α-amylase variants

Abstract

To provide an α-amylase capable of functioning at low temperatures while maintaining or improving stability and / or cleaning performance.SOLUTION: A variant of a parent α-amylase comprising modification of one or more amino acid residues at positions corresponding to the respective positions of G5, S38, T49, Q96, N126, T129, G140, F153, Q167, G179, W186, E187, N192, M199, Y200, L203, Y205, D206, R211, K215, H240, S241, Y242, G244, E257, F259, K278, H283, S284, A288, H295, Y296, N303, T320, S331, L348, Y360, W408, L429, V430, G433, A434, W439, N471, G476, and G477 of the amino acid sequence set forth in SEQ ID NO: 2. The parent α-amylase or the variant of the parent α-amylase has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4.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 viewpoint of environmental protection and reduction of cleaning costs, it has become important to lower the temperature during dishwashing and laundry washing, especially laundry washing, and shortening the washing time is also desired. However, the optimum temperature for most enzymes, including amylase, is higher than the temperature normally set for low-temperature washing, making it difficult to completely remove many starchy stains. Therefore, it is important to find an α-amylase that retains its cleaning performance and starch-degrading activity even at low temperatures and has a high stain removal effect. [Prior art documents] [Patent documents]

[0005] [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 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention relates to providing α-amylases that are capable of functioning at low temperatures while maintaining or improving stability and / or cleaning performance. [Means for solving the problem]

[0007] The present inventors have succeeded in obtaining α-amylase variants that have improved cleaning performance and / or stability compared to the parent α-amylase, using an α-amylase that functions at low temperatures as a parent.

[0008] That is, the present invention relates to the following. 1) G5, S38, T49, Q96, N126, T129, G140, F153, Q167, G179, W186, E187, N192, M199, Y200, L203, Y205, D206, R211, K215, H240, S241, Y242, G244, E257, F259, K278, H283, S284, A288, H295, Y296, N303, and T320 of the amino acid sequence shown in SEQ ID NO: 2 , S331, L348, Y360, W408, L429, V430, G433, A434, W439, N471, G476 and G477, wherein the parent α-amylase or α-amylase variant has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:4. 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]

[0009] The present invention provides α-amylase variants that have improved cleaning performance and / or stability compared to the parent α-amylase, enabling excellent starch stain removal even at low temperatures. [Brief explanation of the drawings]

[0010] [Figure 1] Stability assessment of two-amino acid deletion mutants. DETAILED DESCRIPTION OF THE INVENTION

[0011] 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).

[0012] 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.

[0013] 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).

[0014] 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". Amino acid "insertions" are designated as [original amino acid, position, original amino acid, inserted amino acid]. For example, the insertion of a lysine after the glycine at position 195 is designated as "G195GK." Multiple amino acid insertions are designated as [original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2; etc.]. For example, the insertion of a lysine and an alanine after the glycine at position 195 is designated as "G195GKA." 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.

[0015] 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.

[0016] 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.

[0017] 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).

[0018] <Mutant> The mutant of the present invention includes G5, S38, T49, Q96, N126, T129, G140, F153, Q167, G179, W186, E187, N192, M199, Y200, L203, Y205, D206, R211, K215, H240, S241, Y242, G244, E257, and F259 in the amino acid sequence shown in SEQ ID NO: 2. , K278, H283, S284, A288, H295, Y296, N303, T320, S331, L348, Y360, W408, L429, V430, G433, A434, W439, N471, G476 and G477. In other words, a "variant" refers to a polypeptide having α-amylase activity in which one or more amino acid residues at specific positions in the amino acid sequence of the parent α-amylase have been modified. The modifications at these specific positions are intended to enhance cleaning performance and / or stability in detergents, and the variant therefore has improved cleaning performance and / or stability compared to the parent α-amylase.

[0019] In the mutant of the present invention, the modified amino acid residue sites (mutation positions) are G5, S38, T49, Q96, N126, T129, G140, F153, Q167, G179, W186, E187, N192, M199, Y200, L203, Y205, D206, R211, K215, H216, L217, L218, L219, L220, L221, L222, L223, L224, L225, L226, L227, L228, L229, L230, L231, L232, L233, L234, L235, L236, L237, L238, L239, L240, L241, L242, L243, L244, L245, L246, L247, L248, L249, L250, L251, L252, L253, L254, L255, L256, L257, L258, L259, L260, L261, L262, L263, L264, L265, L266, L267, L268, L269, L270, L271, L272, L273, L274, L275, L276, L277, L278, L279, L280, L281, L282, L283, L284, L285, L286, L287, L288, L289, L290, L291, L292, L293, L294, L These positions correspond to positions 240, S241, Y242, G244, E257, F259, K278, H283, S284, A288, H295, Y296, N303, T320, S331, L348, Y360, W408, L429, V430, G433, A434, W439, N471, G476 and G477. Here, the amino acid sequence shown in SEQ ID NO: 2 is the amino acid sequence that constitutes α-amylase YR288, and the mutation positions in the mutants of the present invention are numbered based on the amino acid numbering of the amino acid sequence. YR288 is a protein registered as WP_100346362.1 in the NCBI protein sequence database, and has been identified by the present applicant as an α-amylase with high starch-degrading activity and cleaning performance at low temperatures (Patent Application No. 2020-121626).

[0020] The "corresponding position" on an amino acid sequence can be determined by aligning the target sequence with the reference sequence (the amino acid sequence shown in SEQ ID NO: 2 in this 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 from, for example, the European Bioinformatics Institute. These are available on the websites of the 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 alignment is considered to be the "position corresponding to" that position.

[0021] 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.

[0022] In the present invention, "parent α-amylase" refers to a reference α-amylase to which modification is made to result in a variant of the invention. The parent may be a naturally occurring (wild-type) polypeptide or a variant thereof.

[0023] In the present invention, the parent α-amylase or α-amylase variant has an amino acid sequence that is 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% identical to the amino acid sequence set forth in SEQ ID NO:4. Here, the α-amylase consisting of the amino acid sequence shown in SEQ ID NO: 4 is an α-amylase mutant (R178Δ+T180Δ) in which the amino acid residues corresponding to R178 and T180 in the α-amylase (YR288) consisting of the amino acid sequence shown in SEQ ID NO: 2 have been deleted. As shown in the Examples below, mutants with YR288 as the parent α-amylase, which contain deletions of two amino acid residues corresponding to R178, G179, T180, and G181 in the amino acid sequence shown in SEQ ID NO: 2, have significantly improved stability in detergents compared to YR288. Therefore, any α-amylase mutant, including an α-amylase consisting of the amino acid sequence shown in SEQ ID NO: 4, in which two or more amino acid residues have been deleted at positions corresponding to R178, G179, T180 and G181 in the amino acid sequence shown in 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, can serve as the parent α-amylase for the mutants of the present invention. Here, deletions of two or more amino acid residues preferably include R178Δ+T180Δ, G179Δ+T180Δ, R178Δ+G179Δ, R178Δ+G181Δ, G179Δ+G181Δ, etc., with R178Δ+T180Δ being more preferred.

[0024] Other examples of α-amylases having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 4 include Bacillus flexus ( Examples include DE0178, an α-amylase derived from Bacillus flexus, and RU2C, an α-amylase derived from Bacillus sp. (Patent Application No. 2020-121626).

[0025] The modification of amino acid residues at one or more of the predetermined positions may be substitution, insertion, and / or deletion of amino acid residues, where "substitution" refers to replacing an amino acid at a position with a different amino acid, "deletion" refers to removing an amino acid at a position, and "insertion" refers to adding an amino acid immediately adjacent to the amino acid at a position.

[0026] In the present invention, the mutation site may be one or more, preferably two or more, more preferably 2 to 15, more preferably 3 to 15, and even more preferably 5 to 10. From the viewpoint of improving cleaning performance or stability, the mutant is preferably an α-amylase having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 4. Furthermore, the mutant may contain any number of conservative amino acid substitutions as long as it retains the properties of the mutant.

[0027] G5, S38, T49, Q96, N126, T129, G140, F153, Q167, G179, W186, E187, N192, M199, Y200, L203, Y205, D206, R211, K215, H240, S241, Y242, G244, E257, F259, K278, H283, S284, A288, H295, and Y299 of the amino acid sequence shown in SEQ ID NO: 2. Among the mutation sites indicated by positions corresponding to positions 6, N303, T320, S331, L348, Y360, W408, L429, V430, G433, A434, W439, N471, G476, and G477, from the viewpoint of improving stability, preferred are positions corresponding to positions N126, E187, N192, F205, R211, H240, S241, and Y242. Among these, preferred modifications at two or more sites include, for example, combinations of the following modifications a) to f). a) A combination of alterations in at least one or more amino acid residues selected from the amino acid residues corresponding to positions E187, F205, and H240, and alterations in at least one or more amino acid residues selected from the amino acid residues corresponding to positions N126, N192, R211, S241, and Y242. b) A combination of alterations in at least one or more amino acid residues selected from the amino acid residues corresponding to positions E187 and H240 and alterations in at least one or more amino acid residues selected from the amino acid residues corresponding to positions N126, N192, F205, R211, S241 and Y242. c) A combination of alterations in at least one or more amino acid residues selected from the amino acid residues corresponding to positions F205 and H240 and alterations in at least one or more amino acid residues selected from the amino acid residues corresponding to positions N126, E187, N192, R211, S241 and Y242. d) A combination of an alteration in the amino acid residue corresponding to position F205 and an alteration in at least one amino acid residue selected from the amino acid residues corresponding to positions N126, E187, N192, R211, H240, S241 and Y242. e) A combination of an alteration in the amino acid residue corresponding to position H240 and an alteration in at least one amino acid residue selected from the amino acid residues corresponding to positions N126, E187, N192, F205, R211, S241 and Y242. f) A combination of an alteration in the amino acid residue corresponding to position R211 and an alteration in at least one amino acid residue selected from the amino acid residues corresponding to positions N126, E187, N192, F205, H240, S241 and Y242.

[0028] Preferred embodiments of modifications of amino acid residues at positions corresponding to each of the following positions are shown below: G5, S38, T49, Q96, N126, T129, G140, F153, Q167, G179, W186, E187, N192, M199, Y200, L203, Y205, D206, R211, K215, H240, S241, Y242, G244, E257, F259, K278, H283, S284, A288, H295, Y296, N303, T320, S331, L348, Y360, W408, L429, V430, G433, A434, W439, N471, G476, and G477. That is, G5 is preferably substituted with E, D, P, R, or K (G5E / D / P / R / K); S38 is preferably substituted with N (S38N); T49 is preferably substituted with Q (T49Q); Q96 is preferably substituted with R or K (Q96R / K); N126 is preferably substituted with Y (N126Y); T129 is preferably substituted with I (T129I); G140 is preferably substituted with Y, F or W (G140Y / F / W); F153 is preferably substituted with W (F153W); Q167 is preferably substituted with E (Q167E); G179 is preferably substituted with D or H (G179D / H); W186 is preferably substituted with L (W186L); E187 is preferably substituted with P (E187P); N192 is preferably substituted with F (N192F); M199 is preferably substituted with L, T, A, N, Q, S, V or I (M199L / T / A / N / Q / S / V / I); Y200 is preferably substituted with G (Y200G); L203 is preferably substituted with Y, M or F (L203Y / M / F); Y205 is preferably substituted with F (Y205F); D206 is preferably substituted with R, E, N, T or G (D206R / E / N / T / G); R211 is preferably substituted with L, V, or I (R211L / V / I); K215 is preferably substituted with F (K215F); H240 is preferably substituted with F (H240F); S241 is preferably substituted with A, Q, D, L, Y, P, or H (S241A / Q / D / L / Y / P / H); Y242 is preferably substituted with F (Y242F); G244 is preferably substituted with K, W, L or R (G244K / W / L / R); E257 is preferably substituted with T (E257T); F259 is preferably substituted with W (F259W); K278 is preferably substituted with L, D, W, I, H, S, T, N, Q, V, A, Y or F (K278L / D / W / I / H / S / T / N / Q / V / A / Y / F); H283 is preferably substituted with Q (H283Q); S284 is preferably substituted with W (S284W); A288 is preferably substituted with F (A288F); H295 is preferably substituted with Y (H295Y); Y296 is preferably substituted with A (Y296A); N303 is preferably substituted with R, E, S, G, V, D, T or A (N303R / E / S / G / V / D / T / A); T320 is preferably substituted with D or E (T320D / E); S331 is preferably substituted with T (S331T); L348 is preferably substituted with I (L348I); Y360 is preferably substituted with C, M, L or V (Y360C / M / L / V); Substitution of P at position W408 (W408P) is preferred; L429 is preferably substituted with V (L429V); V430 is preferably substituted with M (V430M); G433 is preferably an insertion of S after G (G433GS); A434 is preferably substituted with V (A434V); W439 is preferably substituted with R (W439R); N471 is preferably substituted with T (N471T); G476 is preferably substituted with A, P, E, S, F, R or K (G476A / P / E / S / F / R / K); G477 is preferably substituted with E (G477E).

[0029] Next, suitable combinations of mutations that contribute to improved cleaning performance are shown in Tables 1-1 to 1-4 below. Therefore, mutants having at least these combined mutations are mutants that particularly contribute to improved cleaning performance.

[0030] [Table 1-1]

[0031] [Table 1-2]

[0032] [Table 1-3]

[0033] [Table 1-4]

[0034] Next, preferred combinations of mutations that contribute to improved stability are shown in Tables 2-1 to 2-4 below. Therefore, mutants having at least these combinations of mutations are mutants that particularly contribute to improved stability. Furthermore, mutations that combine the combinations of mutations in Tables 1-1 to 1-4 with the combinations of mutations in Tables 2-1 to 2-4 are also preferred from the viewpoint of improving cleaning performance and stability.

[0035] [Table 2-1]

[0036] [Table 2-2]

[0037] [Table 2-3]

[0038] [Table 2-4]

[0039] Next, suitable combinations of mutations that contribute to improved cleaning performance and stability are shown in Table 3. Therefore, mutants having at least these combinations of mutations are mutants that particularly contribute to improved cleaning performance and stability.

[0040] [Table 3]

[0041] <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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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).

[0046] 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:4 is shown in SEQ ID NO:3, and the DNA sequence containing the base sequence encoding the α-amylase (YR288) consisting of the amino acid sequence shown in SEQ ID NO:2 is shown in SEQ ID NO:1.

[0047] 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.

[0048] 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 / ]).

[0049] <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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] <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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] The resulting variants of the invention have improved cleaning performance and / or stability compared to the parent α-amylase. Here, "enhanced cleaning performance" means the ability to provide improved cleaning benefits, eg, stain removal, in a washing or cleaning process compared to the parent α-amylase. Washing performance can be evaluated using methods well known in the art. For example, soiled cloth cut to a predetermined size is inserted into the wells of a 96-well assay plate, and a detergent solution and an enzyme solution are added to perform a washing process under predetermined conditions. After washing, the absorbance of the wash solution at 488 nm is measured, and the difference ΔA488 from the blank is calculated as the washing power. The relative washing power can be calculated by dividing the ΔA488 of the mutant by the ΔA488 of the parent α-amylase.

[0062] Additionally, "improved stability" refers to the ability to maintain α-amylase activity in the presence of detergents, which is improved compared to the parent α-amylase. Stability can be evaluated using methods well known in the art. For example, an enzyme solution is added to a detergent, and after treatment for a predetermined time, α-amylase activity is measured, and the half-life (h) is calculated by calculating the inactivation rate per unit time (h) due to the treatment. Relative stability can be determined by dividing the half-life (h) of the mutant by the half-life (h) of the parent α-amylase.

[0063] 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 suitable for low-temperature washing. Here, "low temperature" includes 40°C or lower, 35°C or lower, 30°C or lower, and 25°C or lower, as well as 5°C or higher, 10°C or higher, and 15°C or higher, and also includes 5 to 40°C, 10 to 35°C, 15 to 30°C, and 15 to 25°C.

[0064] 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.

[0065] 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).

[0066] The detergent composition may contain known detergent components, and examples of such known detergent components include the following:

[0067] (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.

[0068] 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.

[0069] 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.

[0070] 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).

[0071] (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.

[0072] (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.

[0073] (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.

[0074] (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.

[0075] (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.

[0076] (7) 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.

[0077] 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.

[0078] 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 suitable for use at temperatures of 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. The cleaning composition is also suitable for use at temperatures of 5 to 40°C, 10 to 35°C, 15 to 30°C, or 15 to 25°C.

[0079] In relation to the above-described embodiment, the present invention further discloses the following aspects. 1) G5, S38, T49, Q96, N126, T129, G140, F153, Q167, G179, W186, E187, N192, M199, Y200, L203, Y205, D206, R211, K215, H240, S241, Y242, G244, E257, F259, K278, H283, S284, A288, H295, Y296, N303, and T320 of the amino acid sequence shown in SEQ ID NO: 2 , S331, L348, Y360, W408, L429, V430, G433, A434, W439, N471, G476 and G477, wherein the parent α-amylase or α-amylase variant has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:4. 2) The above-mentioned G5, S38, T49, Q96, N126, T129, G140, F153, Q167, G179, W186, E187, N192, M199, Y200, L203, Y205, D206, R211, K215, H240, S241, Y242, G244, E257, F259, K278, H283, S284, A288, H295, Y296, N303, T The modifications of amino acid residues at positions corresponding to positions 320, S331, L348, Y360, W408, L429, V430, G433, A434, W439, N471, G476, and G477 were G5E / D / P / R / K, S38N, T49Q, Q96R / K, N126Y, T129I, G140Y / F / W, F153W, Q167E, G179D / H, W18 6L, E187P, N192F, M199L / T / A / N / Q / S / V / I, Y200G, L203Y / M / F, Y205F, D206R / E / N / T / G, R211V / L / I, K 215F, H240F, S241A / Q / D / L / Y / P / H, Y242F, G244K / W / L / R, E257T, F259W, K278L / D / W / I / H / S / T / N / Q / V / 1) Mutants A / Y / F, H283Q, S284W, A288F, H295Y, Y296A, N303R / E / S / G / V / D / T / A, T320D / E, S331T, L348I, Y360C / M / L / V, W408P, L429V, V430M, G433GS, A434V, W439R, N471T, G476A / P / E / S / F / R / K and G477E. 3) A mutant of 1) or 2), in which the amino acid residues are modified at two or more locations. 4) The mutant of 1), wherein the amino acid residue modifications are modifications of amino acid residues at positions corresponding to N126, E187, N192, F205, R211, H240, S241 and Y242. 5) A mutant of 4), in which the modifications of the amino acid residues at the positions corresponding to N126, E187, N192, F205, R211, H240, S241 and Y242 are N126Y, E187P, N192F, F205Y, R211I, H240F, S241Q and Y242F, respectively. 6) A mutant of 4) or 5), in which the amino acid residues are altered at two or more locations. 7) A variant of 1) or 2), which comprises modifications in at least one or more amino acid residues selected from the amino acid residues corresponding to positions E187, F205, and H240, and modifications in at least one or more amino acid residues selected from the amino acid residues corresponding to positions N126, N192, R211, S241, and Y242. 8) A variant of 1) or 2), which comprises modifications in at least one or more amino acid residues selected from the amino acid residues corresponding to positions E187 and H240, and modifications in at least one or more amino acid residues selected from the amino acid residues corresponding to positions N126, N192, F205, R211, S241, and Y242. 9) A mutant of 1) or 2), which comprises modifications in at least one or more amino acid residues selected from the amino acid residues corresponding to positions F205 and H240, and modifications in at least one or more amino acid residues selected from the amino acid residues corresponding to positions N126, E187, N192, R211, S241, and Y242. 10) A mutant of 1) or 2), comprising a modification in an amino acid residue corresponding to position F205 and a modification in at least one amino acid residue selected from the amino acid residues corresponding to positions N126, E187, N192, R211, H240, S241 and Y242. 11) A mutant of 1) or 2), comprising a modification in an amino acid residue corresponding to position H240 and a modification in at least one amino acid residue selected from the amino acid residues corresponding to positions N126, E187, N192, F205, R211, S241 and Y242. 12) A variant of 1) or 2), comprising a modification in an amino acid residue corresponding to position R211 and a modification in at least one amino acid residue selected from the amino acid residues corresponding to positions N126, E187, N192, F205, H240, S241 and Y242. 13) A mutant of 1) or 2), wherein the parent α-amylase is an α-amylase mutant in which the amino acid residues R178 and G179 in the amino acid sequence shown in SEQ ID NO: 2 have been deleted. 14) The mutant of 1), 2) or 13), which contains at least a mutation selected from the combinations of mutations shown in Tables 1-1 to 1-4. 15) A mutant of 1), 2) or 13) which contains at least a mutation selected from the combinations of mutations shown in Tables 2-1 to 2-4. 16) A mutant of 1), 2) or 13), which comprises a mutation selected from the combinations of mutations shown in Tables 1-1 to 1-4 and a mutation selected from the combinations of mutations shown in Tables 2-1 to 2-4. 17) The mutant of 1), 2) or 13), which contains at least a mutation selected from the combination of mutations shown in Table 3. 18) A polynucleotide encoding the mutant according to any one of 1) to 17). 19) A vector or DNA fragment containing the polynucleotide of 18). 20) A transformed cell containing the vector or DNA fragment of 19). 21) A transformed cell of 20) which is a microorganism. 22) A cleaning composition comprising a variant of 1) to 17). 23) The detergent composition according to 22), which is a laundry detergent or a dish detergent. 24) The cleaning composition according to 23), which is a powder or liquid. 25) The cleaning composition according to any one of 22) to 24) which is used at low temperatures. 26) The cleaning composition of 25) used at a temperature of 5 to 40°C. [Example]

[0080] (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 expression plasmid pHY-YR288 described in the examples of Japanese Patent Application No. 2020-121626 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.

[0081] (2) Enzyme production culture The recombinant B. subtilis colonies obtained in (1) were inoculated into 300 μL of LB medium supplemented with 15 ppm tetracycline in a 96-well deep-well plate and cultured overnight at 30°C and 210 rpm. The next day, 6 μL of the culture was inoculated into 100 μL of 2× L-maltose medium (2% tryptone, 1% yeast extract, 1% NaCl, 7.5% maltose, 7.5 ppm manganese sulfate pentahydrate, 0.04% calcium chloride dihydrate, 15 ppm tetracycline; % is (w / v)%) in a 96-well deep-well plate and cultured at 30°C and 210 rpm for 2 days. The culture supernatant containing the enzyme produced by the bacteria was collected by centrifugation and used as the enzyme solution.

[0082] (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.

[0083] (4) Activity measurement Non-reducing-end-protected ethylidene-paranitrophenyl-α-D-maltoheptaoside (Et-G7-pNP) was used as a substrate. α-Amylase reacts with Et-G7-pNP to produce maltooligosaccharide-pNP, which is then reacted with α-glucosidase to liberate pNP. α-Amylase activity can be determined by measuring the rate of absorbance increase associated with pNP production. A 2:1 mixture of RI and RII solutions from AMY-EL (Serotec), an α-amylase activity assay reagent containing Et-G7-pNP and α-glucosidase, was used as the substrate solution. 100 μL of the substrate solution and 10 μL of an appropriately diluted enzyme sample were mixed in each well of a 96-well assay plate, and the change in absorbance (OD / min) at 405 nm was measured at 30°C. The activity was measured as the difference ΔOD / min from the blank (sample without enzyme).

[0084] (5) Stability evaluation of the two-amino acid deletion mutant at positions 178-181 The mutant shown in Figure 1 was constructed using YR288 (SEQ ID NO: 2) as the parent polypeptide by the method described in Example (1). The enzyme solution was added to a commercially available liquid laundry detergent (Kao Corporation, Attack 3X) diluted to 10% (v / v) with ion-exchanged water, and the enzyme was incubated at 50°C for 15 minutes before activity measurement. The residual activity (%) was calculated by dividing the activity of the sample after 50°C treatment by the activity of the sample before 50°C treatment and multiplying by 100. Stability was significantly improved by deleting any two residues from R178, G179, T180, and G181 (Figure 1).

[0085] (6) Detergency evaluation CS-26 stained cloth, cut into 5.5 mm diameter circles, was purchased from CFT and used. Two CS-26 stained circular circles were inserted into each well of a 96-well assay plate, and 200 μL of commercially available liquid laundry detergent (Attack Zero, Kao Corporation) diluted 3000-fold with tap water was added. 10 μL of enzyme solution diluted to 3 ppm with tap water was added to each well, sealed, and shaken at 1200 rpm for 15 minutes in a Cute Mixer at 20°C. After washing, 100 μL of the wash solution was transferred to a new 96-well assay plate, and the absorbance at 488 nm was measured. A blank was prepared by adding tap water instead of the enzyme solution, and the difference in ΔA488 between the blank and the blank was calculated as the detergency. Relative detergency was calculated by dividing the ΔA488 of each mutant by the ΔA488 of the parent polypeptide.

[0086] (7) Stability evaluation The enzyme solution was added to a commercially available liquid laundry detergent (Kao Corporation, Attack 3X or Kao Corporation, Attack Zero) diluted to 10% (v / v) with ion-exchanged water, and the enzyme was incubated at 50°C for 30 minutes to 18 hours before activity measurement. The activity value of the sample before 50°C treatment was used as the initial activity, and the deactivation rate per unit time (h) due to 50°C treatment was calculated, from which the half-life (h) was calculated. The relative stability was determined by dividing the half-life (h) of each mutant by the half-life (h) of the parent polypeptide.

[0087] (8) Mutation screening Various mutants were constructed using YR288 R178Δ+T180Δ (SEQ ID NO: 4) as the parent polypeptide by the method described in Example (1). The performance of the mutants was evaluated by the methods described in Examples (6) and (7). Performance was considered improved when the relative detergency and / or relative stability was 1.1 or higher. The following mutants showed improved performance:

[0088] G5E, G5D, G5P, G5R, G5K, S38N, T49Q, Q96R, Q96K, N126Y, T129I, G140Y, G140F, G 140W, F153W, Q167E, G179D, G179H, W186L, E187P, N192F, M199L, M199T, M199A, M 199N, M199Q, M199S, M199V, M199I, Y200G, L203Y, L203M, L203F, Y205F, D206R, D 206E, D206N, D206T, D206G, R211V, R211L, R211I, K215F, H240F, S241A, S241Q, S 241D, Y242F, G244K, G244W, G244L, G244R, E257T, F259W, H283Q, S284W, A288F, H295Y, Y296A, N303R, N303E, N303S, N303G, N303V, N303D, N303T, N303A, T320D, T320E, S331T, L348I, Y360C, Y360M, Y360L, Y360V, W408P, L429V, V430M, G433GS , A434V, W439R, N471T, G476A, G476P, G476E, G476S, G476F, G476R, G476K, G477E

[0089] (9) Evaluation of cleaning power of multiple mutants Using the method described in Example (1), various mutants containing two or more of the mutations obtained in Example (8) were constructed using YR288 R178Δ+T180Δ (SEQ ID NO: 4) as the parent polypeptide. The detergency of the mutants was evaluated using the method described in Example (6). The results are shown below.

[0090] [Table 4-1]

[0091] [Table 4-2]

[0092] [Table 4-3]

[0093] [Table 4-4]

[0094] (10) Evaluation of stability of multiple mutants Using the method described in Example (1), various mutants containing two or more of the mutations obtained in Example (8) were constructed using YR288 R178Δ+T180Δ (SEQ ID NO: 4) as the parent polypeptide. The stability of the mutants was evaluated using the method described in Example (7). The results are shown below.

[0095] [Table 5-1]

[0096] [Table 5-2]

[0097] [Table 5-3]

[0098] [Table 5-4]

[0099] Various mutants were constructed using YR288 R178Δ+T180Δ (SEQ ID NO: 4) as the parent polypeptide by the method described in Example (1). The stability of the mutants was evaluated by the method described in Example (7). The results are shown below.

[0100] [Table 5-5]

[0101] (11) Evaluation of cleaning power and stability of multiple mutants Using the method described in Example (1), various mutants containing the multiple mutations obtained in Example (9) and Example (10) were constructed using YR288 R178Δ+T180Δ (SEQ ID NO: 4) as the parent polypeptide. The performance of the mutants was evaluated using the methods described in Examples (6) and (7). The results are shown below.

[0102] [Table 6]

[0103] (12) Evaluation of cleaning power and stability of multiple mutants The following mutants were constructed using YR288 R178Δ+G179Δ as the parent polypeptide by the method described in Example (1). The performance of the mutants was evaluated by the methods described in Examples (6) and (7). The results are shown below.

[0104] [Table 7]

Claims

1. A variant of a parent α-amylase comprising the following modification (i) or (ii), wherein the parent α-amylase is a polypeptide in which two or more amino acid residues have been deleted at positions corresponding to R178, G179, T180 and G181 of the amino acid sequence shown in SEQ ID NO: 2, and the parent α-amylase or α-amylase variant has at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO:

2. (i) A modification of one or more amino acid residues at positions corresponding to S38, Q96, W186, E257, F259, S284, T320, Y360, W408, and N471 in the amino acid sequence shown in SEQ ID NO: 2, wherein the modifications are S38N, Q96R / K, W186L, E257T, F259W, S284W, T320D / E, Y360C / M / L / V, W408P, and N471T, respectively. (ii) A modification of amino acid residues at positions corresponding to G5 and W439 in the amino acid sequence shown in SEQ ID NO: 2, wherein the modification is a combination of G5R and W439R.

2. The mutant according to claim 1, wherein the amino acid residues are modified at two or more positions.

3. A mutant described in claim 2, which comprises at least a modification selected from the combinations of mutations shown in Tables 1-1 to 1-4 below.

4. A mutant described in claim 2, which comprises at least a modification selected from the combination of mutations shown in Table 3 below.

5. A polynucleotide encoding the variant according to any one of claims 1 to 4.

6. A vector or DNA fragment comprising the polynucleotide of claim 5.

7. A transformed cell containing the vector or DNA fragment according to claim 6.

8. The transformed cell of claim 7, which is a microorganism.

9. A detergent composition comprising the mutant according to any one of claims 1 to 6.

10. The detergent composition according to claim 9, which is a laundry detergent or a dish detergent.

11. The cleaning composition of claim 10, which is a powder or a liquid.

12. The cleaning composition according to claim 10 or 11, which is used at low temperatures.

13. The cleaning composition according to claim 12, which is used at a temperature of 5 to 40°C.

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