origin.

[0143] The host cell can be any cell that is useful in the recombinant-generated variants, such as prokaryotic or eukaryotic cells.

[0144] Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Bacillus aeruginosa, Lactobacillus, Lactococcus, Marine Bacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Coliform, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0145] Inventive Method

[0146] The present invention also relates to methods for producing variants, the methods comprising: (a) culturing a host cell of the present invention under conditions suitable for expression of the variant; and (b) recovering the variant. Therefore, the present invention relates to methods for producing variants comprising substitutions at one or more sites providing oxidative stability of the variant, wherein the method comprises the steps of: a) culturing a host cell according to the present invention under conditions suitable for expression of the variant, and b) recovering the variant.

[0147] As used herein, the term "conditions suitable for expression" refers to the settings in which host cells expressing the variants described in the invention are cultured. These conditions (or settings) can vary depending on the type of host cell. That is, suitable conditions for culturing yeast host cells differ from those for culturing bacterial host cells. Determining which conditions are optimal, i.e. suitable, for a specific host cell is within the knowledge of one skilled in the art.

[0148] In one embodiment, a method for generating a variant (which contains substitutions and / or deletions at two, three, or four positions corresponding to the amino acid sequences described in SEQ ID NO: 3, R181, G182, D183, and G184, and a substitution at position M202 corresponding to the amino acid sequences described in SEQ ID NO: 3) comprises the steps of: a) culturing host cells according to the invention under conditions suitable for the expression of the variant, and b) recovering the variant.

[0149] The host cells are cultured in a nutrient medium suitable for producing the variant using methods known in the art. For example, the cells can be cultured by shake flask culture or by small-scale or large-scale fermentation (including continuous fermentation, batch fermentation, feed-feed fermentation, or solid-state fermentation) in a laboratory or industrial fermenter under conditions that allow for the expression and / or isolation of the variant. The culture occurs using procedures known in the art in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts. Suitable media are available from commercial suppliers or can be prepared according to publicly available compositions (e.g., in the catalogue of the U.S. Center for Type Culture Collection). If the variant is secreted into the nutrient medium, it can be recovered directly from the medium. If the variant is not secreted, it can be recovered from cell lysates.

[0150] The variant can be detected using methods known in the art that are specific to variants having α-amylase activity. These detection methods include, but are not limited to, the use of specific antibodies, the formation of enzyme products, or the disappearance of enzyme substrates. For example, enzyme assays can be used to determine the activity of the variant.

[0151] The variant can be recovered using methods known in the art. For example, the variant can be recovered from the nutrient medium through a variety of routine procedures, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

[0152] Variants can be purified to obtain substantially pure variants by a variety of procedures known in the art, including but not limited to chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatographic focusing, and size exclusion chromatography), electrophoresis procedures (e.g., preparative isoelectric point focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, for example, Protein Purification, edited by Janson and Ryden, VCH Publishers, New York, 1989).

[0153] Alternatively, instead of recycling the variant, the host cell of the present invention expressing the variant is used as the source of the variant.

[0154] In another aspect, the present invention relates to methods for obtaining variants, the methods comprising: a) introducing a substitution at position M202 of a parental α-amylase, wherein the position corresponds to the position of the amino acid sequence set forth in SEQ ID NO: 3; b) optionally introducing a deletion at two or more positions R181, G182, D183, and G184 corresponding to the amino acid sequence set forth in SEQ ID NO: 3; and c) recovering the variant.

[0155] These variants can be prepared using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc.

[0156] Site-directed mutagenesis is a technique that introduces one or more (e.g., several) mutations at one or more designated sites in a polynucleotide encoding the parent.

[0157] Site-directed mutagenesis can be achieved in vitro using PCR involving primers containing oligonucleotides with the desired mutation. Site-directed mutagenesis can also be performed in vitro via cassette mutagenesis, which involves cleavage by a restriction enzyme at a site in a plasmid containing a polynucleotide encoding the parent and subsequent ligation of the oligonucleotide containing the mutation into the polynucleotide. Typically, the restriction enzyme used to digest the plasmid is the same as that used to digest the oligonucleotide to allow 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.

[0158] 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.

[0159] Any site-directed mutagenesis procedure can be used in this invention. Many commercially available kits are available for preparing variants.

[0160] Synthetic gene construction requires the in vitro synthesis of designed polynucleotide molecules to encode polypeptides of interest. Gene synthesis can be performed using a variety of techniques, such as the multi-channel microchip-based technique described by Tian et al. (2004, Nature 432: 1050-1054), and similar techniques involving the synthesis and assembly of oligonucleotides on optically programmable microfluidic chips.

[0161] 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. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409; WO92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

[0162] 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). The mutagenic DNA molecules encoding the active peptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow for the rapid determination of the importance of individual amino acid residues within the peptide.

[0163] Semi-synthetic gene construction is achieved through a combination of various methods, including synthetic gene construction, and / or site-directed mutagenesis, and / or random mutagenesis, and / or shuffling. Semi-synthetic construction typically involves combining the process of synthesizing polynucleotide fragments with PCR technology. Therefore, specific regions of the gene can be synthesized de novo, while other regions can be amplified using site-specific mutagenesis primers, and still others can undergo error-prone or non-error-prone PCR amplification. The polynucleotide subsequence can then be shuffled.

[0164] This invention also relates to a method for improving the oxidative stability of a parental α-amylase having the amino acid sequence of SEQ ID NO: 3 or 4, or having at least 80% sequence identity with it, wherein the method comprises the following steps:

[0165] a) Introduce substitutions at one or more sites that provide oxidative stability in the parental α-amylase; and

[0166] b) Optionally, substitutions and / or deletions are introduced at two, three, or four positions corresponding to positions R181, G182, D183, and G184 of the amino acid sequence described in SEQ ID NO: 3.

[0167] The variant has at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98%, such as at least 99%, but less than 100% sequence identity with the amino acid sequence described in SEQ ID NO: 3, and wherein, compared with its parent α-amylase, the variant has α-amylase activity and improved oxidative stability.

[0168] In one embodiment, a method for improving the oxidative stability of a parental α-amylase (the parental α-amylase having the amino acid sequence of SEQ ID NO: 3 or 4, or having at least 80% sequence identity with it) includes the following steps:

[0169] a) Introducing a substitution at position M202 corresponding to the amino acid sequence described in SEQ ID NO: 3; and

[0170] b) Introduce substitutions and / or deletions at two, three, or four positions corresponding to positions R181, G182, D183, and G184 of the amino acid sequence described in SEQ ID NO: 3.

[0171] The variant has at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98%, such as at least 99%, but less than 100% sequence identity with the amino acid sequence described in SEQ ID NO: 3, and wherein, compared with its parent α-amylase, the variant has α-amylase activity and improved oxidative stability.

[0172] In one embodiment, the variant has at least 50%, such as at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100% activity of the parent polypeptide having the amino acid sequence of SEQ ID NO: 4.

[0173] In another embodiment, the variant has at least 50%, such as at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100% activity of the parent polypeptide having the amino acid sequence of SEQ ID NO: 3.

[0174] In one embodiment, its activity is determined according to Phadebas assay.

[0175] The α-amylase activity can be determined using Phadebas substrates (e.g., from Magle Life Sciences, Lund, Sweden). Phadebas tablets comprise interconnected starch polymers in the form of water-insoluble spherical microspheres. A blue dye is covalently bound to these microspheres. The interconnected starch polymers within the microspheres degrade at a rate proportional to the α-amylase activity. As the α-amylase degrades the starch polymers, the released blue dye is water-soluble, and the dye concentration can be determined by measuring absorbance at 620 nm. The concentration of blue is proportional to the α-amylase activity in the sample.

[0176] Dilute the variant sample to be analyzed in an activity buffer at the desired pH. Suspend both substrate tablets in 5 mL of activity buffer and mix on a magnetic stirrer. While mixing the substrate, transfer 150 μL to a microtiter plate (MTP) or PCR-MTP. Add 30 μL of diluted amylase sample to 150 μL of substrate and mix. Incubate at 37 °C for 15 min. Stop the reaction by adding 30 μL of 1M NaOH and mixing. Centrifuge the MTP at 4000 x g for 5 min. Transfer 100 μL to a fresh MTP and measure the absorbance at 620 nm.

[0177] The α-amylase sample should be diluted such that the absorbance at 620 nm is between 0 and 2.2, and within the linear range of the activity assay.

[0178] Therefore, in one embodiment, its activity is determined by the following method, which includes the following steps:

[0179] a) Incubate the α-amylase variant according to the invention at 37°C for 15 minutes with stained amylose substrate; and

[0180] b) Measure absorbance at OD 620nm.

[0181] In another embodiment, its activity is determined by the following method, which includes the following steps:

[0182] a) Incubate the α-amylase variant according to the invention at 37°C for 15 minutes with stained amylose substrate; and

[0183] b) Centrifuge the sample;

[0184] c) Transfer the supernatant to the reader plate, and

[0185] Absorbance was measured at OD 620nm.

[0186] In another embodiment, its activity was determined according to pNP-G7 assays as described in Example 2.

[0187] Fermentation broth preparations or cell compositions

[0188] The present invention also relates to a fermentation broth formulation or a cell composition comprising variants of the invention. Thus, in one embodiment, the fermentation broth formulation or cell composition comprises a variant containing a substitution at one or more sites providing oxidative stability of the variant. In another embodiment, the fermentation broth formulation or cell composition comprises a polynucleotide encoding a variant comprising a substitution (located at one or more sites providing oxidative stability of the variant), a nucleic acid construct encoding a variant (containing a substitution at one or more sites providing oxidative stability of the variant), or an expression vector encoding a variant (containing a substitution at one or more sites providing oxidative stability of the variant). The fermentation broth product may further comprise additional components for the fermentation process, such as cells (including host cells containing a gene encoding a polypeptide of the invention for producing a polypeptide of interest), cell debris, biomass, fermentation medium, and / or fermentation products. In some embodiments, the composition is a cell-killing whole culture medium comprising one or more organic acids, killed cells and / or cell debris, and a culture medium.

[0189] As used herein, the term "fermentation broth" refers to a preparation produced by cell fermentation that undergoes little or no recovery and / or purification. For example, fermentation broth is produced when a microbial culture is grown to saturation, incubated under carbon-limited conditions to allow protein synthesis (e.g., expression of enzymes by host cells), and secreted into a cell culture medium. This fermentation broth may contain ungraded or graded contents of the fermentation material obtained at the end of fermentation. Typically, fermentation broth is ungraded and includes used culture medium and cell debris remaining after, for example, removal of microbial cells (e.g., filamentous fungal cells) by centrifugation. In some embodiments, fermentation broth contains depleted cell culture medium, extracellular enzymes, and viable and / or non-viable microbial cells.

[0190] In one embodiment, the fermentation broth formulation and cell composition comprise a first organic acid component (comprising at least one 1-5 carbon organic acid and / or its salt) and a second organic acid component (comprising at least one 6- or more carbon organic acid and / or its salt). In a specific embodiment, the first organic acid component is acetic acid, formic acid, propionic acid, its salt, or a mixture of two or more of the foregoing; and the second organic acid component is benzoic acid, cyclohexanecarboxylic acid, 4-methylvaleric acid, phenylacetic acid, its salt, or a mixture of two or more of the foregoing.

[0191] In one embodiment, the composition comprises one or more organic acids and optionally further comprises killed cells and / or cell debris. In one embodiment, the killed cells and / or cell debris are removed from the cell-killing whole culture medium to provide a composition free of these components.

[0192] These fermentation broth formulations or cell compositions may further include preservatives and / or antimicrobial (e.g., bacteriostatic) agents, including but not limited to sorbitol, sodium chloride, potassium sorbate, and other agents known in the art.

[0193] The cell-killing whole culture or composition may contain the ungraded contents of the fermentation material obtained at the end of fermentation. Typically, the cell-killing whole culture or composition contains used culture medium and cell debris present after microbial cells (e.g., filamentous fungal cells) have been grown to saturation and incubated under carbon-limited conditions to allow protein synthesis. In some embodiments, the cell-killing whole culture or composition contains used cell culture medium, extracellular enzymes, and killed filamentous fungal cells. In some embodiments, methods known in the art can be used to permeate and / or lyse the microbial cells present in the cell-killing whole culture or composition.

[0194] The whole culture medium or cell composition described herein is typically a liquid, but may contain insoluble components, such as killed cells, cell debris, culture medium components, and / or one or more insoluble enzymes. In some embodiments, the insoluble components may be removed to provide a clear liquid composition.

[0195] The whole culture medium formulations and cell compositions of the present invention can be produced by the methods described in WO 90 / 15861 or WO 2010 / 096673.

[0196] Composition

[0197] The present invention also relates to compositions comprising variants according to the invention. Therefore, the present invention relates to compositions comprising variants that contain substitutions at one or more sites providing oxidative stability of the variant. Preferably, these compositions are enriched in such variants. The term "rich" means that the α-amylase activity of the composition has been increased, for example, an enrichment factor of 1.1.

[0198] In one embodiment, the composition comprises a variant containing a substitution at position M202 corresponding to the amino acid sequence set forth in SEQ ID NO: 3, and a substitution and / or deletion at two, three, or four positions corresponding to positions R181, G182, D183, and G184 corresponding to the amino acid sequence set forth in SEQ ID NO: 3.

[0199] These compositions can be prepared according to methods known in the art and can be in the form of liquid or dry compositions. For example, the compositions can be in the form of particles or microparticles. Variants can be stabilized according to methods known in the art.

[0200] Accordingly, in one embodiment, the composition is a liquid laundry or liquid dishwashing composition, such as an automatic dishwashing (ADW) liquid detergent composition, or a powdered laundry detergent, such as a soap bar, or a powdered dishwashing composition, such as an ADW detergent composition.

[0201] In one embodiment, the composition further comprises one or more surfactants, one or more sulfonated polymers, one or more chelating agents, one or more bleaching systems, and / or one or more detergent builders.

[0202] The selection of other components is within the capabilities of those skilled in the art and includes conventional ingredients, including the exemplary non-limiting components described below. For fabric care, the selection of components may include considerations such as the type of fabric to be cleaned, the type and / or extent of the stain, the temperature at which cleaning is performed, and the formulation of the detergent product. Although the components mentioned below are categorized under a general heading according to their specific functionality, this is not to be construed as limiting, as a component may include additional functionality as would be understood by one of ordinary skill in the art.

[0203] In one embodiment of the invention, variations of the invention may be added to a detergent composition in amounts corresponding to: 0.001-100 mg of protein per liter of detergent liquid, for example, 0.01-100 mg of protein, preferably 0.005-50 mg of protein, more preferably 0.01-25 mg of protein, even more preferably 0.05-10 mg of protein, most preferably 0.05-5 mg of protein, and even most preferably 0.01-1 mg of protein. The term "protein" is to be understood herein to include variations according to the invention.

[0204] The composition for use in an automatic dishwasher (ADW) may, for example, include 0.0001%-50%, such as 0.001%-20%, such as 0.01%-10%, such as 0.05%-5% of the enzyme protein by weight of the composition.

[0205] The composition used in laundry granulation may, for example, contain 0.0001%-50%, such as 0.001%-20%, such as 0.01%-10%, such as 0.05%-5% of enzyme protein by weight of the composition.

[0206] The composition for use in laundry detergent may, for example, include 0.0001%-10%, such as 0.001%-7%, such as 0.1%-5% of enzyme protein by weight of the composition.

[0207] These variants of the invention, as well as other active components such as additional enzymes, can be stabilized using conventional stabilizers, such as polyols, like propylene glycol or glycerol, sugars or sugar alcohols, lactic acid, boric acid or boric acid derivatives, such as aromatic borate esters, or phenyl boric acid derivatives, such as 4-formylphenylboronic acid, and the composition can be configured as described in, for example, WO92 / 19709 and WO 92 / 19708.

[0208] In some markets, different washing conditions and, in themselves, different types of detergents are used. This is disclosed, for example, in EP 1 025 240. For instance, low detergent concentration systems are used in Asia (Japan), medium detergent concentration systems are used in the United States, and high detergent concentration systems are used in Europe.

[0209] Low detergent concentration systems contain detergents in which less than approximately 800 ppm of detergent components are present in the wash water. Japanese detergents are typically considered low detergent concentration systems because they have approximately 667 ppm of detergent components present in the wash water.

[0210] Medium detergent concentration systems contain detergents with detergent components present in wash water at concentrations between approximately 800 ppm and approximately 2000 ppm. North American detergents are generally considered medium detergent concentration systems because they have approximately 975 ppm of detergent components present in wash water.

[0211] High detergent concentration systems contain detergents with more than approximately 2000 ppm of detergent components in the wash water. European detergents are generally considered high detergent concentration systems because they contain approximately 4500-5000 ppm of detergent components in the wash water.

[0212] Latin American detergents are typically high-foaming phosphate-based detergents, and the range of detergents used in Latin America can fall between medium and high detergent concentrations, as their detergent components in the wash water range from 1500 ppm to 6000 ppm. Such detergent compositions are examples of the present invention.

[0213] Variations of the present invention may also be incorporated into detergent formulations disclosed in WO 97 / 07202, which is incorporated herein by reference.

[0214] Examples of preferred uses of the compositions of the present invention are given herein. The dosage of the composition and other conditions for using the composition can be determined based on methods known in the art.

[0215] In one embodiment, the composition further comprises a bleaching system.

[0216] As used herein, the term "bleaching system" refers to inorganic and organic bleaching agents suitable as cleaning agents. The terms "bleaching system" and "bleaching agent" are used interchangeably herein and have the same meaning and purpose, unless otherwise expressly stated. Inorganic bleaching agents include hydroperoxide salts, such as perborates, percarbonates, superphosphates, persulfates, and persilicates. Inorganic hydroperoxide salts are typically alkali metal salts. Inorganic hydroperoxide salts may be included as crystalline solids without additional protection. Alternatively, the salt may be coated.

[0217] Alkali metal percarbonates, particularly sodium percarbonate, are preferred hydroperoxide compounds for use herein. Most preferably, the percarbonate is incorporated into the product in a coating form, which provides internal stability. Suitable coating materials providing internal stability comprise a mixture of water-soluble alkali metal sulfates and carbonates. Such coatings and coating methods have previously been described in GB 1,466,799. The weight ratio of the mixed salt coating material to the percarbonate is in the range of 1:200 to 1:4, more preferably from 1:99 to 1:9, and most preferably from 1:49 to 1:19. Preferably, the mixed salt is sodium sulfate and sodium carbonate having the general formula Na₂SO₄.n.Na₂CO₃, where n is from 0.1 to 3, preferably from 0.3 to 1.0, and most preferably from 0.2 to 0.5.

[0218] Other suitable coating materials that provide internal stability to the product include sodium silicate, wherein the SiO2:Na2O ratio is from 1.8:1 to 3.0:1, preferably from 1.8:1 to 2.4:1, and / or sodium metasilicate, preferably applied at a SiO2 level of from 2% to 10% (typically from 3% to 5%) by weight of the inorganic hydroperoxide salt. Magnesium silicate may also be included in the coating. Coatings comprising silicates and borates or boric acid or other inorganic substances are also suitable.

[0219] Other coatings, including waxes, oils, and fatty soaps, may also be advantageously used in this invention.

[0220] Potassium persulfate is another inorganic hydrogen peroxide salt that is useful herein. Typical organic bleaching agents are organic peroxy acids, including diacid and tetraacyl peroxides, especially disperoxydodecanoic acid, disperoxytetradecanoic acid, and disperoxyhexadecanoic acid. Benzoyl peroxide is a preferred organic peroxy acid herein. Mono- and dipernonanoic acid, mono- and distearate acid, and N-phthalamide peroxyhexanoic acid are also applicable herein. The diacid peroxide, especially the dibenzoyl peroxide, should preferably be present in particulate form, the particles having a weight-average diameter of from about 0.1 to about 100 micrometers, preferably from about 0.5 to about 30 micrometers, more preferably from about 1 to about 10 micrometers. Preferably, at least about 25%, more preferably at least about 50%, even more preferably at least about 75%, and most preferably at least about 90% of the particles are less than 10 micrometers, preferably less than 6 micrometers. It has been found that diacid peroxides within the aforementioned particle size range provide better stain removal, especially from plastic tableware, compared to larger diacid peroxide particles, while minimizing unwanted deposition and film formation during use in automatic dishwashing machines. This preferred diacid peroxide particle size thus allows the formulator to achieve good stain removal with a low level of diacid peroxide, which reduces deposition and film formation. Conversely, as the diacid peroxide particle size increases, more diacid peroxide is required to achieve good stain removal, which increases the amount of deposition encountered on the surface during dishwashing.

[0221] Other typical organic bleaching agents include peroxy acids, specifically alkyl peroxy acids and aryl peroxy acids. Preferred representatives are (a) peroxybenzoic acid and its cyclic-substituted derivatives, such as alkyl peroxybenzoic acid, but also peroxy-[α]-naphthoic acid and magnesium monoperphthalate, (b) aliphatic or substituted aliphatic peroxy acids, such as peroxylauric acid, peroxystearic acid, [ε]-phthalimide peroxyhexanoic acid [phthaloyliminoperoxyhexanoic acid (PAP)], o-carboxybenzoamide peroxyhexanoic acid, N-nonenyl acyl peroxyacid and N-nonenyl acylamino succinate, and (c) aliphatic and aryliphatic peroxydicarboxylic acids, such as 1,12-disperoxycarboxylic acid, 1,9-disperoxyazelaic acid, disperoxysediic acid, disperoxymethylene acid, disperoxyphthalic acid, 2-decyl-1,4-diacid, and N,N-terephthaloyldi(6-aminoperpropionic acid).

[0222] In one embodiment, the composition further comprises a bleaching activator, a bleaching catalyst, a silicate, and / or one or more metal care agents.

[0223] As used herein, the term "bleaching activator" refers to a typical organic peracid precursor that enhances the bleaching effect during the cleaning process at temperatures of 60°C and below. Bleaching activators suitable for use herein include compounds that, under hydrolytic conditions, yield aliphatic peroxycarboxylic acids having preferably from 1 to 10 carbon atoms, particularly from 2 to 4 carbon atoms, and / or optionally substituted perbenzoic acid. Suitable substances carry O-acyl and / or N-acyl groups with a specified number of carbon atoms, and / or optionally substituted benzoyl groups. Preferred ingredients include polyacylated alkylene diamines (especially tetraacetylethylenediamine (TAED)), acylated triazine derivatives (especially 1,5-diacetyl-2,4-dioxane-1,3,5-triazine (DADHT)), acylated glyurea (especially acyl glyurea (TAGU)), N-acylimides (especially N-nonanoylsuccinimide (NOSI), acylated phenol sulfonates (especially n-nonanoyl- or isonanoyloxybenzenesulfonates (n- or iso-NOBS), carboxylic anhydrides (especially phthalic anhydride), acylated polyols (especially triacetin, ethylene glycol diacetate, and 2,5-diacetoxy-2,5-dihydrofuran, and also triethyl citrate (TEAC)). If included in the compositions of the invention, the bleaching activator is present at a level from about 0.1% to about 10%, preferably from about 0.5% to about 2% by weight of the composition.

[0224] As used herein, the term "bleaching catalyst" refers to manganese-triazacyclononane and related complexes (US-A-4246612, US-A-5227084); Co, Cu, Mn, and Fe dipyridylamines and related complexes (US-A-5114611); and cobalt(III) pentamineacetate and related complexes (US-A-4810410). A complete description of bleaching catalysts suitable for use herein can be found in WO 99 / 06521, page 34, line 26 to page 40, line 16. If included in the compositions of the invention, the bleaching catalyst is present at a level from about 0.1% to about 10%, preferably from about 0.5% to about 2% by weight of the composition.

[0225] According to the present invention, oxidoreductases, such as oxidases, oxygenases, catalases, peroxidases (e.g., halogen-, chlorine-, bromine-, lignin-, glucose, or manganese peroxidases), dioxygenases, or laccases (phenol oxidases, polyphenol oxidases), can also be used to enhance their bleaching effect. Advantageously, additionally, organic, and particularly preferably aromatic compounds that interact with the enzymes are added to enhance the activity of the relevant oxidoreductases (enhancers) or, if there is a large difference in redox potential between these oxidases and the stain, to ensure electron flow (mediators).

[0226] As used herein, the term "silicate" refers to sodium silicates, such as sodium disilicate, sodium metasilicate, and crystalline layered silicates. If present, the silicate content is from about 1% to about 20%, preferably from about 5% to about 15% by weight of the composition.

[0227] As used herein, the term "metal care agent" refers to the prevention or reduction of rust, corrosion, or oxidation of metals, including aluminum, stainless steel, and non-ferrous metals such as silver and copper. Suitable examples include one or more of the following:

[0228] (a) Benzotriazoles, including benzotriazoles or bis-benzotriazoles and their substituted derivatives. Benzotriazole derivatives are those compounds in which the available substitution sites on the aromatic ring are partially or completely substituted. Suitable substituents include linear or branched Ci-C20 alkyl groups and substituents of hydroxyl, sulfur, phenyl, or halogens (such as fluorine, chlorine, bromine, and iodine).

[0229] (b) Metal salts and complexes selected from the group consisting of: zinc, manganese, titanium, zirconium, hafnium, vanadium, cobalt, gallium, and cesium salts and / or complexes, wherein these metals are in one of oxidation states II, III, IV, V, or VI. In one aspect, suitable metal salts and / or metal complexes may be selected from the group consisting of: Mn(II) sulfates, Mn(II) citrates, Mn(II) stearates, Mn(II) acetylacetone compounds, KTiF6, KZrF6, CoSO4, Co(NOs)2, and Ce(NOs)3, zinc salts, for example, zinc sulfate, zinc hydroxide, or zinc acetate; (c) Silicates, including sodium silicate or potassium silicate, sodium disilicate, sodium metasilicate, crystalline layered silicates, and mixtures thereof.

[0230] WO 94 / 26860 and WO 94 / 26859 disclose other suitable organic and inorganic redox active substances for use as silver / copper corrosion inhibitors. Preferably, the compositions of the present invention comprise from 0.1% to 5% by weight of a metal care agent, preferably a zinc salt.

[0231] In one embodiment, the composition further comprises a surfactant.

[0232] As used herein, the term "surfactant" specifically refers to dishwashing components that are nonionic compounds. Suitable nonionic surfactants include, but are not limited to, low-foaming nonionic (LFNI) surfactants. LFNI surfactants are most typically used in automatic dishwashing compositions because of the improved water film effect they impart to the composition (especially from glassware). They may also encompass non-silicone, phosphate, or non-phosphate polymeric materials known to defoam food stains encountered in automatic dishwashing. The LFNI surfactant can have a relatively low cloud point and a high hydrophilic-lipophilic balance (HLB). For optimal control of foaming across the entire range of water temperatures, a 1% solution in water typically has a cloud point below about 32°C, and alternatively, even lower, for example, 0°C. If desired, biodegradable LFNI surfactants with the above properties can be used.

[0233] LFNI surfactants may include, but are not limited to: alkoxylated surfactants, especially ethoxylated compounds derived from primary alcohols, and blends thereof with more complex surfactants such as polyoxypropylene / polyoxyethylene / polyoxypropylene reverse block polymers. Suitable block polyoxyethylene-polyoxypropylene polymers may include those based on ethylene glycol, propylene glycol, glycerol, trimethylolpropane, and ethylenediamine, and mixtures thereof. Polymers made by sequential ethoxylation and propoxylation of an initiator compound with a single reactive hydrogen atom (such as C12-) are fatty alcohols and generally do not provide satisfactory foam control in automatic dishwashing compositions. However, certain block polymeric surfactant compounds named PLURONIC® and TETRONIC® by BASF-Wyandotte, Wyandotte, Michigan are suitable for automatic dishwashing compositions. The LFNI surfactant may optionally include up to about 15% propylene oxide by weight. Other LFNI surfactants may be prepared by the process described in U.S. Patent No. 4,223,163. The LFNI surfactant can also be derived from a straight-chain fatty alcohol containing from about 16 to about 20 carbon atoms (C16-C20 alcohol), alternatively a Ci8 alcohol, condensed at an average of about 6 to about 15 moles, or from about 7 to about 12 moles, and alternatively from about 7 to about 9 moles of ethylene oxide / molar alcohol. Relative to the average, such derived ethoxylated nonionic surfactants can have a narrow ethoxylate distribution.

[0234] In some embodiments, the LFNI surfactant having a cloud point below 30°C may be present in an amount from about 0.01% to about 60% or from about 0.5% to about 10% by weight, and alternatively, in an amount from about 1% to about 5% by weight of the composition.

[0235] In a preferred embodiment, the surfactant is a nonionic surfactant or nonionic surfactant system having a phase transition temperature, measured as measured at a 1% concentration in distilled water, between 40°C and 70°C, preferably between 45°C and 65°C. "Nonionic surfactant system" herein means a mixture of two or more nonionic surfactants. Nonionic surfactant systems are preferred herein. Compared to single nonionic surfactants, they appear to have improved cleaning and finishing properties and stability in products. Suitable nonionic surfactants include: i) ethoxylated nonionic surfactants prepared by reacting a monohydroxylated alkanol or alkylphenol with 6 to 20 carbon atoms, preferably at least 12 moles, specifically preferably at least 16 moles, and even more preferably at least 20 moles of ethylene oxide / moles of alcohol or alkylphenol; ii) alcohol alkoxylated surfactants having 6 to 20 carbon atoms and at least one ethoxy and propoxy group. A mixture of surfactants i) and ii) is preferred herein.

[0236] Another suitable nonionic surfactant is an epoxy-terminated poly(alkoxylated) alcohol represented by the following chemical formula:

[0237] R1O[CH2CH(CH3)O] x [CH2CH2O] y [CH2CH(OH)R2] (I)

[0238] Wherein R1 is a straight-chain or branched aliphatic hydrocarbon group having 4 to 18 carbon atoms; R2 is a straight-chain or branched aliphatic hydrocarbon group having 2 to 26 carbon atoms; x is an integer having an average value of 0.5 to 1.5, more preferably about 1; and y is an integer having a value of at least 15, more preferably at least 20.

[0239] Preferably, the surfactant having Formula I has at least about 10 carbon atoms in the terminal epoxy unit [CH2CH(OH)R2]. A suitable surfactant having Formula I is the POLY-TERGENT(R)SLF-18B nonionic surfactant from Olin Corporation, for example, as described in WO 94 / 22800 published by Olin Corporation on October 13, 1994.

[0240] Preferably, the nonionic surfactants and / or systems described herein have a Draves wetting time of less than 360 seconds, preferably less than 200 seconds, more preferably less than 100 seconds, and especially less than 60 seconds, as measured by the Draves wetting method (using the standard method ISO 8022 under the following conditions: 3-g hook, 5-g cotton skein, 0.1% aqueous solution by weight at 25°C). Amine oxide surfactants are also useful in this invention as anti-reprecipitation surfactants, including linear and branched compounds having the following chemical formulas:

[0241]

[0242] Where R 3 Selected from the following: alkyl, hydroxyalkyl, amide propyl and alkylphenyl groups, or mixtures thereof, comprising from 8 to 26 carbon atoms, preferably from 8 to 18 carbon atoms; R 4 It is an alkylene or hydroxyalkylene group comprising 2 to 3 carbon atoms (preferably 2 carbon atoms), or a mixture thereof; X is from 0 to 5, preferably from 0 to 3; and each R 5 It is an alkyl or hydroxyalkyl group comprising 1 to 3 carbon atoms (preferably 1 to 2 carbon atoms), or a polyethylene oxide group comprising 1 to 3 (preferably 1) ethylene oxide groups. These R 5 Groups can attach to each other, for example, through oxygen or nitrogen atoms, to form a ring structure.

[0243] These amine oxide surfactants specifically include C 10 -C 18 Alkyl dimethyl amine oxide and C8-C 18 Alkoxyethyl dihydroxyethyl amine oxide. Examples of such materials include dimethyl octylamine oxide, diethyl decylamine oxide, bis-(2-hydroxyethylamine)dodecaneamine oxide, dimethyl decylamine oxide, dipropyltetradecaneamine oxide, methyl ethyl hexadecaneamine oxide, dodecylamidopropyl dimethylamine oxide, hexadecyl dimethylamine oxide, octadecyl dimethylamine oxide, tallow dimethylamine oxide, and dimethyl-2-hydroxyoctadecaneamine oxide. C is preferred. 10 -C 18 Alkyl dimethylamine oxide, and C 10 -C 18 Amide alkyl dimethylamine oxide. Surfactants, and especially nonionic surfactants, may be present in amounts from 0 to 10% by weight, preferably from 0.1% to 10%, and most preferably from 0.25% to 6%.

[0244] In one embodiment, the composition further comprises a sulfonated polymer.

[0245] As used herein, the term "sulfonated polymer" refers to a polymer containing sulfonic acid or sulfonate functional groups.

[0246] The polymer (if used) is used in any suitable amount, from about 0.1% to about 20%, preferably from 1% to about 15%, and more preferably from 2% to 10%, based on the weight of the composition. Sulfonated / carboxylated polymers are particularly suitable for the compositions contained in the pouches of the present invention.

[0247] Suitable sulfonated / carboxylated polymers described herein may have the following weight average molecular weights: less than or equal to about 100,000 Da, or less than or equal to about 75,000 Da, or less than or equal to about 50,000 Da, or from about 3,000 Da to about 50,000, preferably from about 5,000 Da to about 45,000 Da.

[0248] As noted herein, the sulfonated / carboxylated polymer may comprise (a) at least one structural unit derived from at least one carboxylic acid monomer having general formula (I):

[0249]

[0250] Where R 1 To R 4 (a) Independently a hydrogen, methyl, carboxylic acid group, or CH2COOH, wherein the carboxylic acid group can be neutralized; (b) Optionally, one or more structural units derived from at least one nonionic monomer having general formula (II):

[0251]

[0252] Where R 5 i is hydrogen, C1 to C6 alkyl, or C1 to C6 hydroxyalkyl, and X is aromatic (wherein, when X is aromatic, R...). 5 (Is it hydrogen or methyl) or X has the general formula (III):

[0253]

[0254] Where R 6 It is (independent of R) 5 Hydrogen, C1 to C6 alkyl, or C1 to C6 hydroxyalkyl, and Y is O or N; and at least one structural unit derived from at least one sulfonic acid monomer having general formula (IV):

[0255]

[0256] Where R 7 It includes at least one sp 2The bond groups, A is O, N, P, S or amide or ester bond, B is a monocyclic or polycyclic aryl group or aliphatic hydrocarbon group, each t is independently 0 or 1, and M + It is a cation. On one hand, R... 7 These are C2 to C6 olefins. On the other hand, R 7 It is ethylene, butene, or propylene.

[0257] Preferred carboxylic acid monomers include one or more of the following: acrylic acid, maleic acid, itaconic acid, isobutylene acid, or ethoxylated esters of acrylic acid, more preferably acrylic acid and methacrylic acid. Preferred sulfonated monomers include one or more of the following: sodium (meth)allyl sulfonate, vinyl sulfonate, sodium phenyl (meth)allyl sulfonate, or 2-acrylamide-methylpropanesulfonic acid. Preferred nonionic monomers include one or more of the following: methyl (meth)acrylate, ethyl (meth)acrylate, tert-butyl (meth)acrylate, methacrylamide, ethyl (meth)acrylamide, tert-butyl (meth)acrylate, styrene, or [α]-methylstyrene.

[0258] Preferably, the polymer comprises monomers in the following levels: a polymer of one or more carboxylic acid monomers, by weight, ranging from about 40% to about 90%, preferably from about 60% to about 90%; a polymer of one or more sulfonic acid monomers, by weight, ranging from about 5% to about 50%, preferably from about 10% to about 40%; and a polymer of one or more nonionic monomers, optionally from about 1% to about 30%, preferably from about 2% to about 20% by weight. Particularly preferred polymers include a polymer of at least one carboxylic acid monomer by weight, ranging from about 70% to about 80%, and a polymer of at least one sulfonic acid monomer by weight, ranging from about 20% to about 30%.

[0259] The carboxylic acid is preferably (meth)acrylic acid. The sulfonic acid monomer is preferably one of the following: 2-acrylamidomethyl-1-propanesulfonic acid, 2-methacryloylamino-2-methyl-1-propanesulfonic acid, 3-methacryloylamino-2-hydroxypropanesulfonic acid, arylsulfonic acid, methylarylsulfonic acid, allyloxybenzenesulfonic acid, methylpropanesulfonic acid, 2-hydroxy-3-(2-propenyloxy)propanesulfonic acid, 2-methyl-2-propen-1-sulfonic acid, styrenesulfonic acid, vinylsulfonic acid, 3-sulfopropyl acrylate, 3-sulfopropyl methacrylate, methyl methacrylate, methyl methacrylate, and their water-soluble salts. The most preferred unsaturated sulfonic acid monomer is 2-acryloylamino-2-propanesulfonic acid (AMPS).

[0260] Preferred commercially available polymers include: Alcosperse 240, Aquatreat AR 540, and Aquatreat MPS from Alco Chemical; Acumer 3100, Acumer 2000, Acusol 587G, and Acusol 588G from Rohm & Haas; Goodrich K-798, K-775, and K-797 from BF Goodrich; and ACP 1042 from ISP Technologies. Particularly preferred polymers are Acusol 587G and Acusol 588G from Rohm & Haas.

[0261] In the polymer, all or some of the carboxylic acid or sulfonic acid groups may exist in a neutralized form, that is, the acidic hydrogen atoms of the carboxylic acid and / or sulfonic acid in some or all of the acid groups may be replaced by metal ions, preferably alkali metal ions and specifically sodium ions.

[0262] In one embodiment, the composition further comprises a water-co-solvent.

[0263] As used herein, the term "co-solvent" refers to a compound that dissolves hydrophobic compounds (or conversely, polar substances in a nonpolar environment) in an aqueous solution. Typically, co-solvents possess both hydrophilic and hydrophobic characteristics (as is known from surfactants as amphiphilic properties); however, the molecular structure of co-solvents generally does not favor spontaneous self-aggregation, see, for example, the review by Hodgdon and Kaler (2007), Current Opinion in Colloid & Interface Science, 12: 121-128. Co-solvents do not exhibit a critical concentration above which self-aggregation, as observed with surfactants, and the formation of micelles, thin layers, or other well-defined intermediate phases by lipids occur. Instead, many co-solvents exhibit a continuous type of aggregation process, where the size of the aggregates increases with increasing concentration. However, many co-solvents alter the phase behavior, stability, and colloidal properties of systems containing both polar and nonpolar substances (including mixtures of water, oils, surfactants, and polymers). Water-soluble solvents are used across a wide range of industries, from pharmaceuticals and personal care to food and technology applications. Their use in detergent compositions allows for, for example, more concentrated surfactant formulations (such as in the process of compressing liquid detergents by removing water) without causing undesirable phenomena such as phase separation or high viscosity.

[0264] Detergents may contain 0-10% by weight, for example 0-5% by weight, such as about 0.5% to about 5%, or about 3% to about 5% of a water-soluble solvent. Any water-soluble solvent known in the art for use in detergents may be used. Non-limiting examples of water-soluble solvents include sodium benzenesulfonate, sodium p-toluenesulfonate (STS), sodium xylenesulfonate (SXS), sodium cumenesulfonate (SCS), sodium cymene sulfonate, amine oxides, alcohols and polyethylene glycol ethers, sodium hydroxynaphthoate, sodium hydroxynaphthoate, sodium ethylhexyl sulfonate, and combinations thereof.

[0265] Specifically, the composition according to the present invention further comprises a chelating agent.

[0266] As used herein, the term "chelating agent" refers to chemicals that form molecules with certain metal ions, passivating these ions and thus preventing them from reacting with other components. Therefore, a chelating agent can be defined as a binding agent that inhibits chemical activity by forming chelates. Chelation is the formation or presence of two or more separate bonds between a ligand and a single central atom. The ligand can be any organic compound, silicate, or phosphate. In the context of this document, the term "chelating agent" includes chelant, chelating agent, chelating agent, complexing agent, or sequestering agent that can form water-soluble complexes with metal ions such as calcium and magnesium. The chelate effect describes the enhanced affinity of a chelating ligand for the same metal ion compared to similar non-chelating ligands. Chelating agents have the ability to bind to metal ions, particularly calcium (Ca2+) ions, and are widely used in detergents and compositions generally used for washing, such as laundry or dishwashing. However, chelating agents themselves exhibit inhibitory effects on enzyme activity. In this application, the term chelating agent is used interchangeably with "complexing agent" or "chelant".

[0267] Since most α-amylases are calcium sensitive, the presence of a chelating agent can affect enzyme activity. The calcium sensitivity of α-amylase can be determined by incubating a given α-amylase in the presence of a strong chelating agent and analyzing the effect of this incubation on the activity of the α-amylase. Calcium-sensitive α-amylases will lose most or all of their activity during incubation. The chelating agent may be present in the composition in an amount from 0.0001 wt% to 20 wt%, preferably from 0.01 wt% to 10 wt%, more preferably from 0.1 wt% to 5 wt%.

[0268] Non-limiting examples of chelating agents are EDTA, DTMPA, HEDP, EDDS, and citrate. Therefore, in one embodiment, the composition comprises a variant according to the invention and a chelating agent such as EDTA, DT, HEDP, EDDS, or citrate.

[0269] As used herein, the term "EDTA" refers to ethylenediaminetetra-acetic acid, which falls under the definition of a "strong chelating agent".

[0270] As used herein, the term "DTMPA" refers to diethylenetriaminetriaminepenta (methylenephosphonic acid). DTMPA can inhibit the scaling of carbonate, sulfate, and phosphate ions.

[0271] The term "HEDP" as used herein refers to hydroxy-ethanediphosphonic acid, which falls under the definition of "strong chelating agent".

[0272] As used herein, the term "EDDS" refers to amino polycarboxylic acid, which falls under the definition of "strong chelating agent".

[0273] The chelate effect (or chelating effect) describes the enhanced affinity of a chelating ligand for a metal ion relative to the affinity of a similar set of non-chelating ligands for the same metal. However, the strength of this chelate effect can be determined by various types of determinations or measurements, thereby allowing chelating agents to be differentiated and ranked according to their chelating effect (or strength).

[0274] In one assay, chelating agents can be characterized by their ability to reduce the concentration of free calcium ions (Ca2+) from 2.0 mM to 0.10 mM or less at pH 8.0, for example by using a test based on the method described by MK Nagarajan et al., JAOCS, Vol. 61, No. 9 (September 1984), pp. 1475-1478.

[0275] For reference, a chelating agent that has the same ability as EDTA at the same concentration to reduce the concentration of free calcium ions (Ca2+) from 2.0 mM to 0.10 mM at pH is called a strong chelating agent.

[0276] The compositions of the present invention can be in any suitable form, such as rods, homogenized tablets, tablets having two or more layers, pouches having one or more compartments, conventional or compressed powders, granules, pastes, gels, or conventional, compressed or concentrated liquids. Various detergent formulations are available, such as layers (same or different phases), pouches, and forms for mechanical feeding devices.

[0277] The pouch can be configured as a single compartment or multiple compartments. It can have any form, shape, and material suitable for preserving the composition, for example, preventing the composition from being released from the pouch before contact with water. The pouch is made of a water-soluble membrane encapsulating the inner volume. The inner volume can be divided into compartments containing the pouch. Preferred membranes are polymeric materials that form membranes or sheets, preferably polymers. Preferred polymers, copolymers, or derivatives thereof are selected from polyacrylates and water-soluble acrylate copolymers, methylcellulose, carboxymethylcellulose, sodium dextrin, ethylcellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, maltodextrin, polymethyl acrylates, and most preferably polyvinyl alcohol copolymers and hydroxypropyl methylcellulose (HPMC). Preferably, the polymer level in the membrane, such as PVA, is at least about 60%. Preferred average molecular weights will typically be from about 20,000 to about 150,000. The membrane can also be a blend composition comprising a hydrolyzable and water-soluble polymer blend, such as polylactic acid and polyvinyl alcohol (known under Trade Reference M8630, as sold by Chris Craft In. Prod., Gary, Indiana, USA), plus plasticizers, such as glycerin, ethylene glycol, propylene glycol, sorbitol, and mixtures thereof. These bags may include solid laundry cleaning compositions or portions thereof and / or liquid cleaning compositions or portions thereof separated by a water-soluble membrane. Chambers for liquid components may be structurally different from those containing solid components. Reference: (US 2009 / 0011970A1).

[0278] Detergent components can be physically separated from each other by compartments in different layers of water-soluble pouches or tablets. This avoids negative storage interactions between components. The different dissolution profiles of each compartment in the washing solution can also cause delayed dissolution of selected components.

[0279] Non-unit-volume liquid or gel detergents may be aqueous, typically containing at least 20% and up to 95% water by weight, such as up to about 70%, about 65%, about 55%, about 45%, or about 35%. Other types of liquids, including but not limited to alkanols, amines, glycols, ethers, and polyols, may be included in aqueous liquids or gels. Aqueous liquid or gel detergents may contain from 0-30% organic solvents. Liquid or gel detergents may be non-aqueous.

[0280] Another form of composition is as follows: soap bars, such as laundry soap bars, which can be used for hand washing clothes, fabrics, and / or textiles. As used herein, the term "soap bar" refers to a combination bar, including laundry bars, soap bars, combo bars, synthetic bars, and detergent bars. The types of bars are generally distinguished by the type of surfactant they contain, and the term "washing soap bar" includes those containing soaps derived from fatty acids and / or synthetic soaps. Washing soap bars have a physical form that is solid at room temperature, rather than liquid, gel, or powder. As used herein, the term "solid" means a physical form that does not change significantly over time, i.e., if a solid object (such as a laundry soap bar) is placed in a container, the solid object will not change in order to fill the container in which it is placed. The bar is typically in the form of a bar when it is solid, but it may also be other solid shapes such as round or oval.

[0281] The soap bar may also contain complexing agents such as EDTA and HEDP, fragrances and / or different types of fillers, surfactants such as anionic synthetic surfactants, detergent builders, polymerized dirt releasers, detergent chelators, stabilizers, fillers, dyes, colorants, dye transfer inhibitors, alkoxylated polycarbonates, defoamers, structural agents, binders, leachants, bleach activators, clay detergents, anti-redeposition agents, polymeric dispersants, brighteners, fabric softeners, fragrances and / or other compounds known in the art.

[0282] The soap bar can be processed in conventional laundry soap bar manufacturing equipment, such as, but not limited to, mixers, pressing machines (e.g., two-stage vacuum pressing machines), extruders, cutters, logo-stamper machines, cooling tunnels, and packaging machines. The invention is not limited to preparing soap bars by any single method. The premix of the invention can be added to the soap at different stages of the process. For example, the premix can be prepared comprising soap, enzymes, optionally one or more additional enzymes, protease inhibitors, and salts of monovalent cations and organic anions, and then the mixture can be extruded. The enzymes and optionally additional enzymes can be added simultaneously, for example, in solid form, as enzyme inhibitors (e.g., protease inhibitors). In addition to the mixing and extrusion steps, the process may further include the following steps: grinding, extrusion, cutting, stamping, cooling, and / or packaging.

[0283] In one embodiment, the composition comprises a detergent builder and / or a co-builder.

[0284] As used herein, the term "builder" refers to a specific type of chelating agent. Accordingly, the composition may contain approximately 0-65% by weight, such as approximately 5% to approximately 50%, of a detergent builder or co-builder, or a mixture thereof. In dishwashing detergents, the level of the builder is typically 40%-65%, particularly 50%-65%. The builder and / or co-builder may be, in particular, a complexing agent that forms a water-soluble complex with Ca and Mg. Any builder and / or co-builder known in the art for use in ADW detergents may be utilized. Non-limiting examples of detergent builders include zeolites, diphosphates (pyrophosphates), triphosphates such as sodium triphosphate (STP or STPP), carbonates such as sodium carbonate, soluble silicates such as sodium silicate, layered silicates (e.g., SKS-6 from Hoechst), ethanolamines such as 2-aminoethanol (MEA), diethanolamine (DEA, also known as 2,2'-iminodiethanol), triethanolamine (TEA, also known as 2,2',2”-nitrotriethanolamine), and carboxymethyl inulin (CMI), and combinations thereof.

[0285] The detergent composition may also contain 0-50% by weight, for example, about 5% to about 30%, of a detergent co-builder. The detergent composition may contain a co-builder, alone or in combination with the co-builder, such as a zeolite co-builder. Non-limiting examples of co-builders include polyacrylate homopolymers or copolymers thereof, such as poly(acrylic acid) (PAA) or copolymers (acrylic acid / maleic acid) (PAA / PMA). Other non-limiting examples include citrates, chelating agents such as aminocarboxylates, aminopolycarboxylates, and phosphonates, and alkyl- or alkenyl succinic acids. Other specific examples include 2,2',2”-N-aminotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N'-disuccinic acid (EDDS), methylglycine diacetic acid (MGDA), glutamic acid-N,N-diacetic acid (GLDA), 1-hydroxyethane-1,1-diphosphonic acid (HEDP), ethylenediaminetetra(methylenephosphonic acid) (EDTMPA), diethylenetriaminepenta(methylenephosphonic acid) (DTMPA or DTPMPA), N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartic-N-monoacetic acid (ASMA), aspartic-N,N-diacetic acid (ASDA), aspartic-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA), N-(2-sulfomethyl)-aspartic acid (SMAS), and N-(2-sulfoethyl)-aspartic acid (SEA). S), N-(2-sulfomethyl)-glutamic acid (SMGL), N-(2-sulfoethyl)-glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA), α-alanine-N,N-diacetic acid (α-ALDA), serine-N,N-diacetic acid (SEDA), isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid (PHDA), anthranilic acid-N,N-diacetic acid (ANDA), sulfanilic acid-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA), sulfonyl-N,N-diacetic acid (SMDA), N-(2-hydroxyethyl)-ethylenediamine-N,N',N'-triacetic acid (HEDTA), diethanolglycine (DEG), diethylenetriaminepenta (DTPMP), aminotris(methylenephosphonic acid) (ATMP), and combinations thereof and their salts. Other exemplary builders and / or co-builders are described, for example, in WO09 / 102854 and US 5977053.

[0286] In one embodiment, the composition comprises a polymer. The composition may contain 0-10% by weight, such as 0.5%-5%, 2%-5%, 0.5%-2%, or 0.2%-1% of a polymer. Any polymer known in the art for use in detergents may be utilized. The polymer may function as a co-adjuvant as mentioned above, or may provide anti-redeposition, fiber protection, dirt release, dye transfer inhibition, oil stain removal, and / or anti-foaming properties. Some polymers may have more than one of the properties mentioned above and / or more than one of the motifs mentioned below. Exemplary polymers include (carboxymethyl) cellulose (CMC), poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(ethylene glycol) or poly(ethylene oxide) (PEG), ethoxylated poly(ethyleneimine), carboxymethyl inulin (CMI), and polycarboxylates such as PAA, PAA / PMA, poly-aspartic acid, and lauryl methacrylate / acrylic acid copolymers, hydrophobically modified CMC (HM-CMC) and silicone, copolymers of terephthalic acid and oligomeric polyethylene glycol, copolymers of poly(ethylene terephthalate) and poly(ethylene oxyterephthalate) (PET-POET), PVP, poly(vinylimidazolium) (PVI), poly(vinylpyridine-N-oxide) (PVPO or PVPNO), and polyvinylpyrrolidone-vinylimidazolium (PVPVI). Other exemplary polymers include sulfonated polycarboxylate esters, polyethylene oxide and polypropylene oxide (PEO-PPO), and diquaternary ammonium ethoxysulfate. Other exemplary polymers are disclosed, for example, in WO 2006 / 130575. Salts of the polymers mentioned above are also considered.

[0287] These compositions according to the invention may include variants of the invention as the main enzyme component, such as single-component compositions. In another embodiment, the composition includes at least one additional enzyme, such as a protease, lipase, cellulose, pectic acid lyase, and / or mannanase. Therefore, these compositions may contain a variety of enzymatic activities, such as one or more (e.g., several) enzymes selected from the group consisting of: hydrolases, isomerases, ligases, lyases, oxidoreductases, or transferases, such as α-galactosidase, α-glucosidase, aminopeptidase, amylase, β-galactosidase, β-glucosidase, β-xylosidase, glycosylase, carboxypeptidase, catalase, cellobiase, cellulase, chitosanase, keratinase, cyclodextrin glucosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucosylamylase, invertase, laccase, lipase, mannosidase, polysaccharidase, oxidase, pectinase, peroxidase, phytase, polyphenol oxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.

[0288] In a specific embodiment, the composition further comprises one or more additional enzymes selected from the group consisting of:

[0289] (i) Compared with the α-amylase in SEQ ID NO: 5, the modified α-amylase is included at the following position: 202;

[0290] (ii) Compared with the α-amylase in SEQ ID NO: 6, it includes one or more modified α-amylases at the following positions: 9, 118, 149, 182, 186, 195, 202, 257, 295, 299, 320, 323, 339, 345 and 458;

[0291] (iii) Compared with the α-amylase in SEQ ID NO: 9, it includes one or more modified α-amylases at the following positions: 405, 421, 422 and 428;

[0292] (iv) α-amylase comprising any of the amino acid sequences set forth in SEQ ID NO: 7, 10, 11, and 12; and / or

[0293] (v) Compared with the protease in SEQ ID NO: 8, it includes one or more modified proteases at the following positions: 32, 33, 48-54, 58-62, 94-107, 116, 123-133, 150, 152-156, 158-161, 164, 169, 175-186, 197, 198, 203-216.

[0294] Generally, the properties of one or more selected enzymes should be compatible with the selected detergent (i.e., optimal pH, compatibility with other enzymes and non-enzyme components, etc.), and the one or more enzymes should be present in an effective amount.

[0295] In one embodiment, compared to the α-amylase in SEQ ID NO: 5, the composition comprises or consists of an α-amylase variant (which includes or is composed of the following modification: G182*+D183*+M202L, wherein the number is according to SEQ ID NO: 3) and an α-amylase (which contains the modification: 202) at the following position.

[0296] In one embodiment, compared to the α-amylase in SEQ ID NO: 5, the composition comprises or consists of an α-amylase variant (which includes or is composed of the following modification: G182*+D183*+N195F+M202L, wherein the number is according to SEQ ID NO: 3) and an α-amylase (which contains the modification: 202) at the following position.

[0297] In one embodiment, compared to the α-amylase in SEQ ID NO: 6, the composition comprises or consists of an α-amylase variant (which includes or is composed of the following modifications: G182*+D183*+M202L, wherein the numbering is according to SEQ ID NO: 3) and an α-amylase (which contains one or more modifications at the following positions: 9, 118, 149, 182, 186, 195, 202, 257, 295, 299, 320, 323, 339, 345, and 458) and the like.

[0298] In one embodiment, compared to the α-amylase in SEQ ID NO: 6, the composition comprises or consists of an α-amylase variant (which includes or is composed of the following modifications: G182*+D183*+N195F+M202L, wherein the numbering is according to SEQ ID NO: 3) and an α-amylase (which contains one or more modifications at the following positions: 9, 118, 149, 182, 186, 195, 202, 257, 295, 299, 320, 323, 339, 345, and 458) or consists of the latter.

[0299] In one embodiment, compared to the α-amylase in SEQ ID NO: 9, the composition comprises or consists of an α-amylase variant (which includes or is composed of the following modifications: G182*+D183*+M202L, wherein the numbering is according to SEQ ID NO: 3) and an α-amylase (which contains one or more modifications at the following positions: 405, 421, 422, and 428).

[0300] In one embodiment, compared to the α-amylase in SEQ ID NO: 9, the composition comprises or consists of an α-amylase variant (which includes or is composed of the following modifications: G182*+D183*+N195F+M202L, wherein the numbering is according to SEQ ID NO: 3) and an α-amylase (which contains one or more modifications at the following positions: 405, 421, 422, and 428).

[0301] In one embodiment, compared to the α-amylase in SEQ ID NO: 5, the composition comprises or consists of an α-amylase variant comprising or consisting of the following modifications: G182*+D183*+M202L, wherein the number is according to SEQ ID NO: 3) and an α-amylase comprising any of the amino acid sequences set forth in SEQ ID NO: 7, 10, 11, and 12.

[0302] In one embodiment, compared to the α-amylase in SEQ ID NO: 5, the composition comprises or consists of an α-amylase variant comprising or consisting of the following modifications: G182*+D183*+N195F+M202L, wherein the numbering is according to SEQ ID NO: 3) and an α-amylase comprising any of the amino acid sequences set forth in SEQ ID NO: 7, 10, 11, and 12.

[0303] In one embodiment, compared to the α-amylase in SEQ ID NO: 8, the composition comprises or consists of an α-amylase variant comprising or consisting of the following modifications: G182*+D183*+M202L, wherein the numbering is according to SEQ ID NO: 3) and a protease comprising one or more modifications at the following positions: 32, 33, 48-54, 58-62, 94-107, 116, 123-133, 150, 152-156, 158-161, 164, 169, 175-186, 197, 198, 203-216.

[0304] In one embodiment, compared to the α-amylase in SEQ ID NO: 8, the composition comprises or consists of an α-amylase variant comprising or consisting of the following modifications: G182*+D183*+N195F+M202L, wherein the numbering is according to SEQ ID NO: 3) and a protease comprising one or more modifications at the following positions: 32, 33, 48-54, 58-62, 94-107, 116, 123-133, 150, 152-156, 158-161, 164, 169, 175-186, 197, 198, 203-216.

[0305] Suitable cellulases include those of bacterial or fungal origin. This includes chemically modified or protein-engineered mutants. Suitable cellulases include those derived from the genera *Bacillus*, *Pseudomonas*, *Pyrophyllus*, *Fusarium*, *Clostridium*, and *Cladosporium*, for example, fungal cellulases produced from specific *Pyrophyllus*, *Thermophyllus*, and *Fusarium* species disclosed in US 4,435,307, US 5,648,263, US 5,691,178, US 5,776,757, and WO 89 / 09259.

[0306] Particularly suitable cellulases are alkaline or neutral cellulases that offer color-care benefits. Examples of such cellulases are those described in EP 0 495 257, EP 0 531 372, WO 96 / 11262, WO 96 / 29397, and WO 98 / 08940. Other examples are cellulase variants, such as those described in WO 94 / 07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95 / 24471, WO 98 / 12307, and PCT / DK 98 / 00299.

[0307] Examples of cellulases exhibiting endo-β-1,4-glucanase activity (EC 3.2.1.4) are those already described in WO 02 / 099091.

[0308] Other examples of cellulases include family 45 cellulases described in WO 96 / 29397, and particularly SEQ ID corresponding to WO 02 / 099091. The following positions in NO:8 contain variations of its substitution, insertion, and / or deletion: 2, 4, 7, 8, 10, 13, 15, 19, 20, 21, 25, 26, 29, 32, 33, 34, 35, 37, 40, 42, 42a, 43, 44, 48, 53, 54, 55, 58, 59, 63, 64, 65, 66, 67, 70, 72, 76, 79, 80, 82, 84, 86, 88, 90, 91, 93, 95, 95d, 95h, 95j, 97, 100, 101, 102, 103, 113, 114, 117, 119, 121, 133, 136. 137, 138, 139, 140a, 141, 143a, 145, 146, 147, 150e, 150j, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160c, 160e, 160k, 161, 162, 164, 165, 168, 170, 171, 172, 173, 175, 176, 178, 181, 183, 184, 185, 186, 188, 191, 192, 195, 196, 200, and / or 20, preferably selected from P19A, G20K, Q44K, N48E, Q119H, or Q146R.

[0309] Commercially available cellulases include Celluzyme and Carezyme (Novozymes A / S), Clazinase and Puradax HA (Genencor International Inc.), and KAC-500(B) (Kao Corporation).

[0310] Suitable lipases and keratins include those of bacterial or fungal origin. This also includes chemically modified or protein-engineered mutant enzymes. Examples include lipases from the genus *Thermomyces* (e.g., *T. lanuginosus*, formerly known as *Humicola lanuginosa*) as described in EP 258 068 and EP 305 216; keratinases from *Pseudomonas* (e.g., *H. insolens* (WO 96 / 13580)); lipases from the genus *Pseudomonas* (some of which are now renamed *Burkholderia*) (e.g., *P. alcaligenes* or *P. pseudoalcaligenes* (EP 218272)); *P. cepacia* (EP 331 376); and strain SD705 (WO 95 / 06720 & WO). 96 / 27002), Pseudomonas wisconsinensis (WO 96 / 12012), GDSL type Streptomyces lipase (WO 10 / 065455), cutinase from rice blast fungus (WO 10 / 107560), cutinase from Mendoza pseudopolycystis (US 5,389,536), lipase from thermophilic schizocarp (WO 11 / 084412), thermophilic stroph ...

[0311] Other examples are lipases sometimes referred to as acyltransferases or perhydrolases, such as an acyltransferase homologous to Candida antarcticis lipase A (WO 10 / 111143), an acyltransferase from Mycobacterium smegmatis (WO 05 / 56782), a perhydrolase from the CE 7 family (WO 09 / 67279), and variants of Mycobacterium smegmatis perhydrolase, particularly the S54V variant (WO 10 / 100028) used in Gentle Power Bleach, a commercial product from Huntsman TextileEffects Pte Ltd.

[0312] Other examples are lipase variants, such as those described in EP 407225, WO 92 / 05249, WO 94 / 01541, WO 94 / 25578, WO 95 / 14783, WO 95 / 30744, WO 95 / 35381, WO 95 / 22615, WO 96 / 00292, WO 97 / 04079, WO 97 / 07202, WO 00 / 34450, WO 00 / 60063, WO 01 / 92502, WO 07 / 87508, and WO 09 / 109500.

[0313] Preferred commercially available lipase products include Lipolase™ and Lipex. TM Lipolex™ and Lipoclean™ (Novozymes), Lumafast (Genencor), and Lipomax (Gist-Brocades).

[0314] Suitable peroxidases / oxidases include those of plant, bacterial, or fungal origin. This includes chemically modified or protein-engineered mutants. Examples of useful peroxidases include peroxidases from the genus *Coprinus*, such as those from *Coprinus spp.*, and their variants, such as those described in WO 93 / 24618, WO 95 / 10602, and WO 98 / 15257.

[0315] Commercially available peroxidases include Guardzyme (Novozymes).

[0316] Detergent enzymes can be incorporated into detergent compositions by adding a single additive containing one or more enzymes, or by adding a combination of additives containing all of these enzymes. The detergent additives of the present invention, i.e., single or combined additives, can be formulated as, for example, granules, liquids, slurries, etc., with preferred detergent additive formulations being granules, particularly dust-free granules; liquids, particularly stabilized liquids; or slurries.

[0317] Dust-free particles can be produced, for example, as disclosed in US 4,106,991 and 4,661,452, and can optionally be coated by methods known in the art. Examples of wax-coated materials are poly(ethylene oxide) products (polyethylene glycol, PEG) with an average molar weight of 1,000 to 20,000; ethoxylated nonylphenol having 16 to 50 ethylene oxide units; ethoxylated fatty alcohols containing 12 to 20 carbon atoms and containing 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and fatty acid monoglycerides, diglycerides, and triglycerides. Examples of film-forming coating materials suitable for application via fluidized bed technology are given in GB 1483591. Liquid enzyme preparations can be stabilized, for example, by adding polyols (such as propylene glycol), sugars or sugar alcohols, lactic acid, or boric acid according to established methods. Protected enzymes can be prepared according to the methods disclosed in EP 238,216.

[0318] These compositions can be prepared according to methods known in the art and can be in the form of liquid or dry compositions. These compositions can be stabilized according to methods known in the art.

[0319] Any detergent components known in the art for use in ADW detergents may also be used. Other optional detergent components include preservatives, shrinkage inhibitors, anti-fouling redeposition agents, anti-wrinkle agents, bactericides, binders, corrosion inhibitors, disintegrants / disintegration agents, dyes, enzyme stabilizers (including boric acid, borates, CMC and / or polyols such as propylene glycol), fabric finishing agents (including clays), fillers / processing aids, optical brighteners / brighteners, foaming agents, foam (foam) conditioners, fragrances, soil suspending agents, softeners, defoamers, dulling inhibitors, and wicking agents, used alone or in combination. Any ingredients known in the art for use in ADW detergents may be used. The selection of such ingredients is entirely within the skill of a person skilled in the art.

[0320] The compositions of the present invention may also contain dispersants. Specifically, powdered detergents may include dispersants. Suitable water-soluble organic materials include homopolymerized or copolymerized acids or salts thereof, wherein the polycarboxylic acid comprises at least two carboxyl groups separated from each other by no more than two carbon atoms. Suitable dispersants are described, for example, in Powdered Detergents, Surfactant Science Series, Volume 71, Marcel Dekker.

[0321] The compositions of the present invention may further comprise one or more dye transfer inhibitors. Suitable polymeric dye transfer inhibitors include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidinone, and polyvinylimidazole or mixtures thereof. When present in the test composition, the dye transfer inhibitor may be present at a level from about 0.0001% to about 10%, from about 0.01% to about 5%, or even from about 0.1% to about 3% by weight of the composition.

[0322] The compositions of the present invention will also preferably contain additional components that can color the item being cleaned, such as optical brighteners or fluorescent whitening agents. The brightening agent is preferably present at a level of about 0.01% to about 0.5%. Any optical brightener suitable for use in laundry detergent compositions can be used in the compositions of the present invention. The most commonly used optical brighteners are those belonging to the following categories: diaminostilbene-sulfonic acid derivatives, diarylpyrazoline derivatives, and diphenyl-bistyryl derivatives. Examples of diaminostilbene-sulfonic acid derivatives of fluorescent whitening agents include sodium salts of the following: 4,4'-bis-(2-diethanolamino-4-anilino-s-triazin-6-ylamino)stilbene-2,2'-disulfonate, 4,4'-bis-(2,4-diphenylamino-s-triazin-6-ylamino)stilbene-2,2'-disulfonate, 4,4'-bis-(2-anilino-4-(N-methyl-N-2-hydroxy-ethylamino)-s-triazin-6-ylamino)stilbene-2,2'-disulfonate, 4,4'-bis-(4-phenyl-1,2,3-triazol-2-yl)stilbene-2,2'-disulfonate, and sodium 5-(2H-naphtho[1,2-d][1,2,3]triazol-2-yl)-2-[(E)-2-phenylvinyl]benzenesulfonate. Preferred fluorescent whitening agents are Tinopal DMS and Tinopal CBS, available from Ciba-Geigy AG (Basel, Switzerland). Tinopal DMS is the disodium salt of 4,4'-bis-(2-morpholino-4-anilino-s-triazine-6-ylamino)stilbene-2,2'-disulfonate. Tinopal CBS is the disodium salt of 2,2'-bis-(phenyl-styryl)-disulfonate. Also preferred is Parawhite KX, commercially available from Paramount Minerals and Chemicals, Mumbai, India. Other fluorescent agents suitable for use in this invention include 1,3-diarylpyrazoline and 7-alkylaminocoumarin.

[0323] Appropriate levels of fluorescent whitening agents include lower levels ranging from about 0.01 wt%, from 0.05 wt%, from about 0.1 wt%, or even from about 0.2 wt% to upper limits of 0.5 wt% or even 0.75 wt%.

[0324] use

[0325] This invention further relates to the use of the variants described according to the invention in cleaning processes (e.g., cleaning of clothing or hard surfaces, including automated dishwashing and industrial cleaning). The dirt and stains important for cleaning consist of many different substances, and a range of different enzymes, all with different substrate specificities, have been developed for use in both cleaning of clothing and hard surfaces (e.g., dishwashing). These enzymes are thought to provide enzymatic washing benefits because they specifically improve stain removal during the cleaning process in which they are applied compared to the same process without enzymes. Cleaning enzymes known in the art include, for example, proteases, amylases, lipases, keratins, cellulases, endoglucanases, xyloglucanases, pectins, pectin lyases, xanthan gums, peroxidases, halogenated peroxidases, catalases, and mannanases.

[0326] In one embodiment, the invention relates to the use of variants of the invention in detergent compositions for cleaning hard surfaces (e.g., dishwashing), or in laundry or for stain removal. In another embodiment, the invention relates to the use of variants according to the invention in cleaning processes (e.g., cleaning of clothing or hard surfaces, including but not limited to automatic dishwashing and industrial cleaning). Thus, in one embodiment, the invention relates to the use of variants that contain substitutions at one or more locations providing oxidative stability of the variant.

[0327] In a specific embodiment, the variant contains a substitution at position M202 corresponding to the amino acid sequence described in SEQ ID NO: 3, and contains substitutions and / or deletions at two, three, or four positions corresponding to positions R181, G182, D183, and G184 corresponding to the amino acid sequence described in SEQ ID NO: 3.

[0328] One embodiment of the invention relates to the use of a composition according to the invention, the composition comprising a variant of the invention together with one or more surfactants and optionally one or more detergent components selected from the list of water-soluble solvents, detergent builders and co-builders, bleaching systems, polymers, fabric colorants and accessories, or any mixture thereof.

[0329] Another embodiment is the use of the composition according to the invention, which comprises a detergent composition comprising the variants of the invention together with one or more surfactants and one or more additional enzymes selected from the group consisting of proteases, lipases, keratins, cellulases, endoglucans, xyloglucans, pectins, pectin lyases, xanthan gums, peroxidases, halogenated peroxidases, catalases, and mannanases or any mixture thereof.

[0330] In another aspect, the present invention relates to a washing method applicable to both household and industrial washing. Furthermore, the present invention relates to a method for washing textiles (e.g., fabrics, garments, clothes, etc.), wherein the method comprises treating the textiles with a washing solution containing a detergent composition and the α-amylase of the present invention. The washing can be performed using a household or industrial washing machine or manually using a detergent composition containing the glucosylase of the present invention.

[0331] In another aspect, the present invention relates to a method for washing tableware, applicable to both household and industrial tableware washing. As used herein, the term "tableware washing" refers to both manual and automated tableware washing. Furthermore, the present invention relates to a method for washing hard surfaces (e.g., tableware such as knives, forks, and spoons; ceramics such as plates, glasses, and bowls; and pans), wherein the method comprises treating the hard surface with a washing solution comprising a detergent composition and an α-amylase variant of the present invention. Hard surfaces can be washed, for example, using a household or industrial dishwasher or manually using a detergent composition comprising the α-amylase of the present invention, optionally together with one or more other enzymes selected from the group consisting of: proteases, amylases, lipases, keratins, cellulases, endoglucanases, xyloglucanases, pectins, pectin lyases, xanthan gums, peroxidases, halogenated peroxidases, catalases, mannanases, or any mixtures thereof.

[0332] In another aspect, the present invention relates to a method for removing stains from a surface, the method comprising contacting the surface with a composition comprising, in a detergent composition and in a detergent application, an α-amylase of the present invention together with one or more surfactants and optionally one or more detergent components selected from the list comprising water-soluble solvents, builder agents and co-builder agents, bleaching systems, polymers, fabric toners and accessories, and any mixture thereof. Another aspect is a method for removing stains from a surface, the method comprising contacting the surface with a composition comprising, in a detergent composition and in a detergent application, an α-amylase variant of the present invention together with one or more surfactants and one or more additional enzymes selected from the group comprising proteases, lipases, keratins, cellulases, endoglucanases, xyloglucanases, pectins, pectin lyases, xanthan gums, peroxidases, halogenated peroxidases, catalases, mannanases, or any mixture thereof.

[0333] The invention is further described through the following embodiments, but should not be construed as limiting the scope of the invention. Example

[0334] Example 1 - Variation Generation

[0335] Variants of the present invention were constructed using a parental α-amylase having the amino acid sequence described in SEQ ID NO: 4. These variants were isolated using standard procedures, specifically by introducing the gene through site-directed mutagenesis, replacing the amino acid at position M200 of the amino acid sequence described in SEQ ID NO: 4 (or, if numbered according to SEQ ID NO: 3, the amino acid position is M202), transforming Bacillus subtilis host cells with the mutated gene, fermenting these transformed cells (e.g., as described in Example 1 of WO 2004 / 111220), and purifying the variants from the fermentation broth. Reference amylases having the amino acid sequence described in SEQ ID NO: 3 or 4, i.e., the parental α-amylase, were recombinantly produced in Bacillus subtilis in a similar manner.

[0336] Example 2 - Determination of starch-degrading activity (α-amylase activity)

[0337] The α-amylase activity of the variants produced as described in Example 1 was tested by pNP-G7 assay.

[0338] α-Amylase activity was determined using the pNP-G7 substrate (PNP-G7 is an abbreviation for 4,6-ethylidene (G7)-p-nitrophenyl (G1)-,D-maltoheptaglycoside, a block oligosaccharide that can be cleaved by endoamylases such as α-amylase).

[0339] The antibody was diluted in phosphate-buffered saline (PBS) (0.010 M phosphate buffer, pH 7.4, 0.0027 M KCl, 0.14 M NaCl) buffer to a concentration of 10 μg / mL. The maxisorp microtiter plate was coated with antibody by adding 100 μl of the diluted antibody (10 μg / mL) to each well and incubating at room temperature (RT) for 1 h and mixing at 800 rpm. After incubation, the microtiter plate was washed (using a Bio-Tek ELx405 ELISA washer) with 3 x 200 μl phosphate-buffered saline and 0.05% Tween (PBST) (0.010 M phosphate buffer, pH 7.4, 0.0027 M KCl, 0.14 M NaCl, 0.05% Tween 20) buffer.

[0340] The microtiter plate containing the α-amylase variant culture medium was centrifuged by rotation, and the supernatant was transferred to a new microtiter plate and diluted 4x in PBST buffer. 100 μl of the diluted supernatant was transferred to an antibody-coated maxisorp microtiter plate and incubated at RT for 1 h, followed by mixing at 800 rpm. After incubation, the microtiter plate was washed in PBST buffer (3 x 200 μl, ELISA washer).

[0341] Following lysis of the pNP-G7 substrate, digestion with the α-glucosidase included in the kit releases free pNP molecules, which are yellow in color and can therefore be measured spectrophotometrically at Abs = 405 nm (400–420 nm). The kit containing pNP-G7 substrate and α-glucosidase is manufactured by Roche / Hitachi (catalog number 11876473). Add 100 μl of pNP-G7 substrate to all wells and mix for 1 minute before measuring absorbance at 405 nm. Determine the slope (absorbance per minute) and use only the linear range of the curve.

[0342] Samples with equivalent or higher activity compared to control samples were considered to have maintained α-amylase activity. The oxidative stability of these variants was further evaluated as described in Example 3.

[0343] This specific α-amylase activity can also be determined by other activity assays, such as the amylazyme activity assay, the Phadebas activity assay, or the glucose-lowering activity assay described below.

[0344] Amylazyme activity assay (From Megazyme, Ireland): Amylazyme tablets comprise interconnected amylose polymers in the form of water-insoluble spherical microspheres. A blue dye is covalently bound to these microspheres. The interconnected amylose polymers within the microspheres degrade at a rate proportional to α-amylase activity. As α-amylase degrades the amylose polymers, the released blue dye is water-soluble, and the dye concentration can be determined by measuring absorbance at 650 nm. The concentration of blue is proportional to the α-amylase activity in the sample.

[0345] Dilute the amylase sample to be analyzed in an activity buffer at the desired pH. Suspend one substrate tablet in 5 mL of activity buffer and mix on a magnetic stirrer. While mixing the substrate, transfer 150 μL to a microtiter plate (MTP). Add 30 μL of the diluted amylase sample to 150 μL of substrate and mix. Incubate at 37 °C for 15 min. Stop the reaction by adding 30 μL of 1M NaOH and mixing. Centrifuge the MTP at 4000 x g for 5 min. Transfer 100 μL to a fresh MTP and measure the absorbance at 620 nm.

[0346] The amylase sample should be diluted so that the absorbance at 650 nm is between 0 and 2.2, and within the linear range of the activity assay.

[0347] Phadebas activity assay (For example, from Magle Life Sciences, Lund, Sweden): Phadebas tablets comprise interconnected starch polymers in the form of water-insoluble spherical microspheres. A blue dye is covalently bound to these microspheres. The interconnected starch polymers within the microspheres degrade at a rate proportional to α-amylase activity. As α-amylase degrades the amylase, the released blue dye is water-soluble, and the dye concentration can be determined by measuring absorbance at 650 nm. The concentration of blue is proportional to the α-amylase activity in the sample.

[0348] Dilute the amylase sample to be analyzed in an activity buffer at the desired pH. Suspend one substrate tablet in 5 mL of activity buffer and mix on a magnetic stirrer. While mixing the substrate, transfer 150 μL to a microtiter plate (MTP). Add 30 μL of the diluted amylase sample to 150 μL of substrate and mix. Incubate at 37 °C for 15 min. Stop the reaction by adding 30 μL of 1M NaOH and mixing. Centrifuge the MTP at 4000 x g for 5 min. Transfer 100 μL to a fresh MTP and measure the absorbance at 620 nm.

[0349] The amylase sample should be diluted so that the absorbance at 650 nm is between 0 and 2.2, and within the linear range of the activity assay.

[0350] Reducing sugar activity assay: The number of reducing ends formed by the hydrolysis of α-1,4-glycosidic bonds in starch by α-amylase was determined by reaction with p-hydroxybenzoic acid hydrazide (PHBAH). After the reaction with PHBAH, the number of reducing ends could be measured by absorbance at 405 nm, and the concentration of reducing ends was proportional to the α-amylase activity in the sample.

[0351] The corn starch substrate (3 mg / mL) was dissolved by boiling in milliQ water for 5 minutes and then cooled before assay. For the stop solution, a Ka-Na-tartrate / NaOH solution was prepared (K-Na-tartrate (Merck 8087) 50 g / L, NaOH 20 g / L) and the stop solution was freshly prepared by adding p-hydroxybenzoic acid hydrazide (PHBAH, Sigma H9882) to the Ka-Na-tartrate / NaOH solution to a concentration of 15 mg / mL.

[0352] In PCR-MTP, mix 50 μl of activity buffer with 50 μl of substrate. Add 50 μl of diluted enzyme and mix. Incubate in a PCR machine at the desired temperature for 5 minutes. Stop the reaction by adding 75 μl of stop solution (Ka-Na-tartrate / NaOH / PHBAH). Incubate in a PCR machine at 95 °C for 10 minutes. Transfer 150 μl to a fresh MTP and measure the absorbance at 405 nm.

[0353] The amylase sample should be diluted so that the absorbance at 405 nm is between 0 and 2.2, and within the linear range of the activity assay.

[0354] Example 3 - Oxidative stability verified by AMSA

[0355] To evaluate the oxidative stability of the variants produced as described in Example 1, the washing performance of these variants in detergent compositions containing oxidants, such as bleaching systems, was determined using automated mechanical stress assay (AMSA). Variants with oxidative stability will have maintained washing performance and may even have improved washing performance.

[0356] The washing performance of these variations of the invention was tested. The washing performance of a large number of small-volume enzyme detergent solutions can be examined using the AMSA test. The AMSA board has numerous slits and caps for the test solution, with the caps forcefully pressing a small piece of textile sample to be washed against all the slit openings. During the washing time, the board, test solution, textile, and caps vibrate violently, causing the test solution to contact the textile and apply mechanical stress in a regular, periodic oscillating manner. For further description, see WO 02 / 42740, especially the “Specific Method Embodiments” paragraph on pages 23-24.

[0357] Overall description of washing performance

[0358] A detergent solution for AMSA testing was prepared, comprising water (15°dH), 8.67 g / L of standard Z detergent composition, and the enzyme of the present invention at concentrations of 0, 0.1, or 0.2 mg / L enzyme protein. Fabrics with starch stains (CS-28 from the BV Center for Test Materials (mailbox 120, 3133KT, Vlardingen, Netherlands)) were added and washed at 55°C for 20 minutes. After thorough rinsing under running tap water and drying in the dark, the light intensity of the stained fabric was then measured as a measure of washing performance. A test with 0 mg / L enzyme protein served as a blank and corresponded to the contribution from the detergent composition. Mechanical action was applied during the washing step, for example by shaking, rotating, or agitating the detergent solution with the fabric. The AMSA washing performance test was conducted under the experimental conditions detailed below:

[0359] Table 1: Experimental Conditions

[0360] Detergent dosage 8.67g / L Test solution volume 160μL pH pH 10.4-10.5 Washing time 20 minutes temperature 55℃ water hardness 15°dH Enzyme concentration in the test 0.1 or 0.2 mg / L Test materials CS-28 (Rice Starch Cotton)

[0361] Table 2: Standard Detergents Containing Bleach (Z)

[0362] LAS, sodium salt 7.03 6.0 soap 1.08 1.0 AEO* 1.51 1.5 Sodium carbonate 20.10 20.0 Sodium silicate 9.99 8.0 Zeolite A 5.00 4.0 HEDP-Na4 0.24 0.2 Sodium citrate 2.01 2.0 Polycarboxylate (PCA) 1.09 1.0 Sodium percarbonate 9.33 8.0 TAED 1.09 1.0 <![CDATA[Na2SO4]]> 41.54 41.5

[0363] Add AEO separately before washing.

[0364] Note the balance up to 100% and the additional contributions to sodium carbonate and sodium sulfate. This balance is: water ≤ approximately 2.6% (from LAS approximately 0.1%; from sodium (di)silicate approximately 1.6%; from zeolite A approximately 0.8%; from polycarboxylate approximately 0.1%); sodium sulfate ≤ approximately 0.9%.

[0365] (From LAS, sodium salt may be about 0.8%; from zeolite A about 0.1%); sodium carbonate may be about 1.1% from sodium percarbonate; and CMC (carboxymethyl cellulose, sodium salt), about 0.2% from TAED.

[0366] By using CaCl2, MgCl2, and NaHCO3

[0367] (Ca 2+ :Mg 2+ :HCO 3 Add a mixture of 4:1:7.5 (4:1:7.5) to the testing system and adjust the water hardness to 15°dH. After washing, rinse the textiles with tap water and dry them.

[0368] Performance of standard detergent Z with bleach relative to parental α-amylase in AMSA, within 20 minutes, at 55°C.

[0369] blank -0.2 0.0 G182*+D183* 1.0 1.0 G182*+D183*+N195F 0.9 1.1 G182*+D183*+N195F+M202L 1.8 1.6

[0370] Specifically, these variations of the invention are considered particularly effective in ADW use. Therefore, to further evaluate this, AMSA testing of ADW performance can be performed. AMSA for ADW can be performed under the following conditions;

[0371] A test solution can be prepared comprising water (15°dH), 8.67 g / L detergent (e.g., a phosphate-containing liquid standard detergent as described below), and a variant of the invention at a concentration of 0 or 0.5 mg enzyme protein / L. Melamine plates with mixed starch stains (DM-177 from Test Material Center BV, Mailbox 120, 3133KT, Vlardingen, Netherlands) are added and washed at 55°C for 20 minutes. After a short rinse under running tap water and drying in the dark, the light intensity value of the stained plates is then measured as a measure of washing performance. A test with 0 mg enzyme protein / L can be used as a blank and corresponds to the contribution from the detergent. Preferably, mechanical action is applied during the washing step, for example, by shaking, rotating, or agitating the washing solution together with the plates. The AMSA automatic tableware washing performance test can be carried out under the experimental conditions detailed below:

[0372] Table 3: Experimental Conditions

[0373] Detergent dosage 5mL / L Test solution volume 160μL pH ～8 Washing time 20 minutes temperature 50℃ water hardness 21°dH Enzyme concentration in the test 0.01-0.24 mg / L Test materials Melamine board (mixed with starch)

[0374] Table 4: Standard Liquid Automatic Dishwashing Detergents Containing Phosphates

[0375] STPP 50.0 Sodium carbonate 20.0 Sodium percarbonate 10.0 Sodium disilicate 5.0 TAED 2.0 Sokalan CP5 (39.5%) 5.0 Surfac 23-6.5 (100%) 2.0 Sodium sulfate 6.0

[0376] By using CaCl2, MgCl2, and NaHCO3 (Ca 2+ :Mg 2+ :HCO3 - Add a 2:1:10 solution to the test system to adjust the water hardness to 21°dH. After washing, rinse the plate with tap water and dry it.

[0377] Table 5: Experimental Conditions

[0378] Detergent dosage 3.94g / L Test solution volume 160μL pH 9.9 Washing time 20 minutes temperature 50℃ water hardness 21°dH Enzyme concentration in the test 0.01-0.24 mg / L Test materials Melamine board (mixed with starch)

[0379] Table 6: Standard Detergent for Powdered Automatic Dishwashers

[0380] MGDA 28,9 20 Sodium citrate 17,1 20 Sodium carbonate 17,1 20 Sodium percarbonate 9,7 10 Sodium silicate 5,3 5 Sodium sulfate 10,2 12 Acusol 588G 4,6 5 TAED 2,8 3 Surfac 23-6.5 (liq) 4,3 5

[0381] Add *Surfac23-6.5 (liq) separately before washing.

[0382] By using CaCl2, MgCl2, and NaHCO3 (Ca 2+ :Mg 2+ :HCO3 - Add the solution (ratio 2:1:10) to the testing system and adjust the water hardness to 21°dH. After washing, rinse the plate with tap water and dry it.

[0383] Evaluation of washing performance

[0384] Washing performance can be measured as brightness, expressed as the intensity of light reflected from a sample when illuminated with white light. When a sample is contaminated, 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 washing performance.

[0385] Color measurements were performed using a professional flatbed scanner (EPSON EXPRESSION 10000XL) to capture images of the washed textiles, and a controlled digital imaging system (ColorVectorAnalyzer) to capture images of the washed melamine boards.

[0386] To extract light intensity values ​​from a scanned image, the values ​​from the image are converted into red, green, and blue (RGB) values. The intensity value (Int) can be calculated by summing the RGB values ​​as a vector and then considering the length of the resulting vector.

[0387]

[0388] Sequence Lists mentioned in this application

[0389]

[0390]

[0391]

[0392]

[0393]

[0394] The invention described and claimed herein is not limited to the specific aspects disclosed herein, as these aspects are intended to illustrate several aspects of the invention. Any equivalent aspects are contemplated to be within the scope of the invention. In fact, various modifications to the invention, other than those shown and described herein, will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In case of conflict, the disclosure including the definition shall prevail.

Claims

1. Use of an α-amylase variant of a parental α-amylase in a cleaning process, wherein the bleaching system is added at a concentration of at least 5% by weight, wherein the parent consists of the amino acid sequence shown in SEQ ID NO: 3, and wherein the mutation in the variant is G182. +D183 +N195F+M202L, when compared with the parental α-amylase, the improvement factor of the variant. > 1.0, this coefficient is used as a measure of washing performance.

2. The use according to claim 1, wherein the bleaching system is added at a concentration of at least 8% by weight.

3. The use according to claim 2, wherein the bleaching system is added at a concentration of at least 10% by weight.

4. The use according to claim 3, wherein the bleaching system is added at a concentration of at least 15% by weight.

5. A composition comprising the variant described in any one of claims 1-4 and a bleaching system with a concentration of at least 5% by weight.

6. The composition according to claim 5, wherein the composition is a detergent composition.

7. The composition according to any one of claims 5 and 6, wherein the composition further comprises one or more surfactants, one or more sulfonated polymers, one or more chelating agents, and / or one or more detergent builders.

8. The composition according to claim 6, wherein the detergent composition is a liquid laundry or liquid tableware washing composition, or a powdered laundry washing composition, or a powdered tableware washing composition.

9. The composition according to claim 8, wherein the liquid dishwashing composition is an automatic dishwashing (ADW) liquid detergent composition.

10. The composition according to claim 5, wherein the composition is in the form of a soap bar.

11. The composition according to claim 8, wherein the powdered dishwashing composition is an ADW detergent composition.

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