Amylase-containing detergent composition

Abstract

To provide an α-amylase-blended detergent composition that shows high starch decomposition activity at a low temperature, and shows high stability even in a composition containing protease.SOLUTION: A detergent composition includes an α-amylase variant and protease, where the α-amylase variant is a variant of a parent α-amylase including a substitution of an amino acid residue at one or more positions selected from specific two positions of a specific amino acid sequence, and furthermore a substitution of an amino acid residue at specific one or more positions, and the parent α-amylase or the α-amylase variant has at least 85% of sequence identity to the specific amino acid sequence.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a detergent composition containing α - amylase and protease.

Background Art

[0002] α - Amylase is used in a wide range of industrial fields such as the starch industry, brewing industry, textile industry, pharmaceutical industry, and food industry. In addition, its suitability for blending into detergents is known, and it is blended into dishwashing detergents for automatic dishwashers and laundry detergents as a component for removing starch - based stains.

[0003] Useful α - amylases for detergents include α - amylase AP1378 derived from Bacillus sp. KSM - 1378 (FERM BP - 3048) (Patent Document 1), Termamyl and Duramyl (registered trademarks), which are α - amylases derived from Bacillus licheniformis. In addition, α - amylase AA560 derived from Bacillus sp. DSM12649 (Patent Document 2), α - amylase SP722 (SEQ ID NO: 4 in Patent Document 3) derived from Bacillus sp. SP722, and α - amylase CspAmy2 derived from the genus Cytophaga (Patent Document 4) are known. Further, these α - amylases have been modified to improve their functions for specific applications. For example, mutants with improved stability in detergents have been reported (Patent Document 5).

[0004] In recent years, from the viewpoints of environmental protection and reduction of washing costs, it is important to lower the temperature during dishwashing, laundry washing, especially laundry washing in a laundry. Also, shortening the washing time is desired. However, the optimum temperature of most enzymes containing amylase is higher than the temperature usually set in low - temperature washing. Therefore, it is difficult to completely remove many starch - based stains. Under such circumstances, the present applicant has found an α - amylase that retains washing performance and starch - degrading activity even at low temperatures and has a high stain - removing effect (Patent Document 6).

[0005] Furthermore, since many starch-containing stains also contain protein components, it is known that using α-amylase and protease together produces an additive-synergistic cleaning effect. Such cleaning agents containing α-amylase and protease are particularly useful for cleaning at low temperatures, where a decrease in cleaning power is a concern. On the other hand, using protease in combination presents the problem of impaired stability of amylase in the cleaning 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] International Publication No. 98 / 044126 [Patent Document 6] Japanese Patent Application No. 2021-112712 [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] The present invention provides an α-amylase-containing detergent composition that exhibits high starch-degrading activity at low temperatures and high stability even in compositions containing proteases. [Means for solving the problem]

[0008] The inventors have discovered that a specific α-amylase variant, which exhibits excellent cleaning performance and stability even at low temperatures, maintains its performance even when used in combination with a protease, and is useful as an enzyme to be incorporated into cleaning agents suitable for low-temperature cleaning.

[0009] In other words, the present invention relates to the following: A detergent composition comprising an α-amylase variant and a protease, wherein the α-amylase variant is a variant of the parent α-amylase comprising the substitution of an amino acid residue at one or more positions selected from positions corresponding to E187 and S241 of the amino acid sequence shown in SEQ ID NO: 2, and further the substitution of an amino acid residue at one or more positions selected from positions corresponding to N192, H240 and K278, and the parent α-amylase or α-amylase variant has at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 4. [Effects of the Invention]

[0010] According to the present invention, it is possible to provide a highly storage-stable detergent composition that contains α-amylase and protease, exhibiting excellent starch stain removal effects even at low temperatures. [Brief explanation of the drawing]

[0011] [Figure 1] Evaluation of amylase washing power after storage in a protease-containing washing composition. [Modes for carrying out the invention]

[0012] In this invention, "amylase" (EC3.2.1.1; 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-glycosidoligosaccharides or polysaccharides. α-amylase activity can be determined by measuring the amount of reducing end produced by the enzymatic degradation of starch. However, it is not limited to this, and can also be determined by measuring the release of dye by the enzymatic degradation of dye-crosslinked 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] In this invention, the identity of amino acid sequences or nucleotide sequences is calculated by the Lipman-Pearson method (Science, 1985, 227:1435-1441). Specifically, it is calculated by performing the analysis using the homology analysis (Search homology) program of the genetic information processing software GENETYX Ver.12 with a unit size to compare (ktup) of 2.

[0014] In the present invention, "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 invention, the positions and variants of amino acids are described using the officially recognized IUPAC single-letter amino acid abbreviations, as follows: The amino acid at a specific position is denoted as [amino acid, position]. For example, threonine at position 226 is shown as "T226". Amino acid substitutions are indicated as [original amino acid, position, substituted amino acid]. For example, the substitution of threonine at position 226 with alanine is shown as "T226A". Amino acid "deletions" are indicated as [original amino acid, position, Δ]. For example, a serine deletion at position 181 is shown as "S181Δ". Regarding amino acid "insertions," they are indicated as [original amino acid, position, original amino acid, inserted amino acid]. For example, the insertion of lysine after glycine at position 195 is shown as "G195GK". Insertions of multiple amino acids are indicated as [original amino acid, position, original amino acid, inserted amino acid 1, inserted amino acid 2; etc.]. For example, the insertion of lysine and alanine after glycine at position 195 is shown as "G195GKA". Mutants containing multiple modifications are denoted by the addition sign ("+"). For example, "R170Y+G195E" represents the substitution of arginine at position 170 with tyrosine and the substitution of glycine at position 195 with glutamate, respectively. When different modifications can be introduced at one position, the different modifications are separated by a slash (" / "). For example, "R170Y / E" represents the substitution of arginine at position 170 with tyrosine or glutamic acid.

[0016] In the present invention, "operable linkage" between a control region such as a promoter and a gene means that the gene and the control region are linked so that the gene can be expressed under the control of the control region. The procedure for "operable linkage" between a gene and a control region is well known to those skilled in the art.

[0017] In the present invention, "upstream" and "downstream" with respect 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 present on the 3'-side of the promoter in the DNA sense strand, and upstream of the gene means the region on the 5'-side of the gene in the DNA sense strand.

[0018] In the present invention, the term "native" used for the functions, properties, and traits of cells is used to represent that the functions, properties, and traits originally exist in the cells. In contrast, the term "foreign" is used to represent functions, properties, and traits that are not originally present in the cells but are introduced from the outside. For example, a "foreign" gene or polynucleotide is a gene or polynucleotide introduced from the outside into a cell. The foreign gene or polynucleotide may be derived from the same species of organism as the cell into which it is introduced or from a different species of organism (i.e., a heterologous gene or polynucleotide).

[0019] The α-amylase variant (hereinafter also referred to as "the variant of the present invention") formulated in the detergent composition of the present invention is a variant of a parent α-amylase, which includes substitution of amino acid residues at one or more positions selected from the positions corresponding to positions E187 and S241 of the amino acid sequence shown in SEQ ID NO: 2, and further substitution of amino acid residues at one or more positions selected from the positions corresponding to positions N192, H240, and K278. In other words, the variant of the present invention refers to a polypeptide having α-amylase activity in which multiple amino acid residues at predetermined positions in the amino acids constituting the parent α-amylase are substituted with other amino acid residues. Such substitution of amino acid residues at predetermined positions is a modification to improve washing performance and / or stability in detergents containing proteases, and therefore, the variant has improved washing performance and / or stability compared to the parent α-amylase.

[0020] In the variant of the present invention, the amino acid residue modification sites (mutation sites) are one or more positions selected from the positions corresponding to E187 and S241 of the amino acid sequence shown in Sequence ID No. 2, and one or more positions selected from the positions corresponding to N192, H240 and K278.

[0021] Furthermore, in addition to the above-mentioned combinations of modifications, the variants of the present invention may also include substitutions of amino acid residues at one or more positions selected from those corresponding to positions F205, R211, and M199 of the amino acid sequence shown in SEQ ID NO: 2. Furthermore, it may also include substitutions of amino acid residues at one or more positions selected from those corresponding to positions G5, S38, Q96, W186, E257, F259, S284, H295, Y296, N303, T320, Y360, W408, L429, V430, G433, A434, W439, N471, G476, and G477 of the amino acid sequence shown in Sequence ID No. 2.

[0022] Here, the amino acid sequence shown in Sequence ID No. 2 is the amino acid sequence that constitutes α-amylase YR288, and the mutation sites in the mutant of the present invention are numbered based on the amino acid numbers of the said amino acid sequence. YR288 is a protein registered as WP_100346362.1 in the NCBI protein sequence database, and has been identified by the applicant as an α-amylase with high starch degradation activity and washing performance at low temperatures (Japanese Patent Application No. 2020-121626).

[0023] The "corresponding position" on the amino acid sequence can be determined by aligning the target sequence and the reference sequence (the amino acid sequence shown in Sequence ID No. 2 in this invention) to give the greatest possible homology. Amino acid sequence alignment can be performed using known algorithms, and the procedure is known to those skilled in the art. For example, alignment can be performed using the Clustal W multiple alignment program (Thompson, J. Det. al., 1994, Nucleic Acids Res. 22:4673-4680) with default settings. Alternatively, revised versions of Clustal W, such as Clustal W2 or Clustal omega, can be used. Clustal W, Clustal W2, and Clustal omega are, for example, developed by the European Bioinformatics Institute (European Bioinformatics Institute). This information can be accessed through the websites of the Bioinformatics Institute (EBI) [www.ebi.ac.uk / index.html] and the DNA Databank 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 method is considered to be the "corresponding position" to that arbitrary position.

[0024] Those skilled in the art can further fine-tune the amino acid sequence alignment obtained above to optimize it. Such an optimal alignment is preferably determined by considering the similarity of the amino acid sequences and the frequency of inserted gaps. Here, similarity of amino acid sequences refers to the ratio (%) of the number of positions where identical or similar amino acid residues exist in both sequences when two amino acid sequences are aligned, relative to the total number of amino acid residues. Similar amino acid residues refer to amino acid residues among the 20 amino acids that make up a protein that have similar properties in terms of polarity and charge, resulting in so-called conservative substitutions. Groups consisting of such similar amino acid residues are well known to those skilled in the art, and examples 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; leucine and isoleucine, etc.

[0025] In this invention, "parent α-amylase" means a reference α-amylase that is modified to produce the variant of the present invention. The parent may be a natural (wild-type) polypeptide or a variant thereof.

[0026] In the present invention, the parent α-amylase or α-amylase mutant has at least 85%, preferably at least 90%, more 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 in its amino acid sequence with the amino acid sequence shown 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 are deleted from the α-amylase (YR288) consisting of the amino acid sequence shown in SEQ ID NO: 2. A mutant of YR288 as the parent α-amylase, which includes the deletion of amino acid residues at two positions selected from the positions corresponding to R178, G179, T180, and G181 in the amino acid sequence shown in SEQ ID NO: 2, exhibits dramatically improved stability in detergents compared to YR288 (see Patent Document 6). Therefore, α-amylase mutants obtained by deleting two or more amino acid residues selected from 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%, and more preferably at least 99% identity therewith, including the α-amylase consisting of the amino acid sequence shown in SEQ ID NO: 4, can all be parent α-amylase mutants of the present invention. Here, examples of deletions of two or more amino acid residues include R178Δ+T180Δ, G179Δ+T180Δ, R178Δ+G179Δ, R178Δ+G181Δ, G179Δ+G181Δ, and so on, with R178Δ+T180Δ being more preferred.

[0027] Other α-amylases that have an amino acid sequence with at least 90% identity to the amino acid sequence shown in Sequence 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. (Japanese Patent Application No. 2020-121626).

[0028] The substitution of amino acid residues at the predetermined positions described above means replacing an amino acid at a certain position with a different amino acid. In the present invention, there are two or more mutation sites, but there is no limit to the number as long as improved cleaning performance and / or stability in detergents containing protease are ensured. Examples include 3, 4, 5, 6, 7, 8 sites, etc., and there may be 10 or more sites. For example, there may be 2 to 15 sites, 2 to 10 sites, 3 to 8 sites, etc. Furthermore, from the viewpoint of improving washing performance or stability, the mutant is preferably an α-amylase that has at least 90% identity with the amino acid sequence shown in SEQ ID NO: 4. In addition, the mutant may contain any number of conservative amino acid substitutions, as long as it retains the above-mentioned mutant properties.

[0029] The following are preferred embodiments of amino acid residue substitutions at positions E187, S241, N192, H240, and K278 of the amino acid sequence shown in Sequence ID No. 2. For example, E187 is preferably substituted with P (E187P); S241 is preferably substituted with A, Q, D, L, Y, P, H (S241A / Q / D / L / Y / P / H); N192 is preferably substituted with F (N192F); H240 is preferably replaced with F (H240F); 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).

[0030] Furthermore, preferred embodiments of amino acid residue substitutions at positions corresponding to F205, R211, and M199 are as follows. For example, F205 is preferably replaced with Y (F205Y); R211 is preferably substituted with L, V, or I (R211L / V / I); M199 is preferably substituted with L, T, A, N, Q, S, V, or I (M199L / T / A / N / Q / S / V / I).

[0031] Furthermore, preferred embodiments of amino acid residue substitutions at positions corresponding to G5, S38, Q96, W186, E257, F259, S284, H295, Y296, N303, T320, Y360, W408, L429, V430, G433, A434, W439, N471, G476, and G477 are as follows. For example, G5 is preferably substituted with E, D, P, R, or K (G5E / D / P / R / K); S38 is preferably substituted with N (S38N); Q96 is preferably substituted with R or K (Q96R / K); W186 is preferably replaced with L (W186L); E257 is preferably substituted with T (E257T); F259 is preferably replaced with W (F259W); S284 is preferably substituted with W (S284W); 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); Y360 is preferably substituted with C, M, L, or V (Y360C / M / L / V); The W408 position is preferably substituted with P (W408P); L429 is preferably replaced with V (L429V); V430 is preferably replaced with M (V430M); In G433, it is preferable to insert S after G (G433GS); A434 is preferably replaced 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).

[0032] Next, Table 1-1 below shows preferred mutation combinations consisting of amino acid residue substitutions at one or more positions selected from those corresponding to E187 and S241, and amino acid residue substitutions at one or more positions selected from those corresponding to N192, H240, and K278. Furthermore, Table 1-2 below shows examples of suitable mutations that combine substitutions of amino acid residues at one or more positions selected from those corresponding to positions F205, R211, and M199.

[0033] [Table 1-1]

[0034] [Table 1-2]

[0035] The mutants of the present invention can be produced using various mutagenesis techniques known in the art. For example, they can be produced by mutating the polynucleotide encoding the amino acid residue to be modified within the parental α-amylase gene (reference α-amylase gene) encoding the reference amino acid sequence to a polynucleotide encoding the modified amino acid residue, and then expressing the mutant from that mutant gene.

[0036] The polynucleotides encoding the variants of the present invention may be in the form of single-stranded or double-stranded DNA, RNA, or artificial nucleic acids, or they may be cDNA or chemically synthesized DNA that does not contain introns.

[0037] In the present invention, various mutagenesis techniques known in the art can be used as means for mutating the amino acid residues of the parent α-amylase. For example, in a polynucleotide encoding the amino acid sequence of the parent α-amylase (hereinafter also referred to as the parent gene), the nucleotide sequence encoding the amino acid residue to be mutated can be changed to the nucleotide sequence encoding the mutated amino acid residue to obtain a polynucleotide encoding the mutant of the present invention.

[0038] The introduction of a desired mutation into a parent gene can, in principle, be carried out using various site-directed mutagenesis methods well known to those skilled in the art. Site-directed mutagenesis methods can be carried out using any method, such as inverse PCR or annealing. Commercially available site-directed mutagenesis kits (for example, Stratagene's QuickChange II Site-Directed Mutagenesis Kit or QuickChange Multi Site-Directed Mutagenesis Kit) can also be used.

[0039] Site-directed mutagenesis into a parent gene can most commonly be performed using a mutation primer containing the nucleotide mutation to be introduced. This mutation primer should be designed to anneal to a region in the parent gene containing the nucleotide sequence encoding the amino acid residue to be mutated, and to include a nucleotide sequence (codon) that encodes the mutated amino acid residue in place of the original nucleotide sequence (codon). Those skilled in the art can appropriately identify and select the nucleotide sequences (codons) encoding the pre-mutation and post-mutation amino acid residues based on standard textbooks. Alternatively, site-directed mutagenesis can also be performed using a method in which two complementary primers containing the nucleotide mutation to be introduced are used separately to amplify the upstream and downstream sides of the mutation site, and the resulting DNA fragments are then ligated together using SOE (splicing by overlap extension)-PCR (Gene, 1989, 77(1): p61-68).

[0040] Template DNA containing the parent gene can be prepared by extracting genomic DNA from the α-amylase-producing microorganisms described above using conventional methods, or by extracting RNA and synthesizing cDNA by reverse transcription. Alternatively, the corresponding nucleotide sequence may be chemically synthesized based on the amino acid sequence of the parent α-amylase and used as template DNA. Sequence ID No. 3 shows a DNA sequence containing the base sequence encoding α-amylase consisting of the amino acid sequence shown in Sequence ID No. 4, and Sequence ID No. 1 shows a DNA sequence containing the base sequence encoding α-amylase (YR288) consisting of the amino acid sequence shown in Sequence ID No. 2.

[0041] Mutation primers can be prepared by well-known oligonucleotide synthesis methods such as the phosphoramidite method (Nucleic Acids R4esearch, 1989, 17:7059-7071). Such primer synthesis can also be carried out using commercially available oligonucleotide synthesizers (e.g., ABI). By using a primer set containing these mutation primers and introducing site-directed mutagenesis as described above with the parental gene as template DNA, polynucleotides encoding the mutant of the present invention having the desired mutation can be obtained.

[0042] The polynucleotide encoding the mutant of the present invention may include 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 include a nucleotide sequence of the untranslated region (UTR) in addition to the open reading frame (ORF). Furthermore, the polynucleotide may be codon-optimized to suit the species of transformant for producing 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 / ]).

[0043] The polynucleotide encoding the obtained 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 plasmids, phages, phagemids, cosmids, viruses, YAC vectors, or shuttle vectors. The vector is, but is not limited, preferably a vector that can be amplified in bacteria, preferably in Bacillus bacteria (e.g., Bacillus subtilis or its mutants), and more preferably an expression vector that can induce the expression of the transgene in Bacillus bacteria. Among these, a shuttle vector, which can replicate in either Bacillus bacteria or other organisms, can be suitably used for recombinant production of the mutant of the present invention. Examples of preferred vectors include, but are not limited to, pHA3040SP64, pHSP64R, or pASP64 (Patent No. 3492935), shuttle vectors such as 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 usable for transforming Bacillus bacteria such as pUB110 (J Bacteriol, 1978, 134:318-329) and pTA10607 (Plasmid, 1987, 18:8-15). Plasmid vectors derived from E. coli (e.g., pET22b(+), pBR322, pBR325, pUC57, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.) can also be used.

[0044] The above vector may include a DNA region containing a DNA replication initiation region or replication origin. Alternatively, in the above vector, a regulatory sequence such as a promoter region, a terminator region, or a secretion signal region for secreting the expressed protein outside the cell may be operably linked upstream of the polynucleotide encoding the variant of the present invention (i.e., the mutant gene). "Operatably linked" means that the gene and the regulatory region are arranged such that the gene can be expressed under the control of the regulatory region.

[0045] The types of regulatory sequences such as promoter regions, terminators, and secretion signal regions mentioned above are not particularly limited, and commonly used promoters and secretion signal sequences can be appropriately selected and used depending on the host to which the vector is introduced. For example, suitable examples of regulatory sequences that can be incorporated into a vector include the promoter and secretion signal sequence of the cellulase gene of Bacllus sp. KSM-S237 strain.

[0046] Alternatively, the vector of the present invention may further incorporate marker genes (e.g., resistance genes for drugs such as ampicillin, neomycin, kanamycin, and chloramphenicol) for selecting a host into which the vector has been appropriately introduced. Alternatively, when using a nutrient-requiring strain as the host, a gene encoding an enzyme for synthesizing the required nutrients may be incorporated into the vector as a marker gene. Or, when using a selective medium that requires specific metabolism for growth, 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.

[0047] The polynucleotide encoding the variant of the present invention described above, along with the regulatory sequence and marker gene, can be linked by methods known in the art, such as SOE (splicing by overlap extension)-PCR (Gene, 1989, 77:61-68). The procedure for introducing the linked fragment into a vector is well known in the art.

[0048] Transformed cells 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 host genome.

[0049] Examples of host cells include microorganisms such as bacteria and filamentous fungi. Examples of bacteria include Escherichia coli, Staphylococcus, Enterococcus, Listeria, and Bacillus, among which Escherichia coli and Bacillus bacteria (for example, Bacillus subtilis Marburg No. 168 (Bacillus subtilis strain 168) or its mutants) are preferred. Examples of Bacillus subtilis mutants include the KA8AX protease 9-fold knockout strain described in J.Biosci.Bioeng.,2007,104(2):135-143, and the D8PA strain, which is an improved protein folding efficiency of the protease 8-fold knockout strain described in Biotechnol.Lett.,2011,33(9):1847-1852. Examples of filamentous fungi include the genera Trichoderma, Aspergillus, and Rizhopus.

[0050] Methods commonly used in this field, such as protoplast synthesis and electroporation, can be used to introduce the vector into the host. By selecting strains in which the vector has been properly introduced based on indicators such as marker gene expression and nutritional requirements, the desired transformants into which the vector has been introduced can be obtained.

[0051] Alternatively, a fragment comprising a polynucleotide encoding the mutant of the present invention, a regulatory sequence, and a marker gene can be directly introduced into the host genome. For example, a DNA fragment can be constructed by adding sequences complementary to the host genome to both ends of the linked fragment using a method such as SOE-PCR. This fragment can then be introduced into the 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 host genome.

[0052] When a transformant into which the polynucleotide encoding the mutant of the present invention or a vector containing the same has been introduced is cultured in a suitable medium, the gene encoding the protein on the vector is expressed, and the mutant of the present invention is produced. The medium used for culturing the transformant can be appropriately selected by a person skilled in the art, according to the type of microorganism of the transformant.

[0053] Alternatively, the mutants of the present invention may be expressed from polynucleotides encoding the mutants of the present invention or their transcripts using a cell-free translation system. A "cell-free translation system" is an in vitro transcription-translation system or in vitro translation system constructed by mechanically disrupting host cells to obtain a suspension and adding reagents such as amino acids necessary for protein translation.

[0054] The mutants of the present invention generated in the culture or cell-free translation system described above can be isolated or purified by common methods used for protein purification, such as centrifugation, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc., either alone or in appropriate combinations. In this case, if the gene encoding the α-amylase mutant of the present invention and the secretion signal sequence are operably linked on the vector in the transformant, the generated protein is secreted extracellularly and can be more easily recovered from the culture. The protein recovered from the culture may be further purified by known means.

[0055] The variant obtained in this way has improved washing performance and / or stability compared to the parent α-amylase. Here, "improved cleaning performance" refers to an improved cleaning effect compared to the parent α-amylase, such as the ability to remove dirt during the washing or cleaning process. The cleaning performance can be evaluated using methods well known in the art. For example, a contaminated 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 cleaning treatment under predetermined conditions. The absorbance of the cleaning solution after cleaning is measured at 488 nm, and the difference ΔA488 from that of the blank is determined as the cleaning power.

[0056] Furthermore, "improved stability" refers to the ability to maintain α-amylase activity in the presence of a protease-containing detergent, which is improved compared to the parent α-amylase. Stability can be evaluated using methods well known in the relevant art. For example, an enzyme solution can be added to a detergent, and the α-amylase activity can be measured before and after treatment for a predetermined time. The activity value of the sample before treatment is taken as the initial activity, and the ratio of the activity value after treatment to the initial activity is taken as the residual activity (%).

[0057] The variant of the present invention is useful as an enzyme for various detergent compositions, and is particularly useful as an enzyme for detergent compositions suitable for low-temperature washing. Here, "low temperature" includes temperatures below 40°C, below 35°C, below 30°C, and below 25°C, as well as temperatures above 5°C, above 10°C, and above 15°C. Additionally, temperatures between 5 and 40°C, 10 and 35°C, 15 and 30°C, and 15 and 25°C are also included.

[0058] The detergent composition of the present invention contains the above-mentioned α-amylase variant and protease. The amount of α-amylase variant incorporated into the detergent composition is not particularly limited as long as the protein exhibits activity. For example, it is preferably 1 mg or more, more preferably 10 mg or more, more preferably 50 mg or more per 1 kg of the detergent composition, and preferably 5000 mg or less, more preferably 1000 mg or less, and more preferably 500 mg or less. It is also preferably 1 to 5000 mg, more preferably 10 to 1000 mg, and more preferably 50 to 500 mg.

[0059] Examples of proteases include proteins that have at least 70%, preferably 80%, more preferably 90%, and more preferably 95% identity with the amino acid sequence shown in SEQ ID NOs. 5, 6, 7, 8, 9, 10, or 11, and that also possess protease activity. Here, the protease consisting of the amino acid sequence shown in SEQ ID NO: 5 is the Bacillus clausii-derived protease Savinase (International Publication No. 2011 / 036263), the protease consisting of the amino acid sequence shown in SEQ ID NO: 6 is the Bacillus amyloliquefaciens-derived protease BPN' (International Publication No. 2011 / 036263), the protease consisting of the amino acid sequence shown in SEQ ID NO: 7 is the Bacillus lentus DSM 5483-derived protease (International Publication No. 92 / 21760), the protease consisting of the amino acid sequence shown in SEQ ID NO: 8 is the Bacillus sp. KSM-KP43-derived protease (International Publication No. 99 / 18218), and the protease consisting of the amino acid sequence shown in SEQ ID NO: 9 is the Bacillus The protease TY145 is derived from sp. (Japanese Patent Publication No. 2019-503404), the protease consisting of the amino acid sequence shown in SEQ ID NO: 10 is Neutrase, a protease derived from Bacillus amyloliquefacience, and the protease consisting of the amino acid sequence shown in SEQ ID NO: 11 is a metalloprotease derived from Bacillus subtilis. Furthermore, the protease may be a commercially available product such as Alcalase, Esperase, Everlase, Kannase, Progress Uno (registered trademark; Novozymes), PREFERENZ, EFFECTENZ, EXCELLENZ (registered trademark; DuPont), or Lavergy (registered trademark; BASF).

[0060] The amount of protease to be included is not particularly limited as long as it is an amount in which the protein exhibits activity, but for example, it is preferably 1 mg or more, more preferably 10 mg or more, more preferably 50 mg or more per 1 kg of the detergent composition, and preferably 5000 mg or less, more preferably 1000 mg or less, and more preferably 500 mg or less. It is also preferably 1 to 5000 mg, more preferably 10 to 1000 mg, and more preferably 50 to 500 mg.

[0061] In addition to the α-amylase variant and protease mentioned above, various other enzymes may also be used in combination with the detergent composition. Examples of such enzymes include amylases, cellulases, keratinases, esterases, cutinases, lipases, pullulanases, pectinases, mannanases, glucosidases, glucanases, cholesterol oxidases, peroxidases, laccases, etc., different from the α-amylase variant, and preferably cellulases, amylases, and lipases. Examples of cellulases include Celluclean, Carezyme (registered trademark; Novozymes), KAC, alkaline cellulase produced by Bacillus SP KSM-S237 strain described in Japanese Patent Publication No. 10-313859, and mutant alkaline cellulase described in Japanese Patent Publication No. 2003-313592 (all from Kao Corporation). Examples of amylases include Teramyl, Duramyl, Stainzyme, Stainzyme Plus, Amplify Prime (registered trademark; Novozymes), PREFERENZ, EFFECTENZ (registered trademark; DuPont), and KAM (Kao). Examples of lipases include Lipolase and Lipex (registered trademark; Novozymes).

[0062] The detergent composition of the present invention may contain known detergent components, and examples of such known detergent components include the following: (1) Surfactants The surfactant is incorporated into the detergent composition at a concentration of 0.5 to 60% by mass, and is preferably incorporated at a concentration of 10 to 45% by mass for powder detergent compositions and 20 to 90% by mass for liquid detergent compositions. Furthermore, when the detergent composition of the present invention is a laundry detergent or a detergent for automatic dishwashers, the surfactant is generally incorporated at a concentration of 1 to 10% by mass, preferably 1 to 5% by mass.

[0063] Examples of surfactants used in detergent compositions include anionic surfactants, nonionic surfactants, amphoteric surfactants, and cationic surfactants, one or a combination thereof, but anionic surfactants and nonionic surfactants are preferred.

[0064] As anionic surfactants, sulfate esters of alcohols having 10 to 18 carbon atoms, sulfate esters of alkoxylated alcohols having 8 to 20 carbon atoms, alkylbenzene sulfonates, paraffin sulfonates, α-olefin sulfonates, internal olefin sulfonates, α-sulfo fatty acid salts, α-sulfo fatty acid alkyl esters, or fatty acid salts are preferred. In the present invention, one or more anionic surfactants selected from linear alkylbenzene sulfonates having 10 to 14 carbon atoms in the alkyl chain, more preferably 12 to 14, and internal olefin sulfons having 12 to 20 carbon atoms in the alkylene chain, more preferably 16 to 18, are preferred, and as counterions, alkali metal salts and amines are preferred, with sodium and / or potassium, monoethanolamine, and diethanolamine being particularly preferred. For internal olefin sulfonic acid, see, for example, WO2017 / 098637.

[0065] As nonionic surfactants, polyoxyalkylene alkyl (8-20 carbon atoms) ethers, alkyl polyglycosides, polyoxyalkylene alkyl (8-20 carbon atoms) phenyl ethers, polyoxyalkylene sorbitan fatty acid (8-22 carbon atoms) esters, polyoxyalkylene glycol fatty acid (8-22 carbon atoms) esters, and polyoxyethylene polyoxypropylene block polymers are preferred. In particular, as nonionic surfactants, polyoxyalkylene alkyl ethers obtained by adding 4-20 moles of alkylene oxide such as ethylene oxide or propylene oxide to an alcohol with 10-18 carbon atoms [such as having an HLB value (calculated by the Griffin method) of 10.5-15.0, preferably 11.0-14.5] are preferred.

[0066] (2) Divalent metal ion scavenger The divalent metal ion scavenger is blended in an amount of 0.01 to 50% by mass, preferably 5 to 40% by mass. Examples of divalent metal ion scavengers used in the detergent composition of the present invention include condensed phosphates such as tripolyphosphate, pyrophosphate, and orthophosphate, aluminosilicates such as zeolites, synthetic layered crystalline silicates, nitrilotriacetate, ethylenediaminetetraacetate, citrate, isocitrate, and polyacetal carboxylate. Of these, crystalline aluminosilicate (synthetic zeolite) is particularly preferred, and among type A, type X, and type P zeolites, type A is particularly preferred. Synthetic zeolites with an average primary particle size of 0.1 to 10 μm, and particularly 0.1 to 5 μm, are preferably used.

[0067] (3) Alkaline agents 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 framework during detergent drying, resulting in a relatively hard detergent with excellent fluidity. Other alkalis include sodium sesquicarbonate and sodium bicarbonate, and phosphates such as tripolyphosphate also act as alkaline agents. In addition to the above alkaline agents, sodium hydroxide and mono, di, or triethanolamine can be used as alkaline agents in liquid detergents, and can also be used as counterions for surfactants.

[0068] (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. Of these, carboxylic acid polymers have the function of capturing metal ions in addition to their anti-redeposition ability, and the effect of dispersing solid particulate dirt from clothing into the washing bath. Carboxylic acid polymers are homopolymers or copolymers of acrylic acid, methacrylic acid, itaconic acid, etc., and copolymers obtained by copolymerizing the above monomers with maleic acid are preferred, with molecular weights of several thousand to 100,000 being preferred. In addition to the above carboxylic acid polymers, polymers such as polyglycidyl salts, cellulose derivatives such as carboxymethylcellulose, and aminocarboxylic acid polymers such as polyaspartic acid are also preferred because they have metal ion capturing, dispersing, and anti-redeposition abilities.

[0069] (5) Bleach For example, bleaching agents such as hydrogen peroxide and percarbonates are preferably added in an amount of 1 to 10% by mass. When using bleaching agents, bleaching activators such as tetraacetylethylenediamine (TAED) or those described in Japanese Patent Publication No. 6-316700 can be added in an amount of 0.01 to 10% by mass.

[0070] (6) Fluorescent agents Examples of fluorescent agents used in detergent compositions include biphenyl-type fluorescent agents (e.g., Chinopearl CBS-X) and stilbene-type fluorescent agents (e.g., DM-type fluorescent dyes). It is preferable to include 0.001 to 2% by mass of the fluorescent agent.

[0071] (7) Other ingredients The detergent composition may contain builders, softeners, reducing agents (such as sulfites), antifoaming agents (such as silicones), fragrances, antibacterial and antifungal agents (such as Proxel [trade name], benzoic acid, etc.), and other additives known in the field of laundry detergents.

[0072] The detergent composition can be manufactured by combining the α-amylase variant and protease of the present invention obtained by the above method with the above known detergent components, according to conventional methods. The form of the detergent can be selected according to the application, for example, it can be a liquid, powder, granules, paste, solid, etc.

[0073] The resulting detergent composition can be used as a laundry detergent, dishwashing detergent, bleach, hard surface cleaner, drain cleaner, denture cleaner, or disinfectant cleaner for medical instruments, but is more preferably used as a laundry detergent or dishwashing detergent, and more preferably as a laundry detergent (laundry detergent), a hand-washing dishwashing detergent, or a dishwashing machine detergent. Furthermore, the cleaning agent composition is suitable for use at temperatures below 40°C, below 35°C, below 30°C, below 25°C, and above 5°C, above 10°C, and above 15°C. It is also suitable for use at temperatures between 5 and 40°C, 10 and 35°C, 15 and 30°C, and 15 and 25°C.

[0074] With regard to the embodiments described above, the present invention further discloses the following embodiments. <1> A detergent composition comprising an α-amylase variant and a protease, wherein the α-amylase variant is a variant of the parent α-amylase comprising the substitution of an amino acid residue at one or more positions selected from positions corresponding to E187 and S241 of the amino acid sequence shown in SEQ ID NO: 2, and further the substitution of an amino acid residue at one or more positions selected from positions corresponding to N192, H240 and K278, and the parent α-amylase or α-amylase variant has at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 4. <2> The amino acid residue substitutions at positions E187, S241, N192, H240, and K278 of the aforementioned α-amylase mutants are E187P, S241Q, N192F, H240F, and K278F / Y / W, respectively. <1> The cleaning agent composition described above. <3> The α-amylase variant includes at least one mutation selected from the following combinations a) to g): <1> or <2> The cleaning agent composition described above. a) E187P+N192F b) E187P+H240F c) E187P+K278F d) E187P+K278Y e) E187P + K278W f) H240F + S241Q g) N192F + H240F + S241Q <4> The α-amylase variant further comprises the substitution of one or more amino acid residues at positions selected from those corresponding to positions F205, R211, and M199 of the amino acid sequence shown in SEQ ID NO: 2. <1> ~ <3> A detergent composition as described in any of the following. <5> The amino acid residue substitutions at positions F205, R211, and M199 of the aforementioned α-amylase mutant are F205Y, R211V / L / I, and M199L / T / A / N / Q / S / V / I, respectively. <4> The cleaning agent composition described above. <6> The α-amylase mutant further comprises the substitution of one or more amino acid residues selected from positions corresponding to G5, S38, Q96, W186, E257, F259, S284, H295, Y296, N303, T320, Y360, W408, L429, V430, G433, A434, W439, N471, G476 and G477 of the amino acid sequence shown in SEQ ID NO: 2. <4> or <5> The cleaning agent composition described above. <7> The amino acid residue substitutions at positions G5, S38, Q96, W186, E257, F259, S284, H295, Y296, N303, T320, Y360, W408, L429, V430, G433, A434, W439, N471, G476, and G477 of the aforementioned α-amylase mutants are G5E / D / P / R / K and S38N , Q96R / K, W186L, E257T, F259W, S284W, H295Y, Y296A, N303R / E / S / G / V / D / T / A, T320D / E, Y360C / M / L / V, W408P, L429V, V430M, G433GS, A434V, W439R, N471T, G476A / P / E / S / F / R / K, G477E, <6> The cleaning agent composition described above. <8> The parent α-amylase is an α-amylase mutant in which two or more amino acid residues are deleted, preferably two to three, and more preferably two, selected from the positions corresponding to R178, G179, T180, and G181 of the amino acid sequence shown in SEQ ID NO: 2. <1> ~ <7> A detergent composition as described in any of the following. <9> The deletion of the two amino acid residues is preferably a combination selected from R178Δ+T180Δ, G179Δ+T180Δ, R178Δ+G179Δ, R178Δ+G181Δ, and G179Δ+G181Δ, and more preferably R178Δ+T180Δ. <8> The cleaning agent composition described above. <10> The protease is a protein that has at least 70% identity with the amino acid sequence shown in SEQ ID NOs. 5, 6, 7, 8, 9, 10, or 11, and possesses protease activity. <1> ~ <9> A detergent composition as described in any of the following. <11> It is a laundry detergent or a dishwashing detergent. <1> ~ <10> A detergent composition as described in any of the following. <12> It is a laundry detergent or a dishwashing detergent for hand washing or automatic dishwashers. <11> The cleaning agent composition described above. <13> It is either a powder or a liquid. <11> or <12> The cleaning agent composition described above. <14> Used at low temperatures, <11> ~ <13> A detergent composition as described in any of the following. <15> Used at temperatures below 40℃, below 35℃, below 30℃, below 25℃, and above 5℃, above 10℃, above 15℃, or used at temperatures between 5 and 40℃, 10 and 35℃, 15 and 30℃, and 15 and 25℃. <14> The cleaning agent composition described above. <16> It is used in low-temperature (15-30°C) washing in laundry, or in low-temperature (15-30°C) washing in automatic dishwashers. <11> The cleaning agent composition described above. [Examples]

[0075] (1) Construction of YR288 mutant expression plasmid The method for constructing the YR288 mutant described in the following example is as follows. A forward primer containing the mutant sequence and having a 15-base complementary sequence with the reverse primer at its 5' end, and a reverse primer with the base immediately preceding the mutant sequence at its 5' end were used as the mutant primer pair. PCR was performed using the mutant primer pair with the YR288 R178Δ T180Δ expression plasmid described in the example of Japanese Patent Application No. 2020-121626 or the YR288 mutant expression plasmid prepared in this example as a template. When multiple fragments were ligated, an In-Fusion reaction was performed using each PCR product according to the In-Fusion, HD Cloning kit (Clontech) protocol. The PCR product or In-Fusion reaction solution was transformed into Bacillus subtilis by the protoplast method to obtain transformants holding the target YR288 mutant expression plasmid.

[0076] (2) Enzyme production culture Recombinant Bacillus subtilis colonies obtained in (1) were inoculated into 96-well deep-well plates dispensed with 500 μL of LB medium supplemented with 10 ppm tetracycline, and then incubated overnight at 32°C and 1500 rpm. The following day, 20 μL of the culture solution was inoculated into 96-well deep-well plates dispensed with 500 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, 10 ppm tetracycline; % is (w / v)%), and incubated at 32°C and 1500 rpm for 2 days. The culture supernatant containing enzymes produced from the bacterial cells was collected by centrifugation to obtain the enzyme solution.

[0077] (3) Measurement of protein concentration in culture supernatant The Protein Assay Rapid Kit Wako II (Fujifilm Wako Pure Chemical Corporation) was used to measure the protein concentration of the culture supernatant. The concentration of α-amylase in the culture supernatant was calculated by using the protein concentration of the culture supernatant of a pHY300PLK (Takara Bio) strain without an α-amylase expression cassette as a blank.

[0078] (4) Stability evaluation of α-amylase mutants (1) Various mutants were constructed using YR288 R178Δ+T180Δ (SEQ ID NO: 4) as the parent polypeptide by the method described in Example (1), and the storage stability of these mutants in protease-containing detergents was evaluated. Commercial liquid laundry detergents (Kao Corporation's Attack 3X or Lion Corporation's Top Clear Liquid, labeled Detergent 1 and Detergent 2 respectively in Table 2) were left to stand in a boiling water bath for 30 minutes to deactivate the enzymes they contained. Either Savinase (SIGMA, P3111) or KAP8.0Q-L (Kao) protease was added to these detergents to a final concentration of 300 ppm. After thorough mixing, an amylase solution was added, and the mixture was incubated at 40°C for 2 weeks before activity measurement. The activity value of the sample before 40°C treatment was defined as the initial activity, and the percentage of the activity value after treatment relative to the initial activity was defined as the residual activity (%). The results are shown in Table 2. The mutants showed high stability in all protease-containing detergents.

[0079] (5) Stability evaluation of α-amylase mutants (2) Commercial liquid laundry detergent (Kao Corporation, Attack 3X, labeled as Detergent 1 in Table 3) was left to stand in a boiling water bath for 30 minutes to deactivate the enzymes it contained. Savinase was added to these detergents to a final concentration of 300 ppm, and after thorough mixing, an amylase solution was added. The samples were incubated at 40°C for 2 weeks, and then the activity was measured. The activity value of the sample before 40°C treatment was defined as the initial activity, and the percentage of the activity value after treatment to the initial activity was defined as the residual activity (%). The results are shown in Table 3. All mutants showed high stability in protease-containing detergents.

[0080] (6) Evaluation of amylase washing power after storage in protease-containing washing compositions CS-26 contaminated cloth, cut into 5.5 mm diameter circles, was obtained from CFT and used. Savinase (SIGMA, P3111) and amylase culture supernatant were added to Attack 3X (treated in a boiling water bath for 30 minutes) to a final concentration of 300 ppm each, mixed well, and incubated at 40°C for 2 weeks before being used for the washing test. CS-26 contaminated cloth was inserted into each well of a 96-well assay plate, and 200 μL of the aforementioned protease and amylase-containing detergent, diluted 1200-fold with tap water, was added to each well. The plates were sealed and shaken at 1200 rpm for 15 minutes at 20°C using a Cute Mixer. After washing, 100 μL of the washing solution was transferred to a new 96-well assay plate, and the absorbance at 488 nm was measured. A blank was prepared by adding deionized water instead of amylase solution, and the difference in absorbance ΔA488 between the blank and the blank was determined as the α-amylase washing power. Figure 1 shows the washing power of the parent α-amylase (YR288 R178Δ+T180Δ) and the α-amylase mutant. Compared to the washing composition containing the α-amylase mutant and protease, the excellent low-temperature washing power characteristic of α-amylase YR288 was maintained even after storage at 40°C for two weeks.

[0081] Table 2-1

[0082] Table 2-2

[0083] Table 2-3

[0084] Table 3-1

[0085] Table 3-2

[0086] Table 3-3

Claims

1. A detergent composition comprising an α-amylase mutant and a protease, wherein the α-amylase mutant is a mutant of parent α-amylase comprising a mutation selected from the following combinations a) to g) in the amino acid sequence shown in SEQ ID NO: 2, and two or more deletions selected from positions corresponding to R178, G179, T180 and G181 in the amino acid sequence shown in SEQ ID NO: 2, and the α-amylase mutant has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4 and has amylase activity. a) E187P+N192F b) E187P+H240F c) E187P+K278F d) E187P+K278Y e) E187P+K278W f) H240F+S241Q g) N192F+H240F+S241Q

2. The detergent composition according to claim 1, wherein the detergent performance and / or stability in a detergent containing the α-amylase mutant protease is improved compared to that of the parent α-amylase.

3. The detergent composition according to claim 1 or 2, wherein the α-amylase mutant further comprises the substitution of one or more amino acid residues at positions corresponding to F205, R211, and M199 of the amino acid sequence shown in SEQ ID NO:

2.

4. The detergent composition according to claim 3, wherein the substitutions of amino acid residues at positions F205, R211, and M199 of the α-amylase mutant are F205Y, R211V / L / I, and M199L / T / A / N / Q / S / V / I, respectively.

5. The detergent composition according to any one of claims 1 to 4, wherein the protease is a protein that has at least 90% identity with the amino acid sequence shown in SEQ ID NOs. 5, 6, 7, 8, 9, 10, or 11, and has protease activity.

6. A detergent composition according to any one of claims 1 to 5, which is a laundry detergent or a dishwashing detergent.

7. The detergent composition according to claim 6, which is a laundry detergent or a dishwashing detergent for hand washing or automatic dishwashers.

8. The cleaning agent composition according to claim 6 or 7, which is a powder or a liquid.

9. A detergent composition according to any one of claims 6 to 8, to be used at a temperature of 5 to 40°C.

10. A method for cleaning dirt, comprising contacting a cleaning agent composition containing an α-amylase variant and a protease with the dirt at a temperature of 5 to 40°C, wherein the α-amylase variant is a variant of parent α-amylase containing mutations selected from combinations corresponding to a) to g) below in the amino acid sequence shown in SEQ ID NO: 2, and two or more deletions selected from positions corresponding to R178, G179, T180 and G181 in the amino acid sequence shown in SEQ ID NO: 2, and the α-amylase variant has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4 and has amylase activity. a) E187P+N192F b) E187P+H240F c) E187P+K278F d) E187P+K278Y e) E187P+K278W f) H240F+S241Q g) N192F+H240F+S241Q

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