Α-amylase variants

Abstract

To provide α-amylase variants having excellent starch degrading activity at low temperature and excellent stability at low pH and / or in the presence of chelating agents.SOLUTION: Provided herein are α-amylase variants A1, B1, C1, D1 and E1 derived from respective parent α-amylase A, B, C, D and E those having at least 90% sequence identity to a specific amino acid sequence, where the amylase variants A1 to E1 include amino acid deletion at two positions selected from positions corresponding to the positions 178-181 of the specific amino acid sequence, and amino acid substitution at three or more positions selected from positions corresponding to the positions 178, 181, 238, 239, 240, 126, 129 and 201 of the specific amino acid sequences.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 (Patent Document 3, SEQ ID NO: 4), α-amylase CspAmy2 derived from the genus Cytophaga (Patent Document 4), and LABM, a chimera of the A and B domains of α-amylase AAI10 derived from the genus Bacillus and an α-amylase derived from an Alicyclobacillus species (Patent Document 5, SEQ ID NO: 13). Furthermore, these α-amylases have been modified to improve their functions for specific applications, and for example, mutants with improved stability in detergents have been reported (Patent Document 6). However, when the parent enzymes are different, mutations do not necessarily produce the same effects, and obtaining multiple mutants with significantly improved specific properties requires a great deal of effort tailored to each parent enzyme.

[0004] On the other hand, in recent years, it has become known that cleaning in a low pH environment is gentle on items being cleaned and not as strong as commonly used alkaline compositions, thereby keeping items such as glass and patterned tableware looking new for a long time. It also reduces fabric odors, promotes the release of calcium soaps that tend to trap dirt on fabrics, improves performance against pH-sensitive stains, and even benefits the feel of fabrics. Furthermore, dishwashing detergents are often used to clean stainless steel and resin sinks in addition to cleaning the main target of cleaning: tableware. Therefore, they often contain high concentrations of chelating agents, such as citric acid, which exerts cleaning power against limescale. Therefore, adding enzymes such as α-amylase to such detergents is expected to further improve cleaning performance.

[0005] Therefore, there is a demand for improved stability of the above-mentioned α-amylases for use in detergents in acidic detergents or detergents containing a chelating agent, particularly detergents that are both acidic and contain a chelating agent. [Prior art documents] [Patent documents]

[0006] [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] Special Publication No. 2018-516553 [Patent Document 6] International Publication No. 98 / 044126 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention relates to providing an α-amylase that exhibits excellent stability at low pH and / or in the presence of a chelating agent. [Means for solving the problem]

[0008] The present inventors have found that when a specific amino acid modification common to various detergent amylases is performed, it brings about a remarkable stabilizing effect in the presence of a low pH and / or a chelating agent.

[0009] That is, the present invention relates to the following. 1) α-amylase A having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4, α-amylase B having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6, α-amylase C having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8, α-amylase D having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10, or α-amylase D having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 12 The α-amylase variants A1, B1, C1, D1 and E1 each have as their parent α-amylase E having the formula: a) For A1, substitution of the amino acid residue at the position corresponding to position 178 with H, substitution of the amino acid residue at the position corresponding to position 181 with E or Q, and substitution of the amino acid residue at the position corresponding to position 239 with Q b) For B1, substitution of the amino acid residue at the position corresponding to position 178 with H, substitution of the amino acid residue at the position corresponding to position 181 with E or Q, and substitution of the amino acid residue at the position corresponding to position 239 with Q c) For C1, three or more substitutions selected from the following: substitution of the amino acid residue at the position corresponding to position 178 with H; substitution of the amino acid residue at the position corresponding to position 181 with E or Q; substitution of the amino acid residue at the position corresponding to position 238 with F; and substitution of the amino acid residue at the position corresponding to position 239 with Q. d) For D1, three or more substitutions selected from the following: substitution of the amino acid residue at the position corresponding to position 178 with H; substitution of the amino acid residue at the position corresponding to position 181 with E or Q; substitution of the amino acid residue at the position corresponding to position 238 with F; and substitution of the amino acid residue at the position corresponding to position 239 with Q. e) For E1, three or more substitutions selected from the following: substitution of the amino acid residue at position 178 with H; substitution of the amino acid residue at position 181 with E or Q; substitution of the amino acid residue at position 238 with F; substitution of the amino acid residue at position 239 with Q; and substitution of the amino acid residue at position 240 with F. 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]

[0010] 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. [Brief explanation of the drawings]

[0011] [Figure 1] Alignment of five parent α-amylases with SEQ ID NO:1 and SEQ ID NO:2. [Figure 2] Alignment of SEQ ID NO:2 with five 2-amino acid deleted α-amylases. 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 variants of the present invention are α-amylase variants A1, B1, C1, D1 and E1, which have as their parent amylase α-amylase A having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 4, α-amylase B having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 6, α-amylase C having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 8, α-amylase D having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 10, or α-amylase E having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 12, and each of A1 to E1 contains a deletion of two amino acid residues selected from positions 178-181 in the numbering of SEQ ID NO: 1, and contains substitutions of at least the following amino acid residues at three or more positions selected from positions 178, 181, 238, 239, 240, 126, 129 and 201 in the numbering of SEQ ID NO: 2. a) For A1, substitution of the amino acid residue at the position corresponding to position 178 with H, substitution of the amino acid residue at the position corresponding to position 181 with E or Q, and substitution of the amino acid residue at the position corresponding to position 239 with Q b) For B1, substitution of the amino acid residue at the position corresponding to position 178 with H, substitution of the amino acid residue at the position corresponding to position 181 with E or Q, and substitution of the amino acid residue at the position corresponding to position 239 with Q c) For C1, three or more substitutions selected from the following: substitution of the amino acid residue at the position corresponding to position 178 with H; substitution of the amino acid residue at the position corresponding to position 181 with E or Q; substitution of the amino acid residue at the position corresponding to position 238 with F; and substitution of the amino acid residue at the position corresponding to position 239 with Q. d) For D1, three or more substitutions selected from the following: substitution of the amino acid residue at the position corresponding to position 178 with H; substitution of the amino acid residue at the position corresponding to position 181 with E or Q; substitution of the amino acid residue at the position corresponding to position 238 with F; and substitution of the amino acid residue at the position corresponding to position 239 with Q. e) For E1, three or more substitutions selected from the following: substitution of the amino acid residue at position 178 with H; substitution of the amino acid residue at position 181 with E or Q; substitution of the amino acid residue at position 238 with F; substitution of the amino acid residue at position 239 with Q; and substitution of the amino acid residue at position 240 with F.

[0020] In other words, a "variant" refers to a polypeptide having α-amylase activity in which, in the amino acid sequence constituting the parent α-amylases A to E, two amino acid residues selected from positions corresponding to 178-181 in the numbering of SEQ ID NO: 1 have been deleted, and at least one of the amino acid residues a) to e) above has been substituted at three or more positions selected from positions corresponding to 178, 181, 238, 239, 240, 126, 129, and 201 in the numbering of SEQ ID NO: 2. The deletion and substitution of amino acid residues at such specific positions are modifications intended to improve stability at low pH and / or in the presence of chelating agents, and therefore the mutant has improved stability at low pH and / or in the presence of chelating agents 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, and in the present invention is α-amylase A having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4, α-amylase B having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6, α-amylase C having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8, α-amylase D having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10, or α-amylase E having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 12. Such parent amylases may be native (wild-type) polypeptides or variants thereof.

[0022] α-Amylase A consisting of the amino acid sequence shown in SEQ ID NO: 4 is an α-amylase LABM derived from the A and B domains of α-amylase AAI10 derived from the genus Bacillus and the α-amylase C domain of Alicyclobacillus species. The α-amylase B consisting of the amino acid sequence shown in SEQ ID NO: 6 is the α-amylase CspAmy2 derived from the genus Cytophaga. The α-amylase C consisting of the amino acid sequence shown in SEQ ID NO: 8 is α-amylase AA560 derived from Bacillus sp. DSM12649 strain. The α-amylase D consisting of the amino acid sequence shown in SEQ ID NO: 10 is α-amylase SP722 derived from Bacillus sp. SP722 strain. The α-amylase E consisting of the amino acid sequence shown in SEQ ID NO: 12 is α-amylase AP1378 derived from Bacillus sp. KSM-1378 (FERM BP-3048) strain.

[0023] Parent α-amylases include polypeptides having 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: 4, 6, 8, 10 or 12.

[0024] In mutants A1, B1, C1, D1 and E1 of the present invention, the modified amino acid residue sites (mutation positions) are deletion of two amino acid residues selected from positions corresponding to positions 178-181 in the numbering of SEQ ID NO: 1, and substitution of amino acid residues at three or more positions selected from positions corresponding to positions 178, 181, 238, 239, 240, 126, 129 and 201 in the numbering of SEQ ID NO: 2. Here, SEQ ID NO: 1 is the amino acid sequence constituting α-amylase YR288 (International Publication No. 2022 / 017728) registered as WP_100346362.1 in the NCBI protein sequence database, and SEQ ID NO: 2 is the amino acid sequence constituting α-amylase in which the amino acid residues corresponding to R178 and T180 in the amino acid sequence shown in SEQ ID NO: 1 are deleted (R178Δ+T180Δ). Figure 1 shows an alignment of the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2 with the amino acid sequences of the parent α-amylases A, B, C, D, and E. Figure 2 also shows an alignment of the amino acid sequence of SEQ ID NO: 2 with the amino acid sequences of the parent α-amylases A, B, C, D, and E, in which two amino acid residues corresponding to positions 178 and 180 in the numbering of SEQ ID NO: 1 have been deleted. For parent α-amylases A to E, the correspondence between the amino acid residues at positions 178-181 in SEQ ID NO: 1 and the amino acid residues at positions 178, 181, 238, 239, 240, 126, 129, and 201 in SEQ ID NO: 2 is shown in Table 1 below.

[0025] [Table 1]

[0026] 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.

[0027] 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.

[0028] In the mutants A1, B1, C1, D1 and E1 of the present invention, the deletion of two amino acid residues selected from positions corresponding to positions 178-181 in the numbering of SEQ ID NO: 1 is preferably 178Δ+180Δ, 179Δ+180Δ, 178Δ+179Δ, 178Δ+181Δ, 179Δ+181Δ, etc., with 178Δ+180Δ being more preferred.

[0029] In the mutants A1, B1, C1, D1 and E1 of the present invention, the "substitution" of amino acid residues at three or more positions selected from positions corresponding to 178, 181, 238, 239, 240, 126, 129 and 201 in the numbering of SEQ ID NO: 2 means replacing the amino acid at that position with a specific amino acid, and for A1, B1, C1, D1 and E1, this includes at least the following aspects a) to e). The number of amino acid residue substitutions may be three or more, but may also be four or more, five or more, or six or more from the viewpoint of stability at low pH and / or in the presence of a chelating agent.

[0030] a) In A1, there are three substitutions (178H+181E or 181Q+239Q): substitution of the amino acid residue at position 178 with H (178H), substitution of the amino acid residue at position 181 with E or Q (181E or 181Q), and substitution of the amino acid residue at position 239 with Q (239Q). b) In B1, there are three substitutions (178H+181E or 181Q+239Q): substitution of the amino acid residue at position 178 with H (178H), substitution of the amino acid residue at position 181 with E or Q (181E or 181Q), and substitution of the amino acid residue at position 239 with Q (239Q). c) In C1, three or more substitutions selected from the following: substitution of the amino acid residue at position 178 with H (178H), substitution of the amino acid residue at position 181 with E or Q (181E or 181Q), substitution of the amino acid residue at position 238 with F (238F), and substitution of the amino acid residue at position 239 with Q (239Q). For example, four substitutions: 178H + 181E or 181Q + 238F + 239Q. d) In D1, three or more substitutions selected from the following: substitution of the amino acid residue at position 178 with H (178H), substitution of the amino acid residue at position 181 with E or Q (181E or 181Q), substitution of the amino acid residue at position 238 with F (238F), and substitution of the amino acid residue at position 239 with Q (239Q). For example, four substitutions: 178H + 181E or 181Q + 238F + 239Q. e) In E1, three or more substitutions selected from the group consisting of substitution of the amino acid residue at position 178 with H (178H), substitution of the amino acid residue at position 181 with E or Q (181E or 181Q), substitution of the amino acid residue at position 238 with F (238F), substitution of the amino acid residue at position 239 with Q (239Q), and substitution of the amino acid residue at position 240 with F. For example, quadruple substitutions of 178H+181E or 181Q+239Q+240F, and quintuple substitutions of 178H+181E+238F+239Q+240F.

[0031] In the above a) to e), if the amino acid residue at position 178 in the numbering of SEQ ID NO: 2 in the corresponding parent amylase A, B, C, D, or E is H, the amino acid residue at position 181 is E or Q, the amino acid residue at position 238 is F, the amino acid residue at position 239 is Q, or the amino acid residue at position 240 is F, the amino acid residues before and after the substitution will be identical, and such embodiments are also encompassed by the mutants of the present invention.

[0032] In addition to the substitutions of the amino acid residues a) to e) above, it is also preferable to further combine substitutions of one or more amino acid residues selected from positions corresponding to 126, 129, and 201, in order to improve stability at low pH and / or in the presence of a chelating agent. Such substitutions include substitution of the amino acid residue at position 126 with Y (126Y), substitution of the amino acid residue at position 129 with I (129I), and substitution of the amino acid residue at position 201 with L or Y (201L / Y). For example, in A1, B1, and C1, preferred examples include those in which two substitutions of 126Y+129I or one substitution of 201L / Y are further added to the above amino acid residue substitutions, In D1, preferred examples include those in which one substitution of 126Y or 201L / Y has been added to the above amino acid residue substitutions.

[0033] 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 variant 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 of the parent amylase. Furthermore, a mutant may contain any number of conservative amino acid substitutions, so long as the above-mentioned properties of the mutant are maintained.

[0034] <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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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).

[0039] Template DNA containing the parent gene can be prepared from the aforementioned α-amylase-producing microorganisms 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 the template DNA. The DNA sequence encoding α-amylase A (LABM) consisting of the amino acid sequence shown in SEQ ID NO:4 is shown in SEQ ID NO:3. The DNA sequence encoding α-amylase B (CspAmy2) consisting of the amino acid sequence shown in SEQ ID NO:6 is shown in SEQ ID NO:5. The DNA sequence encoding α-amylase C (AA560) consisting of the amino acid sequence shown in SEQ ID NO:8 is shown in SEQ ID NO:7. The DNA sequence encoding α-amylase D (SP722) consisting of the amino acid sequence shown in SEQ ID NO:10 is shown in SEQ ID NO:9. The DNA sequence encoding α-amylase E (AP1378) consisting of the amino acid sequence shown in SEQ ID NO:12 is shown in SEQ ID NO:11.

[0040] 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.

[0041] 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 / ]).

[0042] <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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] <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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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 (%).

[0057] The mutant of the present invention is useful as an enzyme to be incorporated into various detergent compositions, and is particularly suitable for low-temperature washing at 40°C or below, and is useful as an enzyme to be incorporated into detergent compositions that are acidic and contain a chelating agent.

[0058] 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.

[0059] 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 alkaline cellulase produced by Bacillus sp. KSM-S237 strain described in JP-A-2003-313592. Examples include mutant alkaline cellulases (all manufactured by Kao). 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).

[0060] The detergent composition may contain known detergent components, and examples of such known detergent components include the following:

[0061] (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.

[0062] 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.

[0063] 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.

[0064] 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).

[0065] (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.

[0066] (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.

[0067] (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.

[0068] (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.

[0069] (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.

[0070] (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.

[0071] (8) Buffering agents 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.

[0072] (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.

[0073] 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.

[0074] 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).

[0075] In relation to the above-described embodiment, the present invention further discloses the following aspects. <1> α-Amylase A having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO:4, α-Amylase B having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO:6, α-Amylase C having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO:8, α-Amylase D having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO:10, or α-Amylase D having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO:12. The α-amylase variants A1, B1, C1, D1 and E1 each have α-amylase E having the above structure as a parent amylase, and each of A1 to E1 contains deletion of two amino acid residues selected from positions corresponding to 178-181 in the numbering of SEQ ID NO: 1, and substitution of amino acid residues at three or more positions selected from positions corresponding to 178, 181, 238, 239, 240, 126, 129 and 201 in the numbering of SEQ ID NO: 2, and contain at least the following a) to e): a) For A1, substitution of the amino acid residue at the position corresponding to position 178 with H, substitution of the amino acid residue at the position corresponding to position 181 with E or Q, and substitution of the amino acid residue at the position corresponding to position 239 with Q b) For B1, substitution of the amino acid residue at the position corresponding to position 178 with H, substitution of the amino acid residue at the position corresponding to position 181 with E or Q, and substitution of the amino acid residue at the position corresponding to position 239 with Q c) For C1, three or more substitutions selected from the following: substitution of the amino acid residue at the position corresponding to position 178 with H; substitution of the amino acid residue at the position corresponding to position 181 with E or Q; substitution of the amino acid residue at the position corresponding to position 238 with F; and substitution of the amino acid residue at the position corresponding to position 239 with Q. d) For D1, three or more substitutions selected from the following: substitution of the amino acid residue at the position corresponding to position 178 with H; substitution of the amino acid residue at the position corresponding to position 181 with E or Q; substitution of the amino acid residue at the position corresponding to position 238 with F; and substitution of the amino acid residue at the position corresponding to position 239 with Q. e) For E1, three or more substitutions selected from the following: substitution of the amino acid residue at position 178 with H; substitution of the amino acid residue at position 181 with E or Q; substitution of the amino acid residue at position 238 with F; substitution of the amino acid residue at position 239 with Q; and substitution of the amino acid residue at position 240 with F. <2> the substitutions for C1 are four substitutions: substitution of the amino acid residue at the position corresponding to position 178 in the numbering of SEQ ID NO:2 with H, substitution of the amino acid residue at the position corresponding to position 181 with E or Q, substitution of the amino acid residue at the position corresponding to position 238 with F, and substitution of the amino acid residue at the position corresponding to position 239 with Q; D1 has four substitutions: an amino acid residue at a position corresponding to position 178 in the numbering of SEQ ID NO:2 is substituted with H, an amino acid residue at a position corresponding to position 181 is substituted with E or Q, an amino acid residue at a position corresponding to position 238 is substituted with F, and an amino acid residue at a position corresponding to position 239 is substituted with Q; the substitutions for E1 are four or more substitutions selected from the group consisting of substitution of the amino acid residue at the position corresponding to position 178 in SEQ ID NO: 2 with H, substitution of the amino acid residue at the position corresponding to position 181 with E or Q, substitution of the amino acid residue at the position corresponding to position 238 with F, substitution of the amino acid residue at the position corresponding to position 239 with Q, and substitution of the amino acid residue at the position corresponding to position 240 with F; <1> α-amylase variants. <3> The substitutions for E1 are five substitutions: a substitution of the amino acid residue at the position corresponding to position 178 in the numbering of SEQ ID NO: 2 with H; a substitution of the amino acid residue at the position corresponding to position 181 with E or Q; a substitution of the amino acid residue at the position corresponding to position 238 with F; a substitution of the amino acid residue at the position corresponding to position 239 with Q; and a substitution of the amino acid residue at the position corresponding to position 240 with F. <2> α-amylase variants. <4> In the above a) to e), the substitution of amino acid residues at one or more positions selected from positions corresponding to 126, 129 and 201 in the numbering of SEQ ID NO: 2 is further included. <1> ~ <3> any of the α-amylase variants. <5> the amino acid residues after substitution at positions corresponding to positions 126, 129, and 201 of the α-amylase are 126Y, 129I, and 201L / Y, respectively; <4> α-amylase variants. <6> <1> ~ <5> A polynucleotide encoding any variant of any of the above. <7> <6> A vector or DNA fragment comprising the polynucleotide described in . <8> <7> A transformed cell containing the vector or DNA fragment described in 1. <9> It is a microorganism, <8> Transformed cells. <10> <1> ~ <5> A detergent composition comprising the variant described in any one of the above. <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> The cleaning composition according to any one of the preceding claims. <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]

[0076] (1) Construction of template α-amylase expression plasmid PCR was performed using the artificially synthesized LABM gene (SEQ ID NO: 3) as a template and the primer pair LABM_fw / LABM_rv (SEQ ID NOs: 15 and 16) and PrimeSTARMax Premix (Takara Bio). PCR was similarly performed using the plasmid pHY-S237 described in Example 7 of International Publication No. 2006 / 068148 as a template and the primer pair S237t_fw / S237s_rv (SEQ ID NOs: 13 and 14). In-Fusion reactions were performed using each PCR product according to the protocol of the In-Fusion, HD Cloning kit (Clontech). Plasmid pHY-LABM was constructed by transforming Bacillus subtilis with the In-Fusion reaction mixture. Furthermore, the plasmids pHY-Cspamy2, pHY-AA560, pHY-SP722, and pHY-AP1378 described in Japanese Patent Application No. 2021-112712 were used to express Cspamy2, AA560, SP722, and AP1378.

[0077] Furthermore, using each of these five plasmids as a template, deletions were made at positions 178 and 180 in SEQ ID NO: 1. Specifically, PCR was performed using pHY-LABM as a template, the primer pair LABM_181-183del_fw / LABM_181-183del_rv (SEQ ID NOs: 17 and 18), and PrimeSTARMax Premix (Takara Bio). Subsequently, an In-Fusion reaction was performed in the same manner as above, and Bacillus subtilis was transformed to construct the plasmid pHY-LABM R181Δ D183Δ. Similarly, PCR and In-Fusion reactions were performed for Cspamy2, AA560, SP722, and AP1378 using pHY-Cspamy2, pHY-AA560, pHY-SP722, and pHY-AP1378 as templates, respectively, and the primer pairs Cspamy2_178-180del_fw / Cspamy2_178-180del_rv (sequence numbers 19 and 20), AA560_181-183del_fw / AA560_181-183del_rv (sequence numbers 21 and 22), SP722_181-183del_fw / SP722_181-183del_rv (sequence numbers 23 and 24), and AP1378_181-183del_fw / AP1378_181-183del_rv (sequence numbers 25 and 26). Bacillus subtilis was transformed with the In-Fusion reaction mixture to construct the plasmids pHY-Cspamy2 R178Δ T180Δ, pHY-AA560 R181Δ D183Δ, pHY-SP722 R181Δ D183Δ, and pHY-AP1378 R181Δ T183Δ.

[0078] (2) Construction of α-amylase mutant expression plasmids Using the above-described pHY-LABM R181Δ D183Δ, pHY-Cspamy2 R178Δ T180Δ, pHY-AA560 R181Δ D183Δ, pHY-SP722 R181Δ D183Δ, or pHY-AP1378 R181Δ T183Δ as templates, α-amylase mutants were constructed as follows. PCR was performed using a pair of mutagenesis primers: a forward primer containing the mutated sequence and a 15-nucleotide sequence complementary to the reverse primer at its 5' end, and a reverse primer with the base immediately preceding the mutated sequence at its 5' end. When multiple fragments were ligated, each PCR product was used in an In-Fusion reaction according to the protocol for the In-Fusion, HD Cloning kit (Clontech). Bacillus subtilis was transformed by the protoplast method using the PCR products or the In-Fusion reaction mixture to obtain transformants harboring the desired α-amylase mutant expression plasmid. Alignments of SEQ ID NO:1 and SEQ ID NO:2 with the five parent α-amylases, created using GENETYX ver. 12, are shown in Figure 1, and alignments of SEQ ID NO:2 with the five two-amino acid deletion α-amylases are shown in Figure 2. Using the numbering of SEQ ID NO:2, residues corresponding to positions 126, 129, 178, 181, 201, 238, 239, and 240 are indicated with red triangles, with the amino acid residue numbers in SEQ ID NO:2 written above. α-Amylase variants shown in Tables 2-1 and 2-2, which contain substitutions at positions selected from these residues, were constructed using the methods described above.

[0079] [Table 2-1]

[0080] [Table 2-2]

[0081] (3) 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.

[0082] (4) Evaluation of amylase mutant stability in the presence of chelating agents The storage stability of the mutants and each parent enzyme listed in Table 2 in the presence of a chelating agent was evaluated. Culture supernatant diluted 5-fold with ion-exchanged water was added to a 10-fold dilution of AATCC High Efficiency Standard Reference Detergent WITHOUT Brightener in 100 mM citrate buffer (pH 5.0). The mixture was incubated at 50°C for 10 minutes to 1 hour, and then diluted 10-fold with ion-exchanged water. Activity was measured using the same method. For stability comparison, a solution of Termamyl 300L (Sigma-Aldrich) diluted to 200 ppm with ion-exchanged water was also evaluated. The activity of the sample before 50°C treatment was used as the initial activity, and the inactivation rate per unit time (h) following 50°C treatment was calculated. From this, 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 or Termamyl. The results are shown in Tables 3-1 and 3-2. All variants were shown to have improved stability compared to the parent polypeptide and Termamyl.

[0083] [Table 3-1]

[0084] [Table 3-2]

[0085] (5) Evaluation of amylase mutant stability in weakly acidic diluted detergents A 10-fold dilution of AATCC High Efficiency Standard Reference Detergent WITHOUT Brightener with 20 mM citrate buffer (pH 5.0) was added to a culture supernatant diluted 5-fold with ion-exchanged water, and the mixture was incubated at 50°C for 30 minutes to 18 hours. The resulting mixture was then diluted 10-fold with 50 mM Tris-HCl (pH 7.5), and activity was measured. The relative stability of the mutants was then determined using the same method as in (4). The results are shown in Tables 4-1 and 4-2. All mutants demonstrated improved stability compared to the parent polypeptide and Termamyl.

[0086] [Table 4-1]

[0087] [Table 4-2]

Claims

1. α-amylase A having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, α-amylase B having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 6, α-amylase C having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 8, α-amylase D having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 10, or α-amylase A having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 12 α-amylase mutants A1, B1, C1, D1, and E1, each having α-amylase E as its parent amylase, wherein A1 to E1 each contain deletions of two amino acid residues selected from positions corresponding to 178-181 in the numbering of SEQ ID NO: 1, and substitutions of three or more amino acid residues selected from positions corresponding to 178, 181, 238, 239, 240, 126, 129, and 201 in the numbering of SEQ ID NO: 2, and each of these mutants contains at least the following a) to e). a) For A1, substitution of an amino acid residue with H at the position corresponding to 178, substitution of an amino acid residue with E or Q at the position corresponding to 181, and substitution of an amino acid residue with Q at the position corresponding to 239. b) For B1, substitution of an amino acid residue with H at the position corresponding to 178, substitution of an amino acid residue with E or Q at the position corresponding to 181, and substitution of an amino acid residue with Q at the position corresponding to 239. c) For C1, three or more substitutions selected from the following: substitution of an amino acid residue with H at the position corresponding to position 178, substitution of an amino acid residue with E or Q at the position corresponding to position 181, substitution of an amino acid residue with F at the position corresponding to position 238, and substitution of an amino acid residue with Q at the position corresponding to position 239. d) For D1, three or more substitutions selected from the following: substitution of an amino acid residue with H at the position corresponding to position 178, substitution of an amino acid residue with E or Q at the position corresponding to position 181, substitution of an amino acid residue with F at the position corresponding to position 238, and substitution of an amino acid residue with Q at the position corresponding to position 239. e) For E1, three or more substitutions selected from the following: substitution of an amino acid residue with H at the position corresponding to 178, substitution of an amino acid residue with E or Q at the position corresponding to 181, substitution of an amino acid residue with F at the position corresponding to 238, substitution of an amino acid residue with Q at the position corresponding to 239, and substitution of an amino acid residue with F at the position corresponding to 240.

2. The substitutions for C1 consist of four substitutions: a substitution of an amino acid residue with H at the position corresponding to position 178 in the numbering of Sequence ID No. 2; a substitution of an amino acid residue with E or Q at the position corresponding to position 181; a substitution of an amino acid residue with F at the position corresponding to position 238; and a substitution of an amino acid residue with Q at the position corresponding to position 239. The substitutions for D1 are four substitutions: a substitution of an amino acid residue with H at the position corresponding to position 178 in the numbering of Sequence ID No. 2, a substitution of an amino acid residue with E or Q at the position corresponding to position 181, a substitution of an amino acid residue with F at the position corresponding to position 238, and a substitution of an amino acid residue with Q at the position corresponding to position 239. The α-amylase mutant according to claim 1, wherein the substitution of E1 is four or more substitutions selected from among the substitution of amino acid residue H at the position corresponding to position 178 in the numbering of Sequence ID No. 2, substitution of amino acid residue E or Q at the position corresponding to position 181, substitution of amino acid residue F at the position corresponding to position 238, substitution of amino acid residue Q at the position corresponding to position 239, and substitution of amino acid residue F at the position corresponding to position 240.

3. The α-amylase mutant according to claim 2, wherein the substitutions for E1 are five substitutions: substitution with amino acid residue H at the position corresponding to position 178 in the numbering of Sequence ID No. 2, substitution with amino acid residue E or Q at the position corresponding to position 181, substitution with amino acid residue F at the position corresponding to position 238, substitution with amino acid residue Q at the position corresponding to position 239, and substitution with amino acid residue F at the position corresponding to position 240.

4. The α-amylase mutant according to claim 1, further comprising substitution of an amino acid residue at one or more positions selected from positions corresponding to positions 126, 129, and 201 in the numbering of Sequence ID No. 2, in a) to e) above.

5. The α-amylase mutant according to claim 2, further comprising substitution of an amino acid residue at one or more positions selected from positions corresponding to positions 126, 129 and 201 in the numbering of Sequence ID No. 2, in a) to e) above.

6. The α-amylase mutant according to claim 3, further comprising substitution of an amino acid residue at one or more positions selected from positions corresponding to positions 126, 129 and 201 in the numbering of Sequence ID No. 2, in a) to e) above.

7. The α-amylase mutant according to claim 4, wherein the substituted amino acid residues at the positions corresponding to positions 126, 129, and 201 of the α-amylase are 126Y, 129I, and 201L / Y, respectively.

8. The α-amylase mutant according to claim 5, wherein the substituted amino acid residues at the positions corresponding to positions 126, 129, and 201 of the α-amylase are 126Y, 129I, and 201L / Y, respectively.

9. The α-amylase mutant according to claim 6, wherein the substituted amino acid residues at the positions corresponding to positions 126, 129, and 201 of the α-amylase are 126Y, 129I, and 201L / Y, respectively.

10. A polynucleotide encoding the mutant according to any one of claims 1 to 9.

11. A vector or DNA fragment comprising the polynucleotide described in claim 10.

12. A transformed cell containing the vector or DNA fragment described in claim 11.

13. A transformed cell according to claim 12, which is a microorganism.

14. A detergent composition comprising the variant described in any one of claims 1 to 9.

15. The detergent composition according to claim 14, which is a laundry detergent or a dishwashing detergent.

16. The detergent composition according to claim 15, which is a powder or a liquid.

17. The detergent composition according to claim 15, which is weakly acidic.

18. A detergent composition according to claim 17, comprising a chelating agent.

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