α-amylase variants

Abstract

To provide α-amylase exhibiting excellent starch degrading activity at a low temperature and excellent stability at low pH and / or in the presence of a chelating agent.SOLUTION: Provided is an α-amylase variant which is a variant of a parent α-amylase, the α-amylase variant including the amino acid residue substitution at a position corresponding to H238-position or E185-position in the amino acid sequence represented by SEQ ID NO:2 and the amino acid residue substitution mentioned in (A) or (B) below, in which the parent α-amylase or the α-amylase variant has at least 80% sequence identity to the amino acid sequence represented by SEQ ID NO:2 (excluding an α-amylase variant having amino acid residue substitutions at three positions corresponding to H238-position, S239-position and G178-position in the aforementioned amino acid sequence, an α-amylase variant having amino acid residue substitutions at three positions corresponding to H238-position, R209-position and G178-position in the aforementioned amino acid sequence and an α-amylase variant having amino acid residue substitutions at three positions corresponding to E185-position, N190-position and G178-position in the aforementioned amino acid sequence); (A) The amino acid residue substitution at at least one position selected from positions respectively corresponding to T116-position, A181-position, A199-position, A275-position, V277-position, A286-position and L323-position in the amino acid sequence represented by SEQ ID NO:2; and (B) the amino acid residue substitution at a position corresponding to G178-position in the amino acid sequence represented by SEQ ID NO:2 and the amino acid residue substitution at at least one position selected from N126-position, T129-position, N190-position, R209-position and S239-position in the aforementioned amino acid sequence.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to variants of α-amylase. [Background technology]

[0002] α-Amylase is used in a wide range of industrial fields, including the starch, brewing, textile, pharmaceutical, and food industries. It is also known for its suitability for incorporation into cleaning agents, and is incorporated into dishwashing detergents for automatic dishwashers and laundry detergents as an ingredient for removing starchy stains.

[0003] Known α-amylases useful for detergents include α-amylase AP1378 derived from Bacillus sp. KSM-1378 (FERM BP-3048) (Patent Document 1), α-amylases derived from Bacillus licheniformis such as Termamyl and Duramyl (registered trademark), α-amylase AA560 derived from Bacillus sp. DSM12649 (Patent Document 2), α-amylase SP722 derived from Bacillus sp. SP722 (SEQ ID NO: 4 in Patent Document 3), and α-amylase CspAmy2 derived from the genus Cytophaga (Patent Document 4). These α-amylases have also been modified to improve their function for specific applications, and mutants with improved stability in detergents, for example, have been reported (Patent Document 5).

[0004] In recent years, from the perspectives of environmental protection and reducing cleaning costs, lowering the temperature during dishwashing and laundry washing, especially in laundries, has become important, and shortening the washing time is also desired. However, the optimum temperature for most enzymes, including amylase, is higher than the temperature typically set for low-temperature washing, making it difficult to completely remove many starchy stains. Therefore, it is important to find an α-amylase that maintains its cleaning performance and starch-degrading activity even at low temperatures and has a high stain removal effect.

[0005] In recent years, it has also become known that washing in a low pH environment is gentler on items being washed and is not as strong as commonly used alkaline compositions, which can help keep items such as glass and patterned tableware looking new for longer, reduce unpleasant odors on fabrics, help release calcium soaps which tend to trap dirt on fabrics, improve performance against pH-sensitive stains, and even benefit the feel of fabrics. In addition, dishwashing detergents are used not only to clean the dishes that are the main target of cleaning, but also to clean the areas around stainless steel and plastic sinks, so they may contain high concentrations of chelating agents such as citric acid, which have cleaning power against limescale. Therefore, it can be said that detergents containing acids and / or chelating agents have various advantages in the cleaning process, and it is thought that adding enzymes such as α-amylase to such detergents can be expected to further improve their cleaning properties.

[0006] Patent Document 6 describes amylases that are stable in starch liquefaction processes under acidic conditions or in detergents containing chelating agents. Patent Document 7 describes the SP722 α-amylase variant that is stable in detergents containing chelating agents. Furthermore, a detergent composition containing α-amylase YR288, which retains its cleaning performance and starch-degrading activity even at low temperatures and has a high stain-removing effect, has also been reported (Patent Document 8). However, there remains a challenge in providing an α-amylase that is highly stable and can exert a high level of detergency in detergents containing acidic or chelating agents, particularly detergents containing both acidic and chelating agents. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 94 / 26881 [Patent Document 2] International Publication No. 00 / 60060 [Patent Document 3] International Publication No. 06 / 002643 [Patent Document 4] International Publication No. 2014 / 164777 [Patent Document 5] International Publication No. 98 / 044126 [Patent Document 6] Special Publication No. 2021-505167 [Patent Document 7] Patent No. 6185243 [Patent Document 8] International Publication No. 2022 / 017728 Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention relates to providing an α-amylase that exhibits excellent amylolytic activity at low temperatures and further exhibits excellent stability at low pH and / or in the presence of a chelating agent. [Means for solving the problem]

[0009] The inventors have succeeded in obtaining an α-amylase mutant that uses an α-amylase that functions at low temperatures as a parent and has improved stability at low pH and / or in the presence of a chelating agent compared to the parent α-amylase.

[0010] That is, the present invention relates to the following. 1) A mutant of a parent α-amylase comprising a substitution of an amino acid residue at a position corresponding to positions H238 or E185 of the amino acid sequence shown in SEQ ID NO: 2 and a substitution of an amino acid residue shown in (A) or (B) below, wherein the parent α-amylase or α-amylase mutant has at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 2 (excluding α-amylase mutants in which amino acid residues are substituted at three positions corresponding to positions H238, S239, and G178, three positions corresponding to positions H238, R209, and G178, and three positions corresponding to positions E185, N190, and G178 of the amino acid sequence). (A) Substitution of amino acid residues at one or more positions selected from positions corresponding to T116, A181, A199, A275, V277, A286, and L323 of the amino acid sequence shown in SEQ ID NO: 2 (B) Substitution of an amino acid residue at a position corresponding to position G178 of the amino acid sequence shown in SEQ ID NO: 2, and substitution of amino acid residues at one or more positions selected from positions corresponding to positions N126, T129, N190, R209, and S239 of the amino acid sequence shown in SEQ ID NO: 2. 2) A polynucleotide encoding a variant of 1). 3) A vector or DNA fragment containing the polynucleotide of 2). 4) A transformed cell containing the vector or DNA fragment of 3). 5) A cleaning composition comprising the variant of 1). [Effects of the Invention]

[0011] The present invention provides an α-amylase variant that has improved stability at low pH and / or in the presence of a chelating agent compared to the parent α-amylase, enabling cleaning that consistently removes starch stains even at low pH and in the presence of a chelating agent. DETAILED DESCRIPTION OF THE INVENTION

[0012] As used herein, the term "amylase" (EC 3.2.1.1; α-D-(1→4)-glucan glucanohydrolase) refers to a group of enzymes that catalyze the hydrolysis of starch and other linear or branched 1,4-glycosidic oligosaccharides or polysaccharides. α-Amylase activity can be determined by measuring the amount of reducing ends produced by enzymatic degradation of starch. It can also be determined by, but is not limited to, measuring the release of pigment from cross-linked starch, such as Phadebas (Soininen, K., M. Ceska, and H. Adlercreutz. "Comparison between a new chromogenic α-amylase test (Phadebas) and the Wohlgemuth amyloclastic method in urine." Scandinavian Journal of Clinical and Laboratory Investigation 30.3 (1972): 291-297).

[0013] Herein, the identity of an amino acid sequence or a nucleotide sequence is calculated by the Lipman-Pearson method (Science, 1985, 227:1435-1441). Specifically, it is calculated by performing analysis using the homology analysis (Search homology) program of the genetic information processing software GENETYX Ver. 12, with the unit size to compare (ktup) set to 2.

[0014] As used herein, the term "amino acid residue" refers to the 20 amino acid residues that make up proteins: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0015] In this specification, amino acid positions and variant descriptions are designated using the accepted IUPAC single-letter amino acid abbreviations as follows: The amino acid at a given position is designated as [amino acid, position], e.g., threonine at position 226 is designated as "T226." Amino acid "substitutions" are indicated as [original amino acid, position, substituted amino acid]. For example, a substitution of threonine at position 226 with alanine would be indicated as "T226A." Amino acid "deletions" are denoted as [original amino acid, position, Δ]. For example, a deletion of serine at position 181 is designated as "S181Δ". Variants containing multiple alterations are designated by a plus sign ("+"), e.g., "R170Y+G195E" represents a substitution of arginine at position 170 with tyrosine and a substitution of glycine at position 195 with glutamic acid, respectively. When different modifications can be introduced at one position, the different modifications are separated by a slash (" / "), for example, "R170Y / E" represents a substitution of arginine at position 170 with tyrosine or glutamic acid.

[0016] As used herein, the term "operably linked" between a gene and a regulatory region such as a promoter means that the gene and regulatory region are linked in such a way that the gene can be expressed under the control of the regulatory region. Procedures for "operably linking" a gene to a regulatory region are well known to those skilled in the art.

[0017] As used herein, "upstream" and "downstream" in relation to a gene refer to upstream and downstream in the transcription direction of the gene. For example, "a gene located downstream of a promoter" means that the gene is located on the 3' side of the promoter on the DNA sense strand, and "upstream" of a gene means the 5' region of the gene on the DNA sense strand.

[0018] As used herein, the term "native" when used with respect to a cellular function, property, or trait is used to indicate that the function, property, or trait is inherently present in the cell. In contrast, the term "exogenous" is used to indicate that the function, property, or trait is not inherently present in the cell but is introduced from outside. For example, an "exogenous" gene or polynucleotide is a gene or polynucleotide that is introduced into a cell from outside. An exogenous gene or polynucleotide may be derived from the same organism as the cell into which it is introduced, or from a different organism (i.e., a heterologous gene or polynucleotide).

[0019] <Mutant> The mutants of the present invention are mutants of a parent α-amylase comprising a substitution of an amino acid residue at a position corresponding to positions H238 or E185 of the amino acid sequence shown in SEQ ID NO: 2 and a substitution of an amino acid residue shown in (A) or (B) below, wherein the parent α-amylase or α-amylase mutant has at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 2. However, α-amylase mutants in which amino acid residues are substituted at three positions corresponding to positions H238, S239, and G178, three positions corresponding to positions H238, R209, and G178, and three positions corresponding to positions E185, N190, and G178 of the amino acid sequence are excluded. (A) Substitution of amino acid residues at one or more positions selected from positions corresponding to T116, A181, A199, A275, V277, A286, and L323 of the amino acid sequence shown in SEQ ID NO: 2 (B) Substitution of an amino acid residue at a position corresponding to position G178 of the amino acid sequence shown in SEQ ID NO: 2, and substitution of amino acid residues at one or more positions selected from positions corresponding to positions N126, T129, N190, R209, and S239 of the amino acid sequence shown in SEQ ID NO: 2.

[0020] That is, a "variant" refers to a polypeptide having α-amylase activity in which an amino acid residue at a position corresponding to H238 or E185 of the amino acid sequence set forth in SEQ ID NO: 2 in the parent α-amylase has been substituted in combination with an amino acid residue substitution set forth in (A) or (B) above. The substitution of amino acid residues at such predetermined positions is a modification intended to improve stability at low pH and / or in the presence of a chelating agent, and therefore the variant has improved stability at low pH and / or in the presence of a chelating agent compared to the parent α-amylase.

[0021] "Parent α-amylase" refers to a reference α-amylase that is modified to yield a variant of the invention, which in the present invention is an α-amylase that has at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 2. The parent may be a naturally occurring (wild-type) polypeptide or a variant thereof. Such parent α-amylases include those that preferably have at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:2.

[0022] Here, the α-amylase consisting of the amino acid sequence shown in SEQ ID NO: 2 is an α-amylase in which the amino acid residues corresponding to R178 and T180 are deleted (R178Δ + T180Δ) in the amino acid sequence (SEQ ID NO: 4) constituting α-amylase YR288 (International Publication No. 2022 / 017728), which is registered as WP_100346362.1 in the NCBI protein sequence database. Such α-amylase has dramatically improved stability in detergents compared to YR288 (Patent Application No. 2021-135746). Therefore, any α-amylase mutant, including an α-amylase consisting of the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identity thereto, in which two or more amino acid residues selected from positions corresponding to R178, G179, T180 and G181 of the amino acid sequence of SEQ ID NO: 4 have been deleted, can serve as the parent α-amylase for the mutant of the present invention. Here, preferred deletions of two or more amino acid residues include R178Δ+T180Δ, G179Δ+T180Δ, R178Δ+G179Δ, R178Δ+G181Δ, G179Δ+G181Δ, etc., with R178Δ+T180Δ being more preferred. The mutation positions in the α-amylase mutants of the present invention are numbered based on the amino acid numbers in the amino acid sequence shown in SEQ ID NO:2. Other α-amylases with an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 2 include DE0178, an α-amylase derived from Bacillus flexus, and RU2C, an α-amylase derived from Bacillus sp. (Patent Application No. 2020-121626).

[0023] In the present invention, a "corresponding position" on an amino acid sequence can be determined by aligning a target sequence with a reference sequence (the amino acid sequence shown in SEQ ID NO: 2 in the present invention) to maximize homology. Alignment of amino acid sequences can be performed using known algorithms, and the procedures are well known to those skilled in the art. For example, alignment can be performed using the Clustal W multiple alignment program (Thompson, J.D. et al., 1994, Nucleic Acids Res. 22:4673-4680) with default settings. Alternatively, Clustal W2 or Clustal omega, which are revised versions of Clustal W, can also be used. Clustal W, Clustal W2, and Clustal omega are available, for example, on the websites of the European Bioinformatics Institute (EBI [www.ebi.ac.uk / index.html]) and the DNA Data Bank of Japan (DDBJ [www.ddbj.nig.ac.jp / searches-j.html]), operated by the National Institute of Genetics. The position of the target sequence aligned to any position in the reference sequence by the above-mentioned alignment is considered to be the "position corresponding to" that position.

[0024] Those skilled in the art can further fine-tune the alignment of amino acid sequences obtained above to optimize it. Such optimal alignment is preferably determined taking into account the similarity of the amino acid sequences, the frequency of inserted gaps, and other factors. Here, amino acid sequence similarity refers to the percentage (%) of the number of positions at which identical or similar amino acid residues exist in both aligned amino acid sequences relative to the total number of amino acid residues in the two sequences. Similar amino acid residues refer to amino acid residues among the 20 amino acids that constitute proteins that have similar properties in terms of polarity and charge, resulting in so-called conservative substitutions. Groups of such similar amino acid residues are well known to those skilled in the art, and include, but are not limited to, arginine and lysine or glutamine; glutamic acid and aspartic acid or glutamine; serine and threonine or alanine; glutamine and asparagine or arginine; and leucine and isoleucine.

[0025] The "substitution" of an amino acid residue at a given position means replacing the amino acid at a certain position with a different amino acid. In the present invention, the substitution sites of amino acid residues may be two or more, but from the viewpoint of stability at low pH and / or in the presence of a chelating agent, the substitution sites are preferably three or more, more preferably 2 to 20, and even more preferably 3 to 19. In addition, from the standpoint of improving cleaning performance and stability at low pH and / or in the presence of a chelating agent, it is preferable that the mutant is an α-amylase that has at least 80%, preferably at least 85%, more preferably at least 90%, preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% identity to the amino acid sequence shown in SEQ ID NO: 2. Furthermore, a mutant may contain any number of conservative amino acid substitutions, so long as the above-mentioned properties of the mutant are maintained.

[0026] The substitution of amino acid residues shown in (A) is at one or more positions selected from positions corresponding to T116, A181, A199, A275, V277, A286, and L323 of the amino acid sequence shown in SEQ ID NO: 2. From the viewpoint of stability at low pH and / or in the presence of a chelating agent, substitutions are preferably made at two or more positions, more preferably at three to six positions. Furthermore, in addition to substituting amino acid residues at one or more positions selected from positions corresponding to the above-mentioned T116, A181, A199, A275, V277, A286, and L323, it is preferable to further combine substitutions of amino acid residues at one or more, preferably two or more, more preferably five or more, and more preferably eight or more positions selected from positions corresponding to each of N126, T129, N190, R209, S239, G178, M197, F203, Y240, E255, Y358, W406, and G474, in terms of improving cleaning performance and stability at low pH and / or in the presence of a chelating agent.

[0027] The substitution of amino acid residues shown in (B) is a substitution of an amino acid residue at a position corresponding to position G178 of the amino acid sequence shown in SEQ ID NO: 2, and a substitution of amino acid residues at one or more positions selected from positions corresponding to positions N126, T129, N190, R209, and S239 of the same amino acid sequence.

[0028] Preferred embodiments of substitutions of amino acid residues at positions corresponding to the above-mentioned H238, E185, T116, A181, A199, A275, V277, A286, L323, G178, N126, T129, N190, R209, S239, M197, F203, Y240, E255, Y358, W406, and G474 in the α-amylase mutant of the present invention are shown below. That is, H238 is preferably substituted with F (H238F); E185 is preferably substituted with P (E185P); T116 is preferably substituted with Q (T116Q); A181 is preferably substituted with T, E or Q (A181T / E / Q); A199 is preferably substituted with E (A199E); A275 is preferably substituted with N (A275N); V277 is preferably substituted with T (V277T); A286 is preferably substituted with V (A286V); L323 is preferably substituted with F (L323F); G178 is preferably substituted with H (G178H); N126 is preferably substituted with Y (N126Y); T129 is preferably substituted with I (T129I); N190 is preferably substituted with F (N190F); R209 is preferably substituted with L, V or I (R209L / V / I); S239 is preferably substituted with A, Q, D, L, Y, P or H (S239A / Q / D / L / Y / P / H); M197 is preferably substituted with L, T, A, N, Q, S, V or I (M197L / T / A / N / Q / S / V / I); F203 is preferably substituted with Y (F203Y); Y240 is preferably substituted with F (Y240F); E255 is preferably replaced by T (E255T); Y358 is preferably substituted with C, M, L or V (Y358C / M / L / V); W406 is preferably substituted with P (W406P); G474 is preferably substituted with A, P, E, S, F, R or K (G474A / P / E / S / F / R / K).

[0029] Next, suitable combinations of mutations that contribute to improved stability at low pH and / or in the presence of a chelating agent are shown in Table 1 (Tables 1-1 to 1-3). Therefore, a mutant having at least this combination of mutations is a mutant that particularly contributes to improved stability at low pH and / or in the presence of a chelating agent.

[0030] [Table 1]

[0031] Next, more suitable combinations of mutations that contribute to improved stability at low pH and / or in the presence of a chelating agent are shown in Table 2. Therefore, a mutant having at least this combination of mutations is a mutant that particularly contributes to improved stability at low pH and / or in the presence of a chelating agent.

[0032] [Table 2]

[0033] <Polynucleotides encoding the mutants of the present invention> The mutants of the present invention can be produced using various mutagenesis techniques known in the art, for example, by mutating a polynucleotide encoding the amino acid residues to be modified in a parent α-amylase gene (reference α-amylase gene) that encodes the reference amino acid sequence into a polynucleotide encoding the modified amino acid residue, and then expressing the mutant from the mutated gene.

[0034] The polynucleotide encoding the variant of the present invention may be in the form of single- or double-stranded DNA, RNA, or an artificial nucleic acid, or may be cDNA, or chemically synthesized DNA containing no introns.

[0035] In the present invention, various mutagenesis techniques known in the art can be used to mutate amino acid residues in a parent α-amylase. For example, a polynucleotide encoding a mutant of the present invention can be obtained by mutating a nucleotide sequence encoding the amino acid residue to be mutated in a polynucleotide encoding the amino acid sequence of the parent α-amylase (hereinafter also referred to as the parent gene) to a nucleotide sequence encoding the mutated amino acid residue.

[0036] Introduction of a desired mutation into a parent gene can basically be carried out using various site-directed mutagenesis methods well known to those skilled in the art. Site-directed mutagenesis can be carried out by any method, such as inverse PCR or annealing. Commercially available site-directed mutagenesis kits (e.g., Stratagene's QuickChange II Site-Directed Mutagenesis Kit or QuickChange Multi Site-Directed Mutagenesis Kit) can also be used.

[0037] Site-directed mutagenesis of a parent gene is most commonly performed using a mutagenesis primer containing the nucleotide mutation to be introduced. The mutagenesis primer is designed to anneal to a region of the parent gene containing a nucleotide sequence encoding the amino acid residue to be mutated, and to contain a nucleotide sequence containing a nucleotide sequence (codon) encoding the mutated amino acid residue in place of the nucleotide sequence (codon) encoding the amino acid residue to be mutated. Those skilled in the art can appropriately recognize and select the nucleotide sequences (codons) encoding the pre- and post-mutation amino acid residues based on standard textbooks. Alternatively, site-directed mutagenesis can be performed using two complementary primers containing the nucleotide mutation to be introduced, each amplifying DNA fragments upstream and downstream of the mutation site, and then joining the resulting fragments together using splicing by overlap extension (SOE)-PCR (Gene, 1989, 77(1): pp. 61-68).

[0038] Template DNA containing the parent gene can be prepared from the above-mentioned α-amylase-producing microorganism by extracting genomic DNA using standard methods, or by extracting RNA and synthesizing cDNA by reverse transcription. Alternatively, a corresponding nucleotide sequence can be chemically synthesized based on the amino acid sequence of the parent α-amylase and used as template DNA. The DNA sequence containing the base sequence encoding the α-amylase consisting of the amino acid sequence shown in SEQ ID NO:2 is shown in SEQ ID NO:1, and the DNA sequence containing the base sequence encoding the α-amylase (YR288) consisting of the amino acid sequence shown in SEQ ID NO:4 is shown in SEQ ID NO:3.

[0039] Mutation primers can be prepared by well-known oligonucleotide synthesis methods, such as the phosphoramidite method (Nucleic Acids Research, 1989, 17:7059-7071). Such primer synthesis can also be performed using, for example, a commercially available oligonucleotide synthesizer (such as that manufactured by ABI). Using a primer set containing the mutation primers, site-specific mutagenesis as described above can be performed using a parent gene as template DNA to obtain a polynucleotide encoding the mutant of the present invention having the desired mutation.

[0040] The polynucleotide encoding the mutant of the present invention may comprise single-stranded or double-stranded DNA, cDNA, RNA, or other artificial nucleic acids. The DNA, cDNA, and RNA may be chemically synthesized. The polynucleotide may also comprise a nucleotide sequence of an untranslated region (UTR) in addition to an open reading frame (ORF). The polynucleotide may also be codon-optimized for the species of the transformant used to produce the mutant polypeptide of the present invention. Information on codons used by various organisms is available from the Codon Usage Database ([www.kazusa.or.jp / codon / ]).

[0041] <Vector or DNA fragment> The obtained polynucleotide encoding the mutant of the present invention can be incorporated into a vector. The type of vector containing the polynucleotide is not particularly limited and may be any vector, such as a plasmid, phage, phagemid, cosmid, virus, YAC vector, or shuttle vector. The vector is preferably, but not limited to, a vector that can be amplified in bacteria, preferably in Bacillus bacteria (e.g., Bacillus subtilis or a mutant thereof), and more preferably an expression vector that can induce expression of an introduced gene in Bacillus bacteria. In particular, shuttle vectors, which are vectors that can replicate in both Bacillus bacteria and other organisms, are suitable for use in recombinantly producing the mutant of the present invention. Preferred examples of vectors include, but are not limited to, shuttle vectors such as pHA3040SP64, pHSP64R, or pASP64 (Japanese Patent No. 3492935), pHY300PLK (an expression vector capable of transforming both Escherichia coli and Bacillus subtilis; Jpn J Genet, 1985, 60:235-243), and pAC3 (Nucleic Acids Res, 1988, 16:8732); and plasmid vectors that can be used to transform Bacillus bacteria, such as pUB110 (J Bacteriol, 1978, 134:318-329) and pTA10607 (Plasmid, 1987, 18:8-15). Plasmid vectors derived from Escherichia coli (e.g., pET22b(+), pBR322, pBR325, pUC57, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.) can also be used.

[0042] The vector may contain a DNA replication origin region or a DNA region containing a replication origin. Alternatively, the vector may have a control sequence, such as a promoter region for initiating transcription of the gene, a terminator region, or a secretion signal region for secreting the expressed protein extracellularly, operably linked upstream of the polynucleotide encoding the mutant of the present invention (i.e., the mutant gene). Note that "operably linked" between a gene and a control sequence means that the gene and the control region are positioned so that the gene can be expressed under the control of the control region.

[0043] The types of the control sequences such as the promoter region, terminator, and secretion signal region are not particularly limited, and commonly used promoters and secretion signal sequences can be appropriately selected and used depending on the host to be introduced into. For example, suitable examples of control sequences that can be incorporated into a vector include the promoter and secretion signal sequence of the cellulase gene of Bacillus sp. KSM-S237 strain.

[0044] Alternatively, the vector of the present invention may further incorporate a marker gene (e.g., a resistance gene to a drug such as ampicillin, neomycin, kanamycin, or chloramphenicol) for selecting a host into which the vector has been appropriately introduced. Alternatively, when an auxotrophic strain is used as the host, a gene encoding an enzyme that synthesizes the required nutrient may be incorporated into the vector as a marker gene. Furthermore, when a selective medium requiring a specific metabolism for growth is used, a gene related to that metabolism may be incorporated into the vector as a marker gene. An example of such a metabolism-related gene is the acetamidase gene for utilizing acetamide as a nitrogen source.

[0045] The polynucleotide encoding the mutant of the present invention can be ligated to a regulatory sequence and a marker gene by methods known in the art, such as splicing by overlap extension (SOE)-PCR (Gene, 1989, 77:61-68). Procedures for introducing the ligated fragment into a vector are well known in the art.

[0046] <Transformed cells> The transformed cell of the present invention can be obtained by introducing a vector containing a polynucleotide encoding the mutant of the present invention into a host, or by introducing a DNA fragment containing a polynucleotide encoding the mutant of the present invention into the genome of the host.

[0047] Host cells include microorganisms such as bacteria and filamentous fungi. Examples of bacteria include Escherichia coli, Staphylococcus, Enterococcus, Listeria, and Bacillus bacteria, among which Escherichia coli and Bacillus bacteria (e.g., Bacillus subtilis Marburg No. 168 (Bacillus subtilis 168 strain) or mutants thereof) are preferred. Examples of Bacillus mutant strains include the KA8AX protease 9-fold deletion strain described in J. Biosci. Bioeng., 2007, 104(2):135-143, and the D8PA strain, an octaprotease deletion strain with improved protein folding efficiency described in Biotechnol. Lett., 2011, 33(9):1847-1852. Examples of filamentous fungi include the genera Trichoderma, Aspergillus, and Rhizopus.

[0048] The vector can be introduced into the host by a method commonly used in the field, such as the protoplast method, electroporation, etc. Strains into which the vector has been appropriately introduced can be selected based on the expression of a marker gene, auxotrophy, etc., to obtain the desired transformant into which the vector has been introduced.

[0049] Alternatively, a fragment comprising a polynucleotide encoding a mutant of the present invention, a regulatory sequence, and a marker gene can be directly introduced into the genome of a host. For example, a DNA fragment in which sequences complementary to the host genome are added to both ends of the above-mentioned ligated fragment using SOE-PCR or other methods can be constructed, and this can be introduced into a host to induce homologous recombination between the host genome and the DNA fragment, thereby introducing the polynucleotide encoding the mutant of the present invention into the genome of the host.

[0050] When the thus obtained transformant into which a polynucleotide encoding the mutant of the present invention or a vector containing the same has been introduced is cultured in an appropriate medium, the gene encoding the protein on the vector is expressed to produce the mutant of the present invention. The medium used to culture the transformant can be appropriately selected by those skilled in the art depending on the type of microorganism used as the transformant.

[0051] Alternatively, the mutant of the present invention may be expressed from a polynucleotide encoding the mutant of the present invention or a transcription product thereof using a cell-free translation system. The "cell-free translation system" is an in vitro transcription / translation system or an in vitro translation system constructed by adding reagents such as amino acids necessary for protein translation to a suspension obtained by mechanically disrupting host cells.

[0052] The mutants of the present invention produced in the above-mentioned culture or cell-free translation system can be isolated or purified by common protein purification methods, such as centrifugation, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc., either alone or in combination. In this case, if a gene encoding the α-amylase mutant of the present invention is operably linked to a secretory signal sequence on a vector in the transformant, the produced protein will be secreted outside the cell and can be more easily recovered from the culture. The protein recovered from the culture may be further purified by known means.

[0053] The resulting variants of the present invention have improved stability at low pH and / or in the presence of chelating agents compared to the parent α-amylase. "Improved stability at low pH and / or in the presence of a chelating agent" refers to an improved ability to maintain α-amylase activity in a low pH environment and / or in the presence of a chelating agent compared to the parent α-amylase. Here, low pH refers to weak acidity, i.e., pH 4 to 7, preferably pH 4.5 to 6.5. Stability in a low pH environment means that α-amylase activity is maintained in a detergent composition having a pH of about 4 to 7, and in wash water having a pH of about 4 to 7 prepared by dissolving or diluting the detergent composition in water.

[0054] Examples of the chelating agent include chelating agents that can be incorporated into the detergent composition, such as aminocarboxylic acid chelating agents, phosphonic acid chelating agents, hydroxycarboxylic acid chelating agents, and polycarboxylic acid chelating agents, which will be described later.

[0055] The stability at such low pH and / or in the presence of a chelating agent can be evaluated using methods well known in the art. For example, an enzyme solution is added to a cleaning composition containing a chelating agent and having a pH of 4 to 7, and α-amylase activity is measured before and after treatment for a predetermined period of time. The activity value of the sample before treatment is defined as the initial activity, and the ratio of the activity value after treatment to the initial activity is defined as the residual activity (%).

[0056] The mutant of the present invention is useful as an enzyme to be incorporated into various detergent compositions, and is particularly useful as an enzyme to be incorporated into detergent compositions that are suitable for low-temperature washing at 40°C or less and that are acidic and contain a chelating agent.

[0057] The amount of the mutant of the present invention to be incorporated into a detergent composition is not particularly limited as long as the protein exhibits activity, but is, for example, preferably 1 mg or more, more preferably 10 mg or more, more preferably 50 mg or more, and preferably 5,000 mg or less, more preferably 1,000 mg or less, more preferably 500 mg or less, per 1 kg of detergent composition. The amount is also preferably 1 to 5,000 mg, more preferably 10 to 1,000 mg, and more preferably 50 to 500 mg.

[0058] The detergent composition can also contain various enzymes in addition to the mutant of the present invention. Examples include hydrolases, oxidases, reductases, transferases, lyases, isomerases, ligases, synthetases, etc. Among these, amylases other than the protein of the present invention, proteases, cellulases, keratinases, esterases, cutinases, lipases, pullulanases, pectinases, mannanases, glucosidases, glucanases, cholesterol oxidases, peroxidases, laccases, etc. are preferred, with proteases, cellulases, amylases, and lipases being particularly preferred. Examples of commercially available proteases include Alcalase, Esperase, Everlase, Savinase, Kannase, Progress Uno (registered trademark; Novozymes), PREFERENZ, EFFECTENZ, EXCELLENZ (registered trademark; DuPont), Lavergy (registered trademark; BASF), and KAP (Kao). Examples of cellulases include Celluclean, Carezyme (registered trademark; Novozymes), KAC, alkaline cellulase produced by Bacillus sp. KSM-S237 strain described in JP-A-10-313859, and mutant alkaline cellulase described in JP-A-2003-313592 (all Kao products). Examples of amylases include Termamyl, Duramyl, Stainzyme, Stainzyme Plus, Amplify Prime (registered trademark; Novozymes), PREFERENZ, EFFECTENZ (registered trademark; DuPont), and KAM (Kao). Examples of lipase include Lipolase and Lipex (registered trademark; Novozymes).

[0059] The detergent composition may contain known detergent components, and examples of such known detergent components include the following:

[0060] (1) Surfactants The surfactant is blended in the detergent composition in an amount of 0.5 to 60% by mass, preferably 10 to 45% by mass for powder detergent compositions and 20 to 90% by mass for liquid detergent compositions. When the detergent composition of the present invention is a laundry detergent or an automatic dishwasher detergent, the surfactant is blended in an amount of generally 1 to 10% by mass, preferably 1 to 5% by mass.

[0061] The surfactant used in the detergent composition may be one or a combination of anionic surfactants, nonionic surfactants, amphoteric surfactants, and cationic surfactants, with anionic surfactants and nonionic surfactants being preferred.

[0062] Preferred anionic surfactants include sulfate salts of alcohols having 10 to 18 carbon atoms, sulfate salts of alkoxylated alcohols having 8 to 20 carbon atoms, alkylbenzenesulfonates, paraffin sulfonates, α-olefinsulfonates, internal olefinsulfonates, α-sulfofatty acid salts, α-sulfofatty acid alkyl ester salts, and fatty acid salts. In the present invention, one or more anionic surfactants selected from linear alkylbenzenesulfonates having an alkyl chain of 10 to 14 carbon atoms, more preferably 12 to 14 carbon atoms, and internal olefinsulfones having an alkylene chain of 12 to 20 carbon atoms, more preferably 16 to 18 carbon atoms, are particularly preferred. The counter ion is preferably an alkali metal salt or an amine, particularly preferably sodium and / or potassium, monoethanolamine, or diethanolamine. For internal olefinsulfonic acids, see, for example, WO2017 / 098637.

[0063] Preferred nonionic surfactants include polyoxyalkylene alkyl (C8-20) ethers, alkyl polyglycosides, polyoxyalkylene alkyl (C8-20) phenyl ethers, polyoxyalkylene sorbitan fatty acid (C8-22) esters, polyoxyalkylene glycol fatty acid (C8-22) esters, and polyoxyethylene polyoxypropylene block polymers. Particularly preferred nonionic surfactants are polyoxyalkylene alkyl ethers in which 4 to 20 moles of alkylene oxide, such as ethylene oxide or propylene oxide, are added to alcohols having 10 to 18 carbon atoms (thus having an HLB value (calculated by the Griffin method) of 10.5 to 15.0, preferably 11.0 to 14.5).

[0064] (2) Divalent metal ion scavenger The divalent metal ion scavenger is blended in an amount of 0.01 to 50 mass%, preferably 5 to 40 mass%. Examples of divalent metal ion scavenger agents used in the detergent composition of the present invention include condensed phosphates such as tripolyphosphates, pyrophosphates, and orthophosphates, aluminosilicates such as zeolites, synthetic layered crystalline silicates, nitrilotriacetates, ethylenediaminetetraacetates, citrates, isocitrates, and polyacetalcarboxylates. Among these, crystalline aluminosilicates (synthetic zeolites) are particularly preferred, with A-type zeolites being particularly preferred among A-, X-, and P-type zeolites. Synthetic zeolites with an average primary particle size of 0.1 to 10 μm, and particularly 0.1 to 5 μm, are preferably used.

[0065] (3) Alkaline agent The alkaline agent is blended in an amount of 0.01 to 80% by mass, preferably 1 to 40% by mass. In the case of powder detergents, examples include alkali metal carbonates such as sodium carbonate, collectively known as dense ash or light ash, and amorphous alkali metal silicates such as JIS No. 1, No. 2, and No. 3. These inorganic alkaline agents are effective in forming the particle skeleton during detergent drying, resulting in a detergent that is relatively hard and has excellent fluidity. Other examples of alkalis include sodium sesquicarbonate and sodium bicarbonate, and phosphates such as tripolyphosphates also function as alkaline agents. In addition to the above alkaline agents, sodium hydroxide and mono-, di-, or triethanolamine can also be used as alkaline agents in liquid detergents, and they can also be used as counterions for the active agent.

[0066] (4) Anti-redeposition agent The anti-redeposition agent is blended in an amount of 0.001 to 10% by mass, preferably 1 to 5% by mass. Examples of anti-redeposition agents used in the detergent composition of the present invention include polyethylene glycol, carboxylic acid polymers, polyvinyl alcohol, and polyvinylpyrrolidone. Among these, carboxylic acid polymers have the ability to prevent redeposition, as well as the ability to capture metal ions and disperse solid particle soils from clothing into the wash bath. The carboxylic acid polymer is a homopolymer or copolymer of acrylic acid, methacrylic acid, itaconic acid, or the like. Suitable copolymers are copolymers of the above monomers with maleic acid, and preferably have a molecular weight of several thousand to 100,000. In addition to the above carboxylic acid polymers, polymers such as polyglycidyl acid salts, cellulose derivatives such as carboxymethylcellulose, and aminocarboxylic acid polymers such as polyaspartic acid are also preferred because they have the ability to capture metal ions, disperse, and prevent redeposition.

[0067] (5) Bleach For example, bleaching agents such as hydrogen peroxide and percarbonate are preferably blended in an amount of 1 to 10% by mass. When using bleaching agents, tetraacetylethylenediamine (TAED) or a bleaching activator such as that described in JP-A-6-316700 can be blended in an amount of 0.01 to 10% by mass.

[0068] (6) Fluorescent agent Fluorescent agents used in the detergent composition include biphenyl-type fluorescent agents (such as Tinopal CBS-X) and stilbene-type fluorescent agents (such as DM-type fluorescent dyes). The fluorescent agent is preferably blended in an amount of 0.001 to 2% by mass.

[0069] (7) Chelating agents The chelating agent is added, for example, to facilitate cleaning of stains and to reduce water hardness during cleaning. Examples of such chelating agents include aminocarboxylic acid chelating agents, phosphonic acid chelating agents, hydroxycarboxylic acid chelating agents, and polycarboxylic acid chelating agents. Examples of aminocarboxylic acid chelating agents include ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), hydroxyethylethylenediaminetriacetic acid (HEDTA), diethylenetriaminepentaacetic acid (DTPA), methylglycine diacetic acid (MGDA), triethylenetetraminehexaacetic acid (TTHA), glutamic acid diacetic acid (GLDA), hydroxyethyliminodiacetic acid (HIDA), dihydroxyethylglycine (DHEG), aspartic acid diacetic acid (ASDA), ethylenediamine succinic acid (EDDS), and salts thereof. Examples of phosphonic acid chelating agents include hydroxyethylidene diphosphonic acid (HEDP), nitrilotrismethylenephosphonic acid (NTMP), phosphonobutanetricarboxylic acid (PBTC), ethylenediaminetetramethylenephosphonic acid (EDTMP), and salts thereof. Examples of the hydroxycarboxylic acid chelating agent include citric acid, malic acid, tartaric acid, gluconic acid, lactic acid, and salts thereof. Examples of polycarboxylic acid chelating agents include succinic acid, oxalic acid, glutaric acid, adipic acid, fumaric acid, malonic acid, and salts thereof.

[0070] (8) Buffer The buffering agent is formulated to create a buffer system capable of maintaining a low pH in the wash solution, and can include a mixture of an organic acid and its salt, such as a mixture of a polycarboxylic acid and its salt, preferably a mixture of citric acid and citrate.

[0071] (9) Other ingredients The detergent composition may contain builders, softeners, reducing agents (such as sulfites), foam inhibitors (such as silicones), fragrances, antibacterial and antifungal agents (such as Proxel (trade name) and benzoic acid), and other additives known in the field of laundry detergents.

[0072] The detergent composition can be produced by combining the protein of the present invention obtained by the above method with the above-mentioned known detergent components according to a conventional method. The form of the detergent can be selected depending on the application, and can be, for example, a liquid, powder, granule, paste, solid, etc.

[0073] The detergent composition thus obtained can be used as a clothing detergent, dish detergent, bleach, detergent for cleaning hard surfaces, drain cleaner, denture cleaner, disinfectant cleaner for medical instruments, etc., but is preferably used as a clothing detergent or dish detergent, and more preferably used as a laundry detergent (laundry laundry detergent), dish detergent for hand washing, or detergent for automatic dishwashers. The cleaning composition is also suitable for use at low temperatures (for example, 40°C or lower, 35°C or lower, 30°C or lower, or 25°C or lower, and 5°C or higher, 10°C or higher, or 15°C or higher).

[0074] In relation to the above-described embodiment, the present invention further discloses the following aspects. <1> An α-amylase variant of a parent α-amylase comprising a substitution of an amino acid residue at a position corresponding to positions H238 or E185 of the amino acid sequence shown in SEQ ID NO: 2 and a substitution of an amino acid residue shown in (A) or (B) below, wherein the parent α-amylase or α-amylase variant has at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 2 (excluding α-amylase variants in which amino acid residues are substituted at three positions corresponding to positions H238, S239 and G178, three positions corresponding to positions H238, R209 and G178, and three positions corresponding to positions E185, N190 and G178 of the amino acid sequence). (A) Substitution of amino acid residues at one or more positions selected from positions corresponding to T116, A181, A199, A275, V277, A286, and L323 of the amino acid sequence shown in SEQ ID NO: 2 (B) Substitution of an amino acid residue at a position corresponding to position G178 of the amino acid sequence shown in SEQ ID NO: 2, and substitution of amino acid residues at one or more positions selected from positions corresponding to positions N126, T129, N190, R209, and S239 of the amino acid sequence shown in SEQ ID NO: 2. <2> The substitution of amino acid residues shown in (A) further includes substitution of amino acid residues at one or more positions selected from the positions corresponding to N126, T129, N190, R209, S239, G178, M197, F203, Y240, E255, Y358, W406, and G474 of the amino acid sequence shown in SEQ ID NO: 2. <1> A mutant of. <3> (A) comprising substitution of amino acid residues at two or more positions selected from positions corresponding to T116, A181, A199, A275, V277, A286, and L323 of the amino acid sequence shown in SEQ ID NO: 2, <1> or <2> A mutant of. <4> the substitutions of amino acid residues at positions corresponding to H238, E185, T116, A181, A199, A275, V277, A286, L323, G178, N126, T129, N190, R209 and S239 are H238F, E185P, T116Q, A181T / E / Q, A199E, A275N, V277T, A286V, L323F, G178H, N126Y, T129I, N190F, R209V / L / I, S239A / Q / D / L / Y / P / H, respectively; <1> ~ <3> Any variant of. <5> The mutant contains at least a mutation selected from the combinations of mutations shown in Table 1-1, Table 1-2, and Table 2. <1> A mutant of. <6> <1> ~ <5> A polynucleotide encoding any variant of any of the above. <7> <6> A vector or DNA fragment comprising the polynucleotide of <8> <7> A transformed cell containing the vector or DNA fragment. <9> It is a microorganism, <8> Transformed cells. <10> <1> ~ <5> A cleaning composition comprising a variant of <11> A laundry detergent or dishwashing detergent, <10> A cleaning composition comprising: <12> It is a powder or a liquid, <11> A cleaning composition comprising: <13> Weakly acidic, preferably pH 4 to 7; <11> or <12> A cleaning composition comprising: <14> Contains chelating agents, <11> ~ <13> Any one of the cleaning compositions described above. <15> The chelating agent is an aminocarboxylic acid chelating agent, a phosphonic acid chelating agent, a hydroxycarboxylic acid chelating agent, or a polycarboxylic acid chelating agent. <14> A cleaning composition comprising: [Example]

[0075] (1) Construction of YR288 mutant expression plasmid The construction method of the YR288 mutant described in the following examples is described. A forward primer containing the mutant sequence and having 15 bases of a sequence complementary to the reverse primer at the 5' end, and a reverse primer with the base immediately preceding the mutant sequence at the 5' end, were used as the primer pair for mutagenesis. PCR was performed using the YR288 R178Δ T180Δ expression plasmid described in the examples of JP 2022-019601 A or the YR288 mutant expression plasmid prepared in this example as a template, and the primer pair for mutagenesis. When multiple fragments were ligated, an In-Fusion reaction was performed using each PCR product according to the protocol of the In-Fusion, HD Cloning kit (Clontech). The PCR product or In-Fusion reaction solution was transformed into Bacillus subtilis by the protoplast method to obtain transformants harboring the desired YR288 mutant expression plasmid.

[0076] (2) Enzyme production culture The recombinant B. subtilis colonies obtained in (1) were inoculated into 500 μL of LB medium supplemented with 10 ppm tetracycline in a 96-well deep-well plate and cultured overnight at 32 °C and 1500 rpm. The next day, 20 μL of the culture was inoculated into 500 μL of 2x L-maltose medium (2% tryptone, 1% yeast extract, 1% NaCl, 7.5% maltose, 7.5 ppm manganese sulfate pentahydrate, 0.04% calcium chloride dihydrate, 10 ppm tetracycline; % is (w / v)%) in a 96-well deep-well plate and cultured at 32 °C and 1500 rpm for 2 days. The culture supernatant containing the enzyme produced by the bacteria was collected by centrifugation and used as the enzyme solution.

[0077] (3) Protein concentration measurement of culture supernatant The protein concentration of the culture supernatant was measured using the Protein Assay Rapid Kit Wako II (Fujifilm Wako Pure Chemical Corporation). The amylase concentration in the culture supernatant was calculated by using the protein concentration of the culture supernatant of a strain introduced with pHY300PLK (Takara Bio) that does not have an amylase expression cassette as a blank.

[0078] (4) Stability evaluation of amylase mutants (1) Various mutants were constructed using YR288 R178Δ+T180Δ (SEQ ID NO: 2) as the parent polypeptide by the method described in Example (1), and the storage stability of these mutants under acidic conditions and in the presence of a high level of chelating agent was evaluated. The amylase solution was added to a mixture of equal volumes of 200 mM EDTA-2NA and 50 mM Britton-Robinson buffer (pH 5.0), and the mixture was incubated at 40°C for 30 minutes. The resulting mixture was then diluted 10-fold with 50 mM Britton-Robinson buffer (pH 8.0), and activity was measured. The activity of the sample before treatment at 40°C was defined as the initial activity, and the percentage of the activity after treatment to the initial activity was defined as the residual activity (%). The results are shown in Table 3. All mutants exhibited high stability under acidic conditions and in the presence of a chelating agent.

[0079] [Table 3]

[0080] (5) Stability evaluation of amylase mutants (2) AATCC High Efficiency Standard Reference Detergent WITHOUT Brightener was diluted 10-fold with 100 mM citrate buffer (pH 5.0), to which the amylase solution was added. After incubation at 50°C for 1 hour, the mixture was diluted 10-fold with 50 mM Britton-Robinson buffer (pH 8.0) and the activity was measured. The activity value of the sample before treatment at 50°C was defined as the initial activity, and the percentage of the activity value after treatment to the initial activity was defined as the residual activity (%). The results are shown in Table 4 (Table 4-1, Table 4-2, and Table 4-3). All mutants exhibited high stability in diluted detergent.

[0081] [Table 4]

[0082] (6) Stability evaluation of amylase mutants (3) AATCC High Efficiency Standard Reference Detergent WITHOUT Brightener was diluted 10-fold with 100 mM citrate buffer (pH 5.0), to which amylase solution was added, and the mixture was incubated at 50°C for 18 hours. The mixture was then diluted 10-fold with 50 mM Britton-Robinson buffer (pH 8.0), and activity was measured. The activity value of the sample before treatment at 50°C was defined as the initial activity, and the percentage of the activity value after treatment to the initial activity was defined as the residual activity (%). The results are shown in Table 5 (Table 5-1, Table 5-2). Both mutants exhibited high stability in diluted detergent.

[0083] (7) Stability evaluation of amylase mutants (4) A 50% w / w citric acid solution was added to AATCC High Efficiency Standard Reference Detergent WITHOUT Brightener to a final concentration of 100 mM, and the pH was adjusted to 5.2 with 1 M NaOH. Amylase solution was added to this detergent, and after incubation at 40°C for 1 week, the detergent was diluted 10-fold with 50 mM Britton-Robinson buffer (pH 8.0) and activity was measured. The activity value of the sample before treatment at 40°C was defined as the initial activity, and the percentage of the activity value after treatment to the initial activity was defined as the residual activity (%). The results are shown in Table 6. All mutants exhibited high stability in the detergent.

[0084] [Table 5]

[0085] [Table 6]

Claims

1. A mutant of a parent α-amylase comprising a substitution of an amino acid residue at a position corresponding to positions H238 or E185 of the amino acid sequence shown in SEQ ID NO: 2 and a substitution of an amino acid residue shown in (A) or (B) below, wherein the parent α-amylase or the α-amylase mutant has at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 2 (excluding α-amylase mutants in which amino acid residues are substituted at three positions corresponding to positions H238, S239, and G178, three positions corresponding to positions H238, R209, and G178, and three positions corresponding to positions E185, N190, and G178 of the amino acid sequence). (A) Substitution of amino acid residues at one or more positions selected from positions corresponding to T116, A181, A199, A275, V277, A286, and L323 of the amino acid sequence shown in SEQ ID NO:

2. (B) Substitution of an amino acid residue at a position corresponding to position G178 of the amino acid sequence shown in SEQ ID NO: 2, and substitution of amino acid residues at one or more positions selected from positions corresponding to positions N126, T129, N190, R209, and S239 of the amino acid sequence shown in SEQ ID NO:

2.

2. The mutant according to claim 1, wherein the substitution of the amino acid residue represented by (A) further includes substitution of an amino acid residue at one or more positions selected from the positions corresponding to N126, T129, N190, R209, S239, G178, M197, F203, Y240, E255, Y358, W406 and G474 of the amino acid sequence represented by SEQ ID NO:

2.

3. The mutant of claim 1, comprising substitutions of amino acid residues at two or more positions selected from positions corresponding to T116, A181, A199, A275, V277, A286 and L323 of the amino acid sequence shown in SEQ ID NO: 2 (A).

4. The mutant of claim 1, wherein the substitutions of amino acid residues at positions corresponding to H238, E185, T116, A181, A199, A275, V277, A286, L323, G178, N126, T129, N190, R209 and S239 are H238F, E185P, T116Q, A181T / E / Q, A199E, A275N, V277T, A286V, L323F, G178H, N126Y, T129I, N190F, R209V / L / I, S239A / Q / D / L / Y / P / H, respectively.

5. The mutant according to claim 1, wherein the mutant comprises at least a mutation selected from the combinations of mutations shown in Tables 1-1, 1-2, 1-3 and 2 below.

6. A polynucleotide encoding the variant according to any one of claims 1 to 5.

7. A vector or DNA fragment comprising the polynucleotide of claim 6.

8. A transformed cell containing the vector or DNA fragment according to claim 7.

9. The transformed cell of claim 8, which is a microorganism.

10. A detergent composition comprising the mutant according to any one of claims 1 to 5.

11. The detergent composition according to claim 10, which is a laundry detergent or a dish detergent.

12. The cleaning composition of claim 11 which is a powder or a liquid.

13. The cleaning composition of claim 11, which is weakly acidic.

14. The cleaning composition of claim 11, comprising a chelating agent.

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