Amylase-containing detergent composition

Abstract

To provide a detergent composition containing α-amylase that exhibits high specific activity at low temperatures.SOLUTION: A detergent composition containing one or more proteins selected from the following (A), (B), (C), and (D). (A) A protein consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2 and having α-amylase activity. (B) A protein consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 4 and having α-amylase activity. (C) A protein consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 6 and having α-amylase activity. (D) A protein consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 8 and having α-amylase activity.SELECTED DRAWING: None

Description

Technical Field

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

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] α-Amylases useful 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 derived from Bacillus sp. SP722 (SEQ ID NO: 4 in Patent Document 3), and α-amylase CspAmy2 derived from the genus Cytophaga (Patent Document 4) are known.

[0004] On the other hand, in recent years, from the viewpoints of environmental protection and reduction of washing costs, it has been considered important to lower the temperature during dishwashing, laundry washing, particularly laundry washing in a laundry. In addition, shortening the washing time is also desired. However, the optimum temperature of most enzymes including amylase is higher than the temperature usually set in low-temperature washing. Therefore, it is difficult to completely remove many starch-based stains. It is important to find an α-amylase that maintains washing performance and starch-degrading activity and has a high stain-removing effect even at low temperatures.

[0005] The washing performance of α-amylase at low temperatures is inversely correlated with the amylase's ability to bind to starch (starch adsorption), and it has been reported that amylases with low starch adsorption have high washing performance at low temperatures (Patent Document 5). Furthermore, Patent Document 5 discloses that starch adsorption can be reduced by introducing mutations into known starch-binding residues and their adjacent residues. [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] Patent No. 6339499 [Overview of the project] [Problems that the invention aims to solve]

[0007] To achieve high cleaning performance at low temperatures, maintaining starch-degrading activity at low temperatures and having low starch-adsorbing properties are considered particularly important. However, maintaining starch-degrading activity at low temperatures is not easy. Conventionally, despite the fact that the strength of activity was considered part of the selection criteria for any existing amylase used in detergents, none of them have adequately maintained starch-degrading activity at low temperatures. Therefore, in order to develop an amylase that exhibits higher cleaning power at low temperatures compared to existing washing amylases, it is important to discover a new amylase that maintains high starch-degrading activity at low temperatures. However, there are no sequence indicators to select an amylase that maintains higher starch-degrading activity at low temperatures than existing amylases, making the search difficult. Furthermore, since many starch-containing stains also contain protein components, it is known that using α-amylase and protease in combination produces an additive synergistic cleaning effect. Detergents containing α-amylase and protease are particularly useful for cleaning at low temperatures, where a decrease in cleaning power is a concern. However, maintaining amylase activity at low temperatures remains a major challenge even with such detergents, and no existing amylase for detergent applications adequately maintains starch-degrading activity at low temperatures. The present invention relates to providing a detergent composition containing α-amylase that exhibits high specific activity (starch-degrading activity) at low temperatures. [Means for solving the problem]

[0008] The inventors of this invention have discovered that a specific α-amylase found among the predicted α-amylase sequences included in the NCBI protein sequence database has high starch-degrading activity at low temperatures of 20-30°C, and that a detergent composition containing this α-amylase is useful as a detergent composition suitable for low-temperature washing.

[0009] In other words, the present invention relates to the following 1) to 2). 1) A detergent composition containing one or more proteins selected from (A), (B), (C), and (D) below. (A) A protein having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in Sequence ID No. 2, and which also has α-amylase activity. (B) A protein having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in Sequence ID No. 4, and which also has α-amylase activity. (C) A protein having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in Sequence ID No. 6, and which also has α-amylase activity. (D) A protein having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in Sequence ID No. 8, and which also has α-amylase activity. 2) A protein selected from (A'), (B'), (C'), and (D') below. (A') A protein having α-amylase activity, comprising an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 2, and having two or more amino acid residues selected from the R178, G179, T180, and G181 positions of the amino acid sequence shown in SEQ ID NO: 2. (B') A protein having α-amylase activity, comprising an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 4, and having two or more amino acid residues selected from the R178, G179, T180, and G181 positions of the amino acid sequence shown in SEQ ID NO: 4. (C') A protein having α-amylase activity, comprising an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 6, and having two or more amino acid residues selected from the R178, G179, T180, and G181 positions of the amino acid sequence shown in SEQ ID NO: 6. (D') A protein having α-amylase activity, comprising an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 8, and having two or more amino acid residues selected from the R178, G179, T180, and G181 positions of the amino acid sequence shown in SEQ ID NO: 8. [Effects of the Invention]

[0010] According to the present invention, a detergent composition that exhibits excellent starch stain removal effect even when used in low-temperature washing can be provided. Furthermore, the protein sequence information of the present invention can be used as a sequence indicator for selecting α-amylase that has high starch decomposition activity at low temperatures. [Brief explanation of the drawing]

[0011] [Figure 1] Starch-degrading activity of each amylase at 20°C and 30°C. [Figure 2] A molecular phylogenetic tree showing the evolutionary relationship between the amylase of the present invention and existing amylases for detergents. [Figure 3] Detergency of each amylase at 20°C. [Figure 4] Starch-degrading activity of the amylase (mutant of YR288) of the present invention at 20°C. [Figure 5] Detergency of the amylase (mutant of YR288) of the present invention at 20°C. [Figure 6] Starch-degrading activity of the amylase (mutant of BCGAmy) of the present invention at 20°C. [Figure 7] Washing performance of a washing composition containing protease (detergency against CS-26 soiled cloth). [Figure 8] Washing performance of a washing composition containing protease (detergency against EMPA117 soiled cloth).

Mode for Carrying Out the Invention

[0012] In the present invention, the identity of nucleotide sequences and amino acid sequences is calculated by the Lipman-Pearson method (Science, 1985, 227: 1435-1441). Specifically, it is calculated by performing an analysis using the homology analysis (Search homology) program of the genetic information processing software Genetyx-Win with Unit size to compare (ktup) being 2.

[0013] In the present invention, "at least 90% identity" with respect to an amino acid sequence or a nucleotide sequence means an identity of 90% or more, preferably 95% or more, more preferably 97% or more, still more preferably 98% or more, and yet more preferably 99% or more. Amino acid sequences having at least 90% identity include amino acid sequences in which one or more amino acids are deleted, inserted, substituted or added. Examples of "amino acid sequences in which one or more amino acids are deleted, inserted, substituted or added" include amino acid sequences in which 1 to 30, preferably 20 or less, more preferably 10 or less, still more preferably 5 or less amino acids are deleted, inserted, substituted or added.

[0014] The protein incorporated into the detergent composition of the present invention is one or more proteins selected from (A), (B), (C), and (D) below. (A) A protein having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in Sequence ID No. 2, and which also has α-amylase activity. (B) A protein having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in Sequence ID No. 4, and which also has α-amylase activity. (C) A protein having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in Sequence ID No. 6, and which also has α-amylase activity. (D) A protein having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in Sequence ID No. 8, and which also has α-amylase activity.

[0015] Such proteins exhibit excellent α-amylase activity at 20-30°C. Here, α-amylase activity refers to the activity that catalyzes the hydrolysis of starch and other linear or branched 1,4-glycosidoligosaccharides or polysaccharides.

[0016] 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 method; it 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.). A correlation has been observed between α-amylase activity measured by Phadebas and the cleaning performance when used as a detergent.

[0017] The proteins consisting of the amino acid sequences shown in SEQ ID NOs: 2, 4, 6, and 8 are proteins that have been identified as α-amylase in the NCBI protein sequence database. Specifically, the protein consisting of the amino acid sequence shown in SEQ ID NO: 2 is registered in the database as accession number WP_138117433.1 (referred to as "DE0178" in this invention), the protein consisting of the amino acid sequence shown in SEQ ID NO: 4 as WP_076512862.1 (referred to as "RU2C" in this invention), the protein consisting of the amino acid sequence shown in SEQ ID NO: 6 as WP_110114708.1 (referred to as "BCGAmy" in this invention), and the protein consisting of the amino acid sequence shown in SEQ ID NO: 8 as WP_100346362.1 (referred to as "YR288" in this invention), but their enzymatic properties have not been reported at all to date. To clarify the evolutionary relationship between DE0178, RU2C, BCGAmy, and YR288 and known detergent amylases such as AP1378 (SEQ ID NO: 10), AA560 (SEQ ID NO: 12), SP722 (SEQ ID NO: 14), CspAmy2 (SEQ ID NO: 16), BAA (SEQ ID NO: 35), BLA (SEQ ID NO: 36), LABM (SEQ ID NO: 37), SP707 (SEQ ID NO: 38), TS23 (SEQ ID NO: 39), Termamyl (SEQ ID NO: 40), and AAI10 (SEQ ID NO: 41), a molecular phylogenetic tree was constructed using the neighbor-joining method with the amino acid sequences of the maturation region of each amylase. This showed that DE0178, RU2C, BCGAmy, and YR288 of the present invention belong to a different group from existing detergent amylases (Figure 2).

[0018] A protein having α-amylase activity and consisting of an amino acid sequence having at least 90% identity with the amino acid sequences shown in SEQ ID NOs: 2, 4, 6, and 8 includes, in one embodiment, a protein variant having α-amylase activity and consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added to the amino acid sequences shown in SEQ ID NOs: 2, 4, 6, and 8. Here, "a few" refers to, for example, 2 to 10 amino acids, preferably 2 to 5, and more preferably 2 to 3.

[0019] Among such mutants, from the viewpoint of starch degradation activity and / or washability at low temperatures, mutants in which two or more amino acid residues selected from arginine at position 178 (R178), glycine at position 179 (G179), threonine at position 180 (T180), and glycine at position 181 (G181) are deleted in the amino acid sequences shown in SEQ ID NOs. 8 are more preferably mutants in which two or more amino acid residues selected from each of the positions R178, G179, T180, and G181 in the amino acid sequence shown in SEQ ID NO. 8 are deleted. Here, examples of deletions of two or more amino acid residues (denoted as [original amino acid, position, Δ]) include R178Δ+T180Δ, G179Δ+T180Δ, R178Δ+G179Δ, R178Δ+G181Δ, G179Δ+G181Δ, etc., with R178Δ+T180Δ being more preferred.

[0020] The above-mentioned variant is a novel protein not described in the literature. Therefore, in one embodiment, the present invention includes a protein selected from (A'), (B'), (C'), and (D') below, and a detergent composition containing said protein. (A') A protein having α-amylase activity, comprising an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 2, and having two or more amino acid residues selected from the R178, G179, T180, and G181 positions of the amino acid sequence shown in SEQ ID NO: 2. (B') A protein having α-amylase activity, comprising an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 4, and having two or more amino acid residues selected from the R178, G179, T180, and G181 positions of the amino acid sequence shown in SEQ ID NO: 4. (C') A protein having α-amylase activity, comprising an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 6, and having two or more amino acid residues selected from the R178, G179, T180, and G181 positions of the amino acid sequence shown in SEQ ID NO: 6. (D') A protein having α-amylase activity, comprising an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 8, and having two or more amino acid residues selected from the R178, G179, T180, and G181 positions of the amino acid sequence shown in SEQ ID NO: 8.

[0021] The protein of the present invention can be produced, for example, by expressing the gene encoding the protein of the present invention. Preferably, the protein of the present invention can be produced from a transformant into which the polynucleotide encoding the protein of the present invention has been introduced. For example, after obtaining a transformant by introducing the polynucleotide encoding the protein of the present invention, or a vector containing it, into a host, the transformant can be cultured in a suitable medium to produce the protein of the present invention from the polynucleotide encoding the protein of the present invention introduced into the transformant. The protein of the present invention can be obtained by isolating or purifying the produced protein from the culture.

[0022] The polynucleotide encoding the protein of the present invention may be in the form of single-stranded or double-stranded DNA, RNA, or artificial nucleic acid, or it may be cDNA or chemically synthesized DNA that does not contain introns. Preferred examples of polynucleotides encoding the protein of the present invention include the polynucleotides represented by (a), (b), (c), or (d) below. (a) Polynucleotides consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in Sequence ID No. 1 (b) Polynucleotides consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in Sequence ID No. 3 (c) A polynucleotide consisting of a base sequence that has at least 90% identity with the base sequence shown in Sequence ID No. 5. (d) Polynucleotides consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in Sequence ID No. 7

[0023] The polynucleotide encoding the protein of the present invention can be synthesized chemically or genetically based on the amino acid sequence of the protein. For example, the polynucleotide can be chemically synthesized based on the amino acid sequence of the protein of the present invention described above. For the chemical synthesis of the polynucleotide, nucleic acid synthesis contract services (e.g., those provided by Medical & Biological Laboratories, Inc., Genscript, etc.) can be used. Furthermore, the synthesized polynucleotide can be amplified by PCR, cloning, etc.

[0024] The polynucleotide encoding the mutant protein is used to produce the polynucleotide encoding the mutated amino acid using various mutagenesis techniques known in the art. Mutation can be introduced using various site-directed mutagenesis methods well known to those skilled in the art. Site-directed mutagenesis can be performed using 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.

[0025] Site-directed mutagenesis can most commonly be performed using mutation primers containing the nucleotide mutation to be introduced. These mutation primers should be designed to anneal to a region in the polynucleotide encoding the parent protein that contains the nucleotide sequence encoding the amino acid residue to be mutated, and to include a nucleotide sequence (codon) that replaces the nucleotide sequence (codon) encoding the amino acid residue to be mutated with a nucleotide sequence (codon) encoding the mutated amino acid residue. The nucleotide sequences (codons) encoding the pre-mutation and post-mutation amino acid residues can be appropriately identified and selected by those skilled in the art based on standard textbooks, etc. 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 by SOE (splicing by overlap extension)-PCR (Gene, 1989, 77(1): p61-68).

[0026] The type of vector containing the polynucleotide encoding the protein of the present invention is not particularly limited and may be any vector such as plasmids, phages, phagemids, cosmids, viruses, YAC vectors, or shuttle vectors. Furthermore, although not limited, the vector is preferably amplified within bacteria, preferably within Bacillus bacteria (e.g., Bacillus subtilis or its mutants), and more preferably an expression vector capable of inducing the expression of a transgene within Bacillus bacteria. Among these, shuttle vectors, which can replicate in either Bacillus bacteria or other organisms, are particularly suitable for recombinant production of the mutants 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.

[0027] 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 for initiating transcription of the protein of the present invention, 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 protein of the present invention (i.e., the α-amylase 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.

[0028] 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.

[0029] 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.

[0030] The polynucleotide encoding the protein 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.

[0031] Examples of hosts for transformants into which the above vector is introduced 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.

[0032] 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.

[0033] Alternatively, a fragment comprising the polynucleotide encoding the protein 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 protein of the present invention into the host genome.

[0034] When a transformant into which the polynucleotide encoding the protein 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 protein 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.

[0035] Alternatively, the protein of the present invention may be expressed from a polynucleotide encoding the protein of the present invention or its transcript 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 adding reagents such as amino acids necessary for protein translation to a suspension obtained by mechanically disrupting host cells.

[0036] The protein of the present invention, produced in the culture or cell-free translation system described above, 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 appropriate combinations. In this case, if the gene encoding the protein of the present invention and the secretion signal sequence are operably linked on the vector in the transformant, the produced 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.

[0037] The protein obtained in this way exhibits significantly higher starch-degrading activity at 20°C and 30°C compared to previously reported detergent amylases (AP1378, AA560, SP722, CspAmy2).

[0038] Furthermore, the protein of the present invention has superior cleaning performance compared to existing detergent amylases. Specifically, when the cleaning performance is measured by the method described below, the relative cleaning performance to AA560 (an amylase consisting of the amino acid sequence shown in SEQ ID NO: 12) is 1 or higher, preferably 1.1 or higher, more preferably 1.2 or higher, more preferably 1.3 or higher, more preferably 1.5 or higher, more preferably 1.7 or higher, and more preferably 1.9 or higher. <Measurement of cleaning performance> Two circular pieces of CS-26 contaminated cloth (CFT Co., Ltd.), cut to a diameter of 5.5 mm, were inserted into each well of a 96-well assay plate. 200 μL of model washing solution (200 ppm linear alkylbenzene sulfonate sodium, 20 mM Tris HCl aqueous solution (pH 7.5)) was added to each well, followed by 10 μL of a 4 ppm diluted test amylase solution. The mixture was then shaken at 20°C, 1200 rpm, for 15 minutes. After washing, the absorbance of the washing solution at 488 nm was measured. A blank was created by adding deionized water instead of the enzyme solution, and the difference ΔA488 from the blank was defined as the washing performance. The relative cleaning performance against AA560 is calculated as (ΔA488 of the test amylase) / (ΔA488 of AA560).

[0039] Therefore, the protein 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.

[0040] The amount of the protein of the present invention incorporated into the detergent composition is not particularly limited as long as the protein exhibits activity. For example, the amount 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.

[0041] The detergent composition of the present invention may also be used in combination with various enzymes other than the protein of the present invention. For example, hydrolases, oxidases, reductases, transferases, lyases, isomerases, ligases, synthetases, etc. Of these, amylases, proteases, cellulases, keratinases, esterases, cutinases, lipases, pullulanases, pectinases, mannanases, glucosidases, glucanases, cholesterol oxidases, peroxidases, laccases, etc., which are different from the protein of the present invention, are preferred, and proteases, cellulases, amylases, and lipases are particularly preferred.

[0042] 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. 42, 43, 44, 45, 46, 47, or 48, and that possess protease activity. Here, the protease consisting of the amino acid sequence shown in SEQ ID NO: 42 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: 43 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: 44 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: 45 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: 46 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: 47 is Neutrase, a protease derived from Bacillus amyloliquefacience, and the protease consisting of the amino acid sequence shown in SEQ ID NO: 48 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).

[0043] 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).

[0044] The detergent composition of the present invention may contain known detergent components, and examples of such known detergent components include the following:

[0045] (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.

[0046] The surfactants used in the detergent composition of the present invention may include one or a combination of anionic surfactants, nonionic surfactants, amphoteric surfactants, and cationic surfactants, but anionic surfactants and nonionic surfactants are preferred.

[0047] 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.

[0048] 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.

[0049] (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.

[0050] (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.

[0051] (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.

[0052] (5) Bleach For example, it is preferable to include bleaching agents such as hydrogen peroxide and percarbonates in an amount of 1 to 10% by mass. When using bleaching agents, tetraacetylethylenediamine (TAED) or bleaching activators such as those described in Japanese Patent Publication No. 6-316700 can be included in an amount of 0.01 to 10% by mass.

[0053] (6) Fluorescent agents Examples of fluorescent agents used in the detergent composition of the present invention 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 incorporate 0.001 to 2% by mass of the fluorescent agent.

[0054] (7) Other ingredients The detergent composition of the present invention 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.

[0055] The detergent composition of the present invention can be manufactured by combining the protein of the present invention obtained by the above method with the above known cleaning 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.

[0056] The detergent composition obtained in this manner can be used as a laundry detergent, dishwashing detergent, bleach, hard surface cleaner, drain cleaner, denture cleaner, disinfectant cleaner for medical instruments, etc. Preferably, it can be used as a laundry detergent or dishwashing detergent, and more preferably as a laundry detergent (laundry detergent), a hand-washing dishwashing detergent, or a detergent for automatic dishwashers. Furthermore, the detergent 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. Preferred uses include low-temperature (15-30°C) washing in laundry facilities and low-temperature (15-30°C) washing in automatic dishwashers.

[0057] With regard to the embodiments described above, the present invention further discloses the following embodiments. <1> A detergent composition containing one or more proteins selected from (A), (B), (C), and (D) below. (A) A protein having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in Sequence ID No. 2, and which also has α-amylase activity. (B) A protein having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in Sequence ID No. 4, and which also has α-amylase activity. (C) A protein having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in Sequence ID No. 6, and which also has α-amylase activity. (D) A protein having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in Sequence ID No. 8, and which also has α-amylase activity. <2> The protein is one or more selected from (A'), (B'), (C'), and (D') below. <1> The cleaning agent composition described above. (A') A protein having α-amylase activity, comprising an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 2, and having two or more amino acid residues selected from the R178, G179, T180, and G181 positions of the amino acid sequence shown in SEQ ID NO: 2. (B') A protein having α-amylase activity, comprising an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 4, and having two or more amino acid residues selected from the R178, G179, T180, and G181 positions of the amino acid sequence shown in SEQ ID NO: 4. (C') A protein having α-amylase activity, comprising an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 6, and having two or more amino acid residues selected from the R178, G179, T180, and G181 positions of the amino acid sequence shown in SEQ ID NO: 6. (D') A protein having α-amylase activity, comprising an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 8, and having two or more amino acid residues selected from the R178, G179, T180, and G181 positions of the amino acid sequence shown in SEQ ID NO: 8. <3> The protein is a protein whose relative washing performance against amylase consisting of the amino acid sequence shown in SEQ ID NO: 12 is 1 or higher, preferably 1.1 or higher, more preferably 1.2 or higher, more preferably 1.3 or higher, more preferably 1.5 or higher, more preferably 1.7 or higher, and more preferably 1.9 or higher. <1> The cleaning agent composition described above. <4> It is a laundry detergent or a dishwashing detergent. <1> ~ <3> A detergent composition as described in any of the following. <5> It is a laundry detergent or a dishwashing detergent for hand washing or automatic dishwashers. <4> The cleaning agent composition described above. <6> It is either a powder or a liquid. <4> or <5> The cleaning agent composition described above. <7> Used at low temperatures, <4> ~ <6> A detergent composition as described in any of the following. <8> 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℃. <7> The cleaning agent composition described above. <9> It is used in low-temperature (15-30°C) washing in laundry, or in low-temperature (15-30°C) washing in automatic dishwashers. <4> The cleaning agent composition described above. <10> A protein selected from (A'), (B'), (C'), and (D') below. (A') A protein having α-amylase activity, comprising an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 2, and having two or more amino acid residues selected from the R178, G179, T180, and G181 positions of the amino acid sequence shown in SEQ ID NO: 2. (B') A protein having α-amylase activity, comprising an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 4, and having two or more amino acid residues selected from the R178, G179, T180, and G181 positions of the amino acid sequence shown in SEQ ID NO: 4. (C') A protein having α-amylase activity, comprising an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 6, and having two or more amino acid residues selected from the R178, G179, T180, and G181 positions of the amino acid sequence shown in SEQ ID NO: 6. (D') A protein having α-amylase activity, comprising an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 8, and having two or more amino acid residues selected from the R178, G179, T180, and G181 positions of the amino acid sequence shown in SEQ ID NO: 8. <11> The relative washing performance against the amylase consisting of the amino acid sequence shown in SEQ ID NO: 12 is 1 or higher, preferably 1.1 or higher, more preferably 1.2 or higher, more preferably 1.3 or higher, more preferably 1.5 or higher, more preferably 1.7 or higher, and more preferably 1.9 or higher. <10> The protein described above. <12> The deletion of two or more amino acid residues selected from the positions R178, G179, T180, and G181 results in R178Δ+T180Δ, G179Δ+T180Δ, R178Δ+G179Δ, R178Δ+G181Δ, or G179Δ+G181Δ, preferably R178Δ+T180Δ. <10> or <11> Protein or <2> or <3> A detergent composition.

[0058] <13> A detergent composition containing one or more proteins having α-amylase activity selected from (A), (B), (C), and (D) below, and a protein having protease activity. (A) A protein having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in Sequence ID No. 2, and which also has α-amylase activity. (B) A protein having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in Sequence ID No. 4, and which also has α-amylase activity. (C) A protein having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in Sequence ID No. 6, and which also has α-amylase activity. (D) A protein having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in Sequence ID No. 8, and which also has α-amylase activity. <14> The protein possessing α-amylase activity is one or more selected from (A'), (B'), (C'), and (D') below. <13> The cleaning agent composition described above. (A') A protein having α-amylase activity, comprising an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 2, and having two or more amino acid residues selected from the R178, G179, T180, and G181 positions of the amino acid sequence shown in SEQ ID NO: 2. (B') A protein having α-amylase activity, comprising an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 4, and having two or more amino acid residues selected from the R178, G179, T180, and G181 positions of the amino acid sequence shown in SEQ ID NO: 4. (C') A protein having α-amylase activity, comprising an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 6, and having two or more amino acid residues selected from the R178, G179, T180, and G181 positions of the amino acid sequence shown in SEQ ID NO: 6. (D') A protein having α-amylase activity, comprising an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 8, and having two or more amino acid residues selected from the R178, G179, T180, and G181 positions of the amino acid sequence shown in SEQ ID NO: 8. <15> The protein having α-amylase activity is a protein whose relative washing performance against the amylase consisting of the amino acid sequence shown in SEQ ID NO: 12 is 1 or higher, preferably 1.1 or higher, more preferably 1.2 or higher, more preferably 1.3 or higher, more preferably 1.5 or higher, more preferably 1.7 or higher, and more preferably 1.9 or higher. <13> or <14> The cleaning agent composition described above. <16> A protein possessing protease activity has at least 70% identity with the amino acid sequence shown in SEQ ID NOs. 42, 43, 44, 45, 46, 47, or 48, and is a protein possessing protease activity. <13> ~ <15> A detergent composition as described in any of the following. <17> It is a laundry detergent or a dishwashing detergent. <13> ~ <16> A detergent composition as described in any of the following. <18> It is a laundry detergent or a dishwashing detergent for hand washing or automatic dishwashers. <17> The cleaning agent composition described above. <19> It is either a powder or a liquid. <17> or <18> The cleaning agent composition described above. <20> Used at low temperatures, <17> ~ <19> A detergent composition as described in any of the following. <21> 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℃. <20> The cleaning agent composition described above. <22> It is used in low-temperature (15-30°C) washing in laundry, or in low-temperature (15-30°C) washing in automatic dishwashers. <17> The cleaning agent composition described above. <23> The deletion of two or more amino acid residues selected from the positions R178, G179, T180, and G181 results in R178Δ+T180Δ, G179Δ+T180Δ, R178Δ+G179Δ, R178Δ+G181Δ, or G179Δ+G181Δ, preferably R178Δ+T180Δ. <14> ~ <22> A detergent composition as described in any of the following. [Examples]

[0059] (1) Construction of an amylase expression plasmid The synthesized DE0178 gene (SEQ ID NO: 1) is used as a template for the primer pair DE0178_fw / DE0178_rv (SEQ ID NOs: 19 and 20) and PrimeSTAR. PCR was performed using Max Premix (Takara Bio). Using plasmid pHY-S237 described in Example 7 of WO2006 / 068148A1 as a template, PCR was performed similarly using the primer pair S237t_fw / S237s_rv (SEQ ID NOs. 17 and 18). In-Fusion reactions were performed using each PCR product according to the In-Fusion, HD Cloning kit (Clontech) protocol. Plasmid pHY-DE0178 was constructed by transforming Bacillus subtilis with the in-fusion reaction solution. Similarly, for RU2C, BCGAmy, YR288, AP1378, AA560, SP722, and CspAmy2, artificially synthesized genes (sequences 3, 5, 7, 9, 11, 13, and 15 respectively) were used as templates, and primer pairs RU2C_fw / RU2C_rv (sequences 21 and 22), BCGAmy_fw / BCGAmy_rv (sequences 23 and 24), and YR288_fw / YR2 PCR and in-fusion reactions were performed using 88_rv (sequences 25 and 26), AP1378_fw / AP1378_rv (sequences 27 and 28), AA560_fw / AA560_rv (sequences 29 and 30), SP722_fw / SP722_rv (sequences 31 and 32), and CspAmy2_fw / CspAmy2_rv (sequences 33 and 34). The in-fusion reaction solutions were transformed into Bacillus subtilis to construct plasmids pHY-RU2C, pHY-BCGAmy, pHY-YR288, pHY-AP1378, pHY-AA560, pHY-SP722, and pHY-CspAmy2, respectively.

[0060] (2) transformation The host organism used was Bacillus subtilis Marburg strain No. 168 (Nature, 390, 1997, p. 249). Bacillus subtilis strain No. 168 was inoculated into 1 mL of LB medium and incubated overnight at 30°C and 200 rpm with shaking. 10 μL of this culture was inoculated into 1 mL of fresh LB medium and incubated at 37°C and 200 rpm for 3 hours. The culture was centrifuged to collect the pellet. 500 μL of SMMP (0.5 M sucrose, 20 mM disodium maleate, 20 mM magnesium chloride hexahydrate, 35% (w / v) Antibiotic medium 3 (Difco)) containing 4 mg / mL of lysozyme (SIGMA) was added to the pellet and incubated at 37°C for 1 hour. The pellet was then recovered by centrifugation and suspended in 400 μL of SMMP. 33 μL of suspension and DNA were mixed, then 100 μL of 40% PEG was added and stirred, followed by 350 μL of SMMP, and the mixture was shaken at 30°C for 1 hour. 200 μL of this solution was spread onto DM3 regenerated agar medium containing tetracycline (15 μg / mL, SIGMA) (0.8% agar (Wako Pure Chemical Industries), 0.5% disodium succinate hexahydrate, 0.5% casamino acid technical (Difco), 0.5% yeast extract, 0.35% monopotassium phosphate, 0.15% dipotassium phosphate, 0.5% glucose, 0.4% magnesium chloride hexahydrate, 0.01% bovine serum albumin (SIGMA), 0.5% carboxymethylcellulose, 0.005% trypan blue (Merck), and amino acid mixture (tryptophan, lysine, methionine, 10 μg / mL each); % is (w / v)); and incubated at 30°C for 3 days to obtain the formed colonies.

[0061] (3) Enzyme production culture Recombinant Bacillus subtilis colonies obtained in (2) were inoculated into 96-well deep-well plates dispensed with 300 μL of LB medium supplemented with 15 ppm tetracycline, and then incubated overnight at 30°C and 210 rpm. The following day, 6 μL of the culture solution was inoculated into 96-well deep-well plates dispensed with 100 μL of 2×L-maltose medium (2% tryptone, 1% yeast extract, 1% NaCl, 7.5% maltose, 7.5 ppm manganese sulfate pentahydrate, 0.04% calcium chloride dihydrate, 15 ppm tetracycline; % is (w / v)%), and incubated at 30°C and 210 rpm for 2 days. The culture supernatant containing enzymes produced from the bacterial cells was then collected by centrifugation.

[0062] (4) Protein concentration measurement The Protein Assay Rapid Kit Wako II (Fujifilm Wako Pure Chemical Corporation) was used to measure protein concentration. The concentration of amylase in the culture supernatant was calculated by using the protein concentration of the culture supernatant of a pHY300PLK (Takara Bio) strain without an amylase expression cassette as a blank.

[0063] (5) Measurement of starch decomposition activity The starch-degrading activity of each culture supernatant and Termamyl (SIGMA, A4862) was measured using Phadebas (Phadebas AB). Phadebas is a tablet consisting of insoluble starch covalently bonded to a blue pigment. The water-soluble blue pigment is released upon starch degradation by α-amylase. The concentration of the blue pigment, measured by absorbance at 620 nm, is proportional to the amylase activity in the sample. One substrate tablet was suspended in 5 mL of 1 / 15 M phosphate buffer (pH 7.4). 500 μL of the substrate suspension was dispensed into a 96-well deep-well plate. The enzyme solution, appropriately diluted with 1 / 15 M phosphate buffer (pH 7.4), was added and mixed. After standing at 20°C or 30°C for 30 minutes, the reaction was stopped by adding 250 μL of 10% citric acid aqueous solution. The mixture was centrifuged at 3000 rpm for 20 minutes, and 100 μL of the supernatant was transferred to a new 96-well plate. The absorbance was measured at 620 nm. Each measurement value was confirmed to be within the linear range of the activity measurement. The starch-degrading activity ΔA620 was calculated by subtracting the blank (no enzyme added) value, and the specific activity ΔA620 / ppm was determined by dividing by the added amylase concentration (Figure 1). DE0178, RU2C, BCGAmy, and YR288 showed significantly higher starch-degrading activity at 20°C and 30°C compared to previously reported detergent amylases (AP1378, AA560, SP722, CspAmy2, Termamyl).

[0064] (6) Phylogenetic tree Phylogenetic trees were constructed using the amino acid sequences of the mature regions of amylases DE0178, RU2C, BCGAmy, YR288, which maintain high activity at low temperatures, and existing detergent amylases AP1378, AA560, SP722, CspAmy2, BAA (SEQ ID NO: 35), BLA (SEQ ID NO: 36), LABM (SEQ ID NO: 37), SP707 (SEQ ID NO: 38), TS23 (SEQ ID NO: 39), Termamyl (SEQ ID NO: 40), and AAI10 (SEQ ID NO: 41), which maintain high activity at low temperatures. Multiple alignment was performed using clustalW with Genetyx, and the phylogenetic trees were constructed using the neighbor-joining method (NJ method). As a result, amylases DE0178, RU2C, BCGAmy, and YR288, which maintain high activity at low temperatures, formed a different group from existing detergent amylases (Figure 2).

[0065] (7) Evaluation of cleaning power CS-26 contaminated cloths, cut into 5.5 mm diameter circles, were obtained from CFT and used. Two CS-26 circular contaminated cloths were inserted into each well of a 96-well assay plate, and 200 μL of Attack Zero (Kao), diluted 3000 times with tap water, was added to each well. 10 μL of appropriately diluted enzyme solution was added to each well, the plates were sealed, and the plates were 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 created by adding tap water instead of the enzyme solution, and the difference ΔA488 from the blank was calculated as the washing power. Amylase YR288, which maintains high activity at low temperatures, showed significantly higher cleaning power at low temperatures compared to previously reported detergent amylases (AP1378, AA560, SP722, CspAmy2) (Figure 3).

[0066] (8) Construction of amylase mutant expression plasmids 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 mutation-introducing primer pair. PCR was performed using the mutation-introducing primer pair with the amylase expression plasmid prepared in Example (1) as a template. The PCR product was transformed into Bacillus subtilis by the protoplast method to obtain transformants carrying the target mutant expression plasmid.

[0067] (9) Evaluation of two amino acid deletion mutants at positions 178-181 of YR288 The mutant shown in Figure 4 was constructed using YR288 (SEQ ID NO: 8) as the parent polypeptide by the method described in Example (8). The starch degradation specific activity ΔA620 / ppm of the mutant at 20°C was determined by the method described in Example (5), and the relative specific activity was calculated by dividing it by the value of wild-type YR288 (Figure 4). The starch degradation activity of YR288 at low temperatures was further improved by deleting any two residues from R178, G179, T180, and G181. The washing power of the mutant was evaluated using the method described in Example (7). The washing power of YR288 was further improved by deleting any two residues from R178, G179, T180, and G181 (Figure 5).

[0068] (10) Evaluation of a two-amino acid deletion mutant at positions 178-181 of BCGAmy The mutant shown in Figure 6 was constructed using BCGAmy (SEQ ID NO: 6) as the parent polypeptide by the method described in Example (8). The starch degradation specific activity ΔA620 / ppm of the mutant at 20°C was determined by the method described in Example (5), and the relative specific activity was calculated by dividing it by the value of wild-type BCGAmy (Figure 6). The starch degradation activity of BCGAmy at low temperatures was further improved by deleting two residues, R178 and G179, or two residues, R178 and T180.

[0069] (11) Relative washing performance of YR288 and two-amino acid deletion mutants of YR288 against AA560 CS-26 contaminated cloths cut into 5.5 mm diameter circles were obtained from CFT and used. Two CS-26 circular contaminated cloths were inserted into each well of a 96-well assay plate (3881-096, IWAKI), and 200 μL of model washing solution (200 ppm linear alkylbenzene sulfonate sodium (195-07682, wako), 20 mM Tris HCl aqueous solution (pH 7.5)) was added to each well. 10 μL of each enzyme solution listed in Table 1, diluted to 4 ppm with deionized water, was added to each well, and the plates were sealed and shaken at 1200 rpm for 15 minutes at 20°C using a Cute Mixer (CUTE MIXER CM-1000, EYELA). 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 the enzyme solution, and the difference ΔA488 from the blank was calculated. The relative washing performance of each enzyme relative to AA560 was determined by dividing the ΔA488 of each enzyme by the ΔA488 of AA560 (Table 1).

[0070] [Table 1]

[0071] (12) Cleaning performance of a cleaning composition containing amylase and protease CS-26 contaminated cloth, cut into 5.5 mm diameter circles, was obtained from CFT, and EMPA117 contaminated cloth was obtained from EMPA. Savinase (SIGMA, P3111) and KAP8.0Q-L (Kao) were used as proteases. CS-26 or EMPA117 contaminated cloth was inserted into each well of a 96-well assay plate, and 200 μL of either Attack Zero (Kao) diluted 3000-fold with tap water (treated at 90°C for 5 hours) or a model washing solution (200 ppm linear alkylbenzene sulfonate sodium (195-07682, wako), 20 mM Tris HCl aqueous solution (pH 7.5)) was added to each well. 10 μL of 4 ppm diluted amylase solution and 10 μL of 4 ppm diluted protease solution were added to each well, the plates were sealed, and the plates were shaken at 1200 rpm for 15 minutes at 20°C using a Cute Mixer. After washing was complete, 100 μL of the washing solution was transferred to a new 96-well assay plate, and the absorbance at 488 nm was measured for CS-26 contaminated cloths and at 660 nm for EMPA117 contaminated cloths. 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 amylase washing power. Figure 7 shows the cleaning power of CS-26 contaminated cloth, and Figure 8 shows the cleaning power of EMPA117 contaminated cloth. A cleaning composition containing amylase (YR288, YR288 R178Δ T180Δ) and protease that maintain high activity at low temperatures exhibited superior cleaning power at low temperatures compared to conventional cleaning compositions containing amylase and protease.

Claims

1. A detergent composition containing one or more proteins selected from (A'), (B'), (C'), and (D') below. (A') A protein having α-amylase activity, comprising an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2, wherein two or more amino acid residues selected from the R178, G179, T180, and G181 positions of the amino acid sequence shown in SEQ ID NO: 2 are deleted. (B') A protein having α-amylase activity, comprising an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 4, wherein two or more amino acid residues selected from the R178, G179, T180, and G181 positions of the amino acid sequence shown in SEQ ID NO: 4 are deleted. (C') A protein having α-amylase activity, comprising an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 6, wherein two or more amino acid residues selected from the R178, G179, T180, and G181 positions of the amino acid sequence shown in SEQ ID NO: 6 are deleted. (D') A protein having α-amylase activity, comprising an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 8, wherein two or more amino acid residues selected from the R178, G179, T180, and G181 positions of the amino acid sequence shown in SEQ ID NO: 8 are deleted.

2. The detergent composition according to claim 1, further containing a protein having protease activity.

3. The detergent composition according to claim 2, wherein the protein having protease activity has at least 90% identity with the amino acid sequence shown in SEQ ID NOs. 42, 43, 44, 45, 46, 47, or 48, and is a protein having protease activity.

4. A detergent composition according to any one of claims 1 to 3, which is a laundry detergent or a dishwashing detergent.

5. The detergent composition according to claim 4, which is a laundry detergent or a dishwashing detergent for hand washing or automatic dishwashers.

6. The cleaning agent composition according to claim 4 or 5, which is in the form of a powder or liquid.

7. A detergent composition according to any one of claims 4 to 6, for use at low temperatures.

8. The detergent composition according to claim 7, which is used at a temperature of 5 to 40°C.

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