Mutant protease

Abstract

To provide a mutant protease having improved cleaning performance on protein stain in a high-concentration detergent liquid.SOLUTION: Provided is a mutant protease comprising a specific amino acid sequence or an amino acid sequence having at least 90% identity thereto and having at least one amino acid residue selected from the group consisting of (a) to (g): (a) Arg or Lys at a position corresponding to position 191 of the specific sequence; (b) Arg or Lys at a position corresponding to position 17 of the specific sequence; (c) Ala or Phe at a position corresponding to position 141 of the specific sequence; (d) Arg or Lys at a position corresponding to position 243 of the specific sequence; (e) Arg or Lys at a position corresponding to position 300 of the specific sequence; (f) Arg or Lys at a position corresponding to position 302 of the specific sequence; and (g) Arg or Lys at a position corresponding to position 311 of the specific sequence.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to mutant proteases. [Background technology]

[0002] The purpose of incorporating proteases in detergents is to decompose protein stains derived from food, dirt, sweat, etc. attached to clothes, tableware, etc., and promote the removal of stains. Since the pH of detergents is generally on the alkaline side, proteases with an optimal pH on the alkaline side have been developed as detergent proteases. Patent Document 1 discloses a protease with a molecular weight of about 43,000 that has cleaning properties against complex stains containing a mixture of proteins and lipids. Patent Documents 2 to 5 disclose mutant proteases obtained by introducing mutations that improve the cleaning performance of proteases, their stability in liquid detergents, their solubility in liquid detergents, or their stability under acidic conditions. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 99 / 18218 [Patent Document 2] JP 2008-212084 A [Patent Document 3] JP 2010-273672 A [Patent Document 4] JP 2013-233141 A [Patent Document 5] JP 2020-145938 A Summary of the Invention [Problem to be solved by the invention]

[0004] In order to remove stubborn stains such as spots, liquid detergent concentrate is sometimes applied directly to the stain without diluting it with water. It is considered effective to apply liquid detergent containing protease to stains containing protein such as food spills and collar / sleeve stains. However, liquid detergent concentrate contains high concentrations of detergent ingredients such as surfactants, which creates harsh conditions for proteases. It is desirable to improve the cleaning performance of proteases in solutions containing high concentrations of detergent ingredients such as liquid detergent concentrate.

[0005] The present invention relates to providing a mutant protease having improved cleaning performance against proteinaceous stains in a high-concentration detergent solution. [Means for solving the problem]

[0006] The present inventors have discovered that a mutant protease derived from protease KP43 (see Patent Document 1) having a molecular weight of 43,000 has improved cleaning performance (hereinafter simply referred to as cleaning performance) against proteinaceous stains even in the concentrate of a liquid detergent.

[0007] Therefore, the present invention provides a mutant protease consisting of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 90% identity thereto, and having at least one amino acid residue selected from the group consisting of the following (a) to (g): (a) Arg or Lys at a position corresponding to position 191 of SEQ ID NO:1; (b) Arg or Lys at a position corresponding to position 17 of SEQ ID NO:1; (c) Ala or Phe at a position corresponding to position 141 of SEQ ID NO:1; (d) Arg or Lys at a position corresponding to position 243 of SEQ ID NO:1; (e) Arg or Lys at a position corresponding to position 300 of SEQ ID NO:1; (f) Arg or Lys at a position corresponding to position 302 of SEQ ID NO:1; (g) Arg or Lys at a position corresponding to position 311 of SEQ ID NO:1; to provide. The present invention also provides a polynucleotide encoding the mutant protease. The present invention also provides a vector containing the polynucleotide. The present invention also provides a recombinant Bacillus bacterium containing the above-mentioned polynucleotide or the above-mentioned vector. The present invention also provides a method for producing a mutant protease using the recombinant Bacillus bacterium. The present invention also provides a detergent composition containing the mutant protease. The present invention further relates to a method for producing a mutant protease, comprising introducing at least one mutation selected from the group consisting of the following (A) to (G) into a parent protease having the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 90% identity thereto: (A) substitution of the amino acid residue at position 191 of SEQ ID NO:1 or a position equivalent thereto with Arg or Lys; (B) substitution of the amino acid residue at position 17 of SEQ ID NO:1 or a position equivalent thereto with Arg or Lys; (C) substitution of the amino acid residue at position 141 of SEQ ID NO:1 or a position equivalent thereto with Ala or Phe; (D) substitution of the amino acid residue at position 243 of SEQ ID NO:1 or a position equivalent thereto with Arg or Lys; (E) substitution of the amino acid residue at position 300 of SEQ ID NO:1 or a position equivalent thereto with Arg or Lys; (F) substitution of the amino acid residue at position 302 of SEQ ID NO:1 or a position equivalent thereto with Arg or Lys; (G) substitution of the amino acid residue at position 311 of SEQ ID NO:1 or a position equivalent thereto with Arg or Lys; to provide. The present invention also provides a method for improving the cleaning performance of a protease against protein stains in a high-concentration detergent solution, the method comprising introducing at least one mutation selected from the group consisting of (A) to (G) into a parent protease having the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 90% identity thereto. Effect of the Invention

[0008] The mutant protease of the present invention has improved cleaning performance for protein stains in a high-concentration detergent solution (e.g., liquid detergent concentrate). The mutant protease of the present invention is effective as a protease to be used in cleaning with a high-concentration detergent solution, such as spray cleaning or soak cleaning. For example, the mutant protease of the present invention is effective as a protease to be incorporated into a detergent for cleaning stains by directly contacting the liquid detergent concentrate or high-concentration detergent solution with the stains. [Brief description of the drawings]

[0009] [Figure 1] Relative application cleaning power of mutant proteases. [Diagram 2] Improved application and cleaning power due to mutation at position 191. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] As used herein, the term "amino acid residue" refers to the 20 types of amino acid residues constituting 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).

[0011] As used herein, "at least 90% identity" with respect to an amino acid sequence or a nucleotide sequence means identity of 90% or more, preferably 95% or more, more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more.

[0012] In the present specification, 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 analysis using the search homology program of the genetic information processing software Genetyx-Win with the unit size to compare (ktup) set to 2.

[0013] As used herein, "an amino acid sequence in which one or several amino acids have been deleted, inserted, substituted or added" includes an amino acid sequence in which preferably one to ten, more preferably one to five, and even more preferably one to three amino acids have been deleted, inserted, substituted or added. As used herein, "a nucleotide sequence in which one or several nucleotides have been deleted, inserted, substituted or added" includes a nucleotide sequence in which preferably one to thirty, more preferably one to fifteen, and even more preferably one to ten nucleotides have been deleted, inserted, substituted or added. As used herein, "addition" of an amino acid or nucleotide includes addition of one or several amino acids or nucleotides to one end and both ends of a sequence.

[0014] In the present specification, the "corresponding position" on an amino acid sequence or a nucleotide sequence can be determined by aligning a target sequence with a reference sequence (e.g., the amino acid sequence of SEQ ID NO: 1) so as to give maximum homology. Alignment of amino acid sequences or nucleotide sequences can be performed using known algorithms, and the procedures are known to those skilled in the art. For example, alignment can be performed using the Clustal W multiple alignment program (Thompson, J. D. et al., 1994, Nucleic Acids Res. 22: 4673-4680) with default settings. Alternatively, Clustal W2 or Clustal omega, which are revised versions of Clustal W, can be used. Clustal W, Clustal W2 and Clustal omega are available, for example, on the Clustal website operated by University College Dublin [www.clustal.org], the European Bioinformatics Institute (EBI [www.ebi.ac.uk / index.html]), or the website of the DNA Data Bank of Japan operated by the National Institute of Genetics (DDBJ [www.ddbj.nig.ac.jp]). The position of the target sequence aligned to any position of the reference sequence by the above-mentioned alignment is considered to be a "position corresponding to" the any position.

[0015] Those skilled in the art can further fine-tune the alignment of the amino acid sequences obtained above to optimize it. Such an optimal alignment is preferably determined taking into consideration the similarity of the amino acid sequences, the frequency of gaps to be inserted, and the like. Here, the similarity of the amino acid sequences refers to the ratio (%) of the number of positions at which identical or similar amino acid residues exist in both sequences when the two amino acid sequences are aligned to the total number of amino acid residues. The similar amino acid residues refer to amino acid residues that have similar properties in terms of polarity and charge among the 20 types of amino acids that constitute proteins, and that cause so-called conservative substitution. Such groups of similar amino acid residues are well known to those skilled in the art, and examples thereof 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.

[0016] The position of an amino acid residue in the target amino acid sequence that is aligned to a position corresponding to any position in the reference sequence by the above-mentioned alignment is considered to be the "corresponding position" of that arbitrary position, and that amino acid residue is referred to as the "amino acid residue at the corresponding position."

[0017] As used herein, a "parent" polypeptide of a mutant polypeptide refers to a polypeptide that becomes the mutant polypeptide by making a predetermined mutation in its amino acid residues. In other words, a "parent" polypeptide is a polypeptide before the polypeptide mutant is mutated. Similarly, a "parent" polynucleotide of a mutant polynucleotide refers to a polynucleotide that becomes the mutant polynucleotide by making a predetermined mutation in its nucleotides. In other words, a "parent" polynucleotide is a polynucleotide before the mutant polynucleotide is mutated.

[0018] As used herein, the term "operably linked" between a control region such as a promoter and a gene means that the gene and the control region are linked in such a way that the gene can be expressed under the control of the control region. Procedures for "operably linked" between a gene and a control region are well known to those skilled in the art.

[0019] In this specification, "upstream" and "downstream" of a gene refer to the upstream and downstream of 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 a gene means the 5' region of the gene in the DNA sense strand.

[0020] As used herein, the term "native" when referring to a function, property, or trait of a cell is used to indicate that the function, property, or trait is inherently present in the cell. In contrast, the term "exogenous" is used to indicate a function, property, or trait that is not inherently present in the cell, but is introduced from the outside. For example, an "exogenous" gene or polynucleotide is a gene or polynucleotide that is introduced into a cell from the outside. An exogenous gene or polynucleotide may be derived from the same organism as the cell into which it is introduced, or from a different organism (i.e., a heterologous gene or polynucleotide).

[0021] The present invention provides a mutant protease. The mutant protease of the present invention is a mutant protease having at least one mutation selected from the group consisting of the following (A) to (G) with respect to a parent protease having the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 90% identity thereto: (A) substitution of the amino acid residue at position 191 of SEQ ID NO:1 or a position equivalent thereto with Arg or Lys, preferably Arg; (B) substitution of the amino acid residue at position 17 of SEQ ID NO:1 or a position equivalent thereto with Arg or Lys, preferably Arg; (C) substitution of the amino acid residue at position 141 of SEQ ID NO:1, or a position equivalent thereto, with Ala or Phe, preferably Ala; (D) substitution of the amino acid residue at position 243 of SEQ ID NO:1 or a position equivalent thereto with Arg or Lys, preferably Lys; (E) substitution of the amino acid residue at or corresponding to position 300 of SEQ ID NO:1 with Arg or Lys, preferably Lys; (F) substitution of the amino acid residue at position 302 of SEQ ID NO:1, or a position equivalent thereto, with Arg or Lys, preferably Arg; (G) Substitution of the amino acid residue at position 311 of SEQ ID NO:1 or a position equivalent thereto with Arg or Lys, preferably Arg.

[0022] Preferably, the mutant protease of the present invention has the mutation (A) relative to the parent protease. In a preferred embodiment, the mutant protease of the present invention has the mutation (A) and at least one mutation selected from the group consisting of the mutations (B) to (G) relative to the parent protease.

[0023] In one embodiment, the mutant protease of the present invention has, relative to the parent protease, the mutation (A) and at least one mutation selected from the group consisting of the mutations (B) and (D). In another embodiment, the mutant protease of the present invention has, relative to the parent protease, the mutation (A) and any one mutation selected from the mutations (B) and (D). Preferably, the mutation (A) is a substitution of the amino acid residue at position 191 or a position equivalent thereto with Arg, the mutation (B) is a substitution of the amino acid residue at position 17 or a position equivalent thereto with Arg, and the mutation (D) is a substitution of the amino acid residue at position 243 or a position equivalent thereto with Lys.

[0024] In one embodiment, the mutant protease of the present invention has the following mutation (H) in addition to at least one mutation selected from the group consisting of (A) to (G) above: (H) Substitution of the amino acid residue at position 82 of SEQ ID NO:1 or a position equivalent thereto with Gln. By having the mutation (H) in addition to any one of the mutations (A) to (G), the cleaning performance of the mutant protease of the present invention in a high concentration detergent solution is further improved. In a preferred embodiment, the mutant protease of the present invention has the mutation (A) and the mutation (H). Preferably, the mutation (A) is a substitution of the amino acid residue at position 191 or a position equivalent thereto with Arg.

[0025] In one embodiment, the mutant protease of the present invention may have at least one mutation selected from the group consisting of the mutations (B) to (G). In a preferred embodiment, the mutant protease of the present invention has the mutation (E) and the mutation (F) relative to the parent protease. By having the mutations (E) and (F), the cleaning performance of the mutant protease of the present invention in a high-concentration detergent solution is further improved. Preferably, the mutation (E) is a substitution of the amino acid residue at position 300 or a position equivalent thereto with Lys, and the mutation (F) is a substitution of the amino acid residue at position 302 or a position equivalent thereto with Arg.

[0026] Parent proteases of the mutant proteases of the present invention include proteases consisting of the amino acid sequence of SEQ ID NO:1 and proteases consisting of an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:1.

[0027] A preferred example of a protease having an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1 is a protease having 95% or more identity with the amino acid sequence of SEQ ID NO: 1, more preferably 96% or more identity, even more preferably 97% or more identity, even more preferably 98% or more identity, even more preferably 99% or more identity. Another example of a protease having an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1 is a protease having an amino acid sequence in which one or several amino acids are deleted, inserted, substituted, or added to the amino acid sequence of SEQ ID NO: 1. The protease having an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1 used as a parent protease in the present invention may be a mutant protease derived from the protease having the amino acid sequence of SEQ ID NO: 1.

[0028] Preferably, in the amino acid sequence of the parent protease, the amino acid residue at the position corresponding to position 30 in SEQ ID NO: 1 is aspartic acid, the amino acid residue at the position corresponding to position 68 is histidine, and the amino acid residue at the position corresponding to position 255 is serine. More preferably, the parent protease has the amino acid residues shown in Table 1(ii) at positions corresponding to the positions shown in Table 1(i) in SEQ ID NO: 1 below. The amino acid residues shown in Table 1 are highly conserved among the protease KP43 from which the parent protease is derived (Patent Document 1) and mutant proteases derived therefrom (Saeki et al., Journal of bioscience and Bioengineering, 2007, 103:501-508).

[0029] [Table 1]

[0030] The parent protease of the mutant of the present invention is a polypeptide having proteolytic activity on the alkaline side (preferably pH 8 or higher). Preferably, the parent protease is a polypeptide having an optimum pH on the alkaline side (preferably pH 8 or higher). More preferably, the parent protease has any of the following enzymatic properties of the protease consisting of the amino acid sequence of SEQ ID NO: 1 (see Patent Document 1): 1) it is resistant to oxidizing agents, acts on the alkaline side (pH 8 or higher), and is stable. Here, oxidizing agent resistance means that the protease has a residual activity (synthetic substrate method) of at least 50% after being left in a 50 mM hydrogen peroxide (containing 5 mM calcium chloride) solution (20 mM Britton-Robinson buffer, pH 10) at 20°C for 20 minutes; 2) shows a residual activity of 80% or more when treated at 50°C, pH 10 for 10 minutes; 3) activity is inhibited by diisopropylfluorophosphate (DFP) and phenylmethanesulfonyl fluoride (PMSF); and 4) molecular weight by SDS-PAGE is 43,000±2,000. More preferably, the parent protease has all of the enzymatic properties 1) to 4) above.

[0031] The mutant protease of the present invention can be produced by introducing at least one mutation selected from the group consisting of (A) to (G) in the amino acid sequence of SEQ ID NO: 1 into the amino acid sequence of the parent protease. Preferably, the mutant protease of the present invention can be produced by introducing the mutation (H) into the amino acid sequence of the parent protease, in addition to the at least one mutation selected from the group consisting of (A) to (G).

[0032] Thus, the mutant protease of the present invention has at least one amino acid residue selected from the group consisting of the following amino acid residues (a) to (g): (a) Arg or Lys, preferably Arg, at a position corresponding to position 191 of SEQ ID NO:1; (b) Arg or Lys, preferably Arg, at a position corresponding to position 17 of SEQ ID NO:1; (c) Ala or Phe, preferably Ala, at a position corresponding to position 141 of SEQ ID NO:1; (d) Arg or Lys, preferably Lys, at a position corresponding to 243 of SEQ ID NO:1; (e) Arg or Lys, preferably Lys, at a position corresponding to 300 of SEQ ID NO:1; (f) Arg or Lys, preferably Arg, at a position corresponding to 302 of SEQ ID NO:1; (g) Arg or Lys, preferably Arg, at a position corresponding to position 311 of SEQ ID NO:1. Preferably, the variant proteases of the present invention have at least 90% sequence identity with the amino acid sequence of SEQ ID NO:1.

[0033] Preferably, the mutant protease of the present invention has the amino acid residue of (a) above. In a preferred embodiment, the mutant protease of the present invention has the amino acid residue of (a) above and at least one amino acid residue selected from the group consisting of (b) to (g) above.

[0034] In one embodiment, the mutant protease of the present invention has the amino acid residue of (a) and at least one amino acid residue selected from the group consisting of (b) and (d). In another embodiment, the mutant protease of the present invention has the amino acid residue of (a) and any one of the amino acid residues of (b) and (d). Preferably, the amino acid residue of (a) is Arg, the amino acid residue of (b) is Arg, and the amino acid residue of (d) is Lys.

[0035] In one embodiment, the mutant protease of the present invention has the following amino acid residue (h) in addition to at least one amino acid residue selected from the group consisting of (a) to (g) above: (h) Gln at a position corresponding to position 82 of SEQ ID NO:1. In a preferred embodiment, the mutant protease of the present invention has the amino acid residues (a) and (h). Preferably, the amino acid residue (a) is Arg.

[0036] In one embodiment, the mutant protease of the present invention may have at least one amino acid residue selected from the group consisting of (b) to (g). In a preferred embodiment, the mutant protease of the present invention has the amino acid residues (e) and (f). Preferably, the amino acid residue (e) is Lys, and the amino acid residue (f) is Arg.

[0037] The mutant protease of the present invention is a mutant protease with improved cleaning performance against protein stains in a detergent solution containing a high concentration of a surfactant (so-called high-concentration detergent solution). More specifically, the mutant protease of the present invention can exhibit higher cleaning performance in a high-concentration detergent solution than a protease consisting of the amino acid sequence of SEQ ID NO: 1. Preferably, the mutant protease of the present invention can exhibit higher cleaning performance in a concentrated liquid detergent stock solution than a protease consisting of the amino acid sequence of SEQ ID NO: 1. Preferably, the mutant protease of the present invention can exhibit at least the same or higher cleaning performance as a protease consisting of the amino acid sequence of SEQ ID NO: 1 in a detergent solution diluted with water to a level used for normal cleaning (so-called normal detergent solution). Therefore, the mutant protease of the present invention can be used as a detergent enzyme, and in particular, can be used as a detergent enzyme for use in cleaning with a high-concentration detergent solution (for example, coating cleaning or soaking cleaning). For example, the mutant protease of the present invention is effective as a protease to be incorporated into a concentrated liquid detergent for cleaning stains by directly applying the stock solution or high-concentration detergent solution to the stains. Furthermore, for example, the mutant protease of the present invention is effective as a protease to be incorporated into concentrated liquid detergents for cleaning stains by immersing the stains in the undiluted solution or in a high-concentration detergent solution.

[0038] In this specification, the term "protein stains" refers to stains caused by food containing protein, body-derived components containing protein (e.g., blood, urine, dirt, sweat, sebum, etc.), etc. More specific examples of "protein stains" include food spills on clothing, stains on collars and sleeves, etc.

[0039] In the present specification, the term "detergent liquid" includes a liquid detergent concentrate and an aqueous detergent solution obtained by diluting a liquid detergent or a solid detergent (e.g., powder detergent) with water. In the present specification, the term "high-concentration detergent liquid" refers to a detergent liquid containing a surfactant at a high concentration, preferably a detergent liquid containing 10% by mass or more, more preferably 20% by mass or more, for example, 10 to 90% by mass, preferably 20 to 90% by mass, more preferably 20 to 75% by mass of the surfactant. Examples of high-concentration detergent liquids include concentrated liquid detergent concentrates and aqueous solutions containing the same at a high concentration. In the present specification, the term "concentrated liquid detergent" refers to a liquid detergent containing 40% by mass or more, preferably 40 to 90% by mass, more preferably 45 to 90% by mass, and even more preferably 50 to 75% by mass of the surfactant. An example of the "concentrated liquid detergent" is a liquid detergent for clothes that is indicated as a standard usage amount of 7 to 13 g or less per 30 L of water in a water tub washing machine.

[0040] In this specification, "cleaning with a high-concentration detergent solution" refers to a cleaning method in which a high-concentration detergent solution is directly brought into contact with dirt on a cleaning target (e.g., cloth and hard surfaces such as tableware and cooking utensils), and the dirt is left or washed for a predetermined time as necessary, and then the cleaning target is washed with water. Examples of "cleaning with a high-concentration detergent solution" include coating washing and soaking washing. "Coating washing" refers to a cleaning method in which a high-concentration detergent solution is directly applied to the dirt on a cleaning target, and the dirt is left or washed for a predetermined time as necessary, and then the cleaning target is washed with water. "Soaking washing" refers to a cleaning method in which the dirt on a cleaning target is immersed in a high-concentration detergent solution, and the dirt is left or washed in the high-concentration detergent solution as necessary, and then the cleaning target is washed with water. Therefore, in the cleaning with the high-concentration detergent solution, examples of means for directly contacting the dirt on a cleaning target with the high-concentration detergent solution include application of the high-concentration detergent solution to the dirt (e.g., by roll-on, spray, dripping, etc.), soaking of the dirt in the high-concentration detergent solution, etc.

[0041] The mutant protease of the present invention may have, in addition to at least one mutation selected from the group consisting of (A) to (G), a mutation (e.g., deletion, substitution, addition, insertion) at any other position relative to the parent protease, so long as it can maintain a higher cleaning performance than the parent protease in the above-mentioned high-concentration detergent solution. The mutation may be naturally occurring or artificially introduced. However, preferably, the mutant protease of the present invention has the same amino acid residues as the parent protease at least at positions 30, 68, and 255 of SEQ ID NO: 1. More preferably, the mutant protease of the present invention has the same amino acid residues as the parent protease at positions corresponding to each position in SEQ ID NO: 1 listed in Table 1(i).

[0042] In the present invention, various mutagenesis techniques known in the art can be used as a means for mutating amino acid residues of a parent protease. For example, in a polynucleotide encoding the amino acid sequence of a parent protease (hereinafter also referred to as a parent polynucleotide), a nucleotide sequence encoding an amino acid residue to be mutated is mutated to a nucleotide sequence encoding the amino acid residue after mutation, and a protein is expressed from the mutated polynucleotide to obtain a mutant protease of interest.

[0043] The introduction of a desired mutation into a parent polynucleotide can be carried out using various site-directed mutagenesis methods well known to those skilled in the art, for example, based on PCR amplification using the parent polynucleotide as a template DNA or a replication reaction using various DNA polymerases. Site-directed mutagenesis can be carried out by any method, such as inverse PCR or annealing (Muramatsu et al., eds., "New Genetic Engineering Handbook, Revised 4th Edition," Yodosha, pp. 82-88). Various commercially available site-directed mutagenesis kits, such as Stratagene's QuickChange II Site-Directed Mutagenesis Kit and QuickChange Multi Site-Directed Mutagenesis Kit, can also be used.

[0044] Site-specific mutagenesis into a parent polynucleotide can be most commonly performed using a mutagenesis primer containing the nucleotide mutation to be introduced. The mutagenesis primer can be designed to anneal to a region containing a nucleotide sequence encoding the amino acid residue to be mutated in the parent polynucleotide, and to contain a nucleotide sequence having a nucleotide sequence (codon) encoding the mutated amino acid residue instead of the nucleotide sequence (codon) encoding the amino acid residue to be mutated. Those skilled in the art can appropriately recognize and select the nucleotide sequences (codons) encoding the amino acid residues before and after the mutation based on ordinary textbooks, etc. Alternatively, site-specific mutagenesis can be performed by a method in which DNA fragments obtained by amplifying the upstream and downstream sides of the mutation site using two complementary primers containing the nucleotide mutation to be introduced separately are linked together by SOE (splicing by overlap extension)-PCR (Horton et al, Gene, 1989, 77(1): p61-68).

[0045] The DNA containing the parent polynucleotide can be prepared by chemically synthesizing the corresponding nucleotide sequence based on the amino acid sequence of the parent protease. Alternatively, the DNA containing the parent polynucleotide can be extracted from a microbial strain carrying the parent polynucleotide by known means. The mutation primer can be prepared by a known oligonucleotide synthesis method such as the phosphoramidite method (Nucleic Acids Research, 1989, 17:7059-7071). Such primer synthesis can also be performed using, for example, a commercially available oligonucleotide synthesizer (such as ABI). By using a primer set containing the mutation primer and using the parent polynucleotide as a template DNA to perform site-specific mutagenesis as described above, a polynucleotide encoding a mutant protease having a desired mutation introduced therein can be obtained.

[0046] Therefore, the present invention also provides a mutant protease gene. The mutant protease gene of the present invention is a polynucleotide encoding the mutant protease of the present invention. The polynucleotide of the present invention may include single-stranded or double-stranded DNA, cDNA, RNA, or other artificial nucleic acid. The DNA, cDNA, and RNA may be chemically synthesized. The polynucleotide of the present invention may also include a nucleotide sequence of an untranslated region (UTR) in addition to an open reading frame (ORF).

[0047] The present invention also provides a vector containing a polynucleotide encoding the mutant protease of the present invention. The vector can be prepared by inserting and ligating the polynucleotide of the present invention into any vector by a conventional method. The type of the vector is not particularly limited, and may be any vector such as a plasmid, a phage, a phagemid, a cosmid, a virus, a YAC vector, or a shuttle vector. The vector is preferably, but not limited to, a vector that can be amplified in bacteria, particularly in Bacillus bacteria, and more preferably an expression vector that can induce the expression of an introduced gene in Bacillus bacteria. Among them, a shuttle vector that is a vector that can be replicated in both Bacillus bacteria and other organisms can be suitably used for recombinantly producing the mutant protease of the present invention. Examples of preferred vectors include, but are not limited to, shuttle vectors such as pHA3040SP64 (JP Patent Publication No. 2013-233141), pHSP64R or pASP64 (JP Patent No. 3492935), pHA64, pHY300PLK (an expression vector capable of transforming both E. coli and Bacillus subtilis; Ishikawa and Shibahara, Jpn J Genet, 1985, 60: 235-243), and pAC3 (Moriyama et al., Nucleic Acids Res, 1988, 16: 8732); pUB110 (Gryczan et al., J Bacteriol, 1978, 134: 318-329), pTA10607 (Bron et al., J Bacteriol, 1978, 134: 318-329), and pTA10607 (Bron et al., J Bacteriol, 1978, 134: 318-329). Examples of such vectors include plasmids that can be used to transform bacteria of the genus Bacillus, such as plasmids that can be used to transform bacteria of the genus Bacillus (Yamane et al., "Fusion Proteins Obtained by Bacillus Subtilis Secretion Vectors," Starch Science, 34 (1987), 163-170), and the like. Plasmids derived from Escherichia coli (e.g., pET22b(+), pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.) can also be used.

[0048] When the mutant protease of the present invention is produced by recombinant production, the vector is preferably an expression vector, which may contain useful sequences, as necessary, such as various elements essential for expression in a host organism, such as a transcription promoter, a terminator, a ribosome binding site, cis elements, such as a polylinker, an enhancer, a polyA addition signal, a ribosome binding sequence (SD sequence), and a selection marker gene, such as a drug (e.g., ampicillin, neomycin, kanamycin, tetracycline, chloramphenicol, etc.) resistance gene.

[0049] The present invention also provides a transformant comprising a polynucleotide encoding the mutant protease of the present invention or a vector containing the polynucleotide. The transformant can be produced by introducing a polynucleotide encoding the mutant protease of the present invention or a vector containing the polynucleotide (preferably a recombinant expression vector) into a host. Thus, the transformant comprises an exogenous polynucleotide encoding the mutant protease of the present invention.

[0050] Hosts for the transformant include bacteria such as Escherichia coli and Bacillus subtilis, microorganisms including yeast cells, as well as any cells such as insect cells, animal cells (e.g., mammalian cells), and plant cells. The host is preferably a Bacillus bacterium, more preferably Bacillus subtilis or a mutant thereof. Thus, the transformant of the present invention is preferably a recombinant Bacillus bacterium, more preferably a recombinant of Bacillus subtilis or a mutant thereof.

[0051] Well-known transformation techniques such as the calcium phosphate method, electroporation, lipofection, particle gun method, PEG method, etc. can be used to transform the host. For example, transformation methods applicable to Bacillus bacteria include competent cell transformation (J Bacteriol, 1967, 93:1925-1937), electroporation (FEMS Microbiol Lett, 1990, 55:135-138), protoplast transformation (Mol Gen Genet, 1979, 168:111-115), Tris-PEG method (J Bacteriol, 1983, 156:1130-1134), etc.

[0052] The mutant protease of the present invention can be produced by culturing the transformant of the present invention. Thus, the present invention also provides a method for producing a mutant protease using the transformant of the present invention. The culture of the transformant for producing the mutant protease can be performed according to a method generally used by those skilled in the art. For example, a medium for culturing a transformant based on a microbial host such as Escherichia coli or yeast cells may be any medium that contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the microbial host and that can efficiently culture the transformant. The medium may be either a natural medium or a synthetic medium. For example, LB medium, 2×YT medium, 2×L-maltose medium, CSL fermentation medium, etc. can be used for culturing a Bacillus subtilis transformant for producing a recombinant protein. A drug corresponding to the type of drug resistance gene (selection marker gene) introduced into the transformant may be added to the medium. In addition, when culturing a microorganism transformed with an expression vector using an inducible promoter, an inducer may be added to the medium as necessary. For example, when culturing a microorganism transformed with an expression vector using the Lac promoter, isopropyl-1-thio-β-D-galactoside (IPTG) or the like can be added to the medium, and when culturing a microorganism transformed with an expression vector using the trp promoter, indoleacetic acid (IAA) or the like can be added to the medium.

[0053] Alternatively, the mutant protease of the present invention may be expressed from a polynucleotide encoding the mutant protease of the present invention or a transcription product thereof using a cell-free translation system. The "cell-free translation system" is an in vitro transcription / translation system or an in vitro translation system that is constructed by adding reagents such as amino acids necessary for protein translation to a suspension obtained by mechanically disrupting host cells.

[0054] The mutant protease of the present invention produced by the transformant or cell-free translation system can be separated or purified from the culture solution, cell lysate, reaction solution of the cell-free translation system, etc., by using a general method used for protein purification, such as centrifugation, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc., alone or in appropriate combination. Alternatively, a solution such as a culture supernatant or a lysate supernatant separated or concentrated using centrifugation or an ultrafiltration filter can be used as it is as a crude enzyme solution. When the expressed mutant protease is not secreted from within the cells, the cells can be disrupted before separation and purification of the protein.

[0055] The procedures used in the present invention, such as DNA extraction, preparation of mRNA, preparation of cDNA, PCR, RT-PCR, preparation of a library, ligation into a vector, cell transformation, determination of DNA base sequence, chemical synthesis of nucleic acid, determination of amino acid sequence at the N-terminus of a protein, mutagenesis, and protein extraction, can be performed by the methods described in ordinary laboratory manuals. Examples of such laboratory manuals include Molecular Cloning, A Laboratory Manual, 2001, 3rd Ed., Sambrook, J. & Russell, DW. Cold Spring Harbor Laboratory Press, by Sambrook et al. Furthermore, for genetic recombination experiments of Bacillus subtilis, general laboratory manuals on genetic manipulation of Bacillus subtilis, such as Yoshikawa Hirofumi, "7.2 Bacillus subtilis System," "Continued Biochemical Experiment Lectures 1. Genetic Research Methods II," 1986, Tokyo Kagaku Dojinsha (Tokyo), pp. 150-169, can be referred to.

[0056] The mutant proteases obtained by the production method of the present invention have improved cleaning performance in a high-concentration detergent solution compared to the parent protease, and are useful as proteases to be used in detergents for cleaning with a high-concentration detergent solution (for example, spray cleaning or soak cleaning). Therefore, another aspect of the present invention can be a method for improving the cleaning performance of a protease against proteinaceous stains in a high-concentration detergent solution, which comprises introducing at least one mutation selected from the group consisting of (A) to (G) into the amino acid sequence of the parent protease as described above.

[0057] The mutant protease of the present invention is useful as a detergent enzyme, and is particularly suitable as an enzyme to be incorporated into detergents for cleaning with a high-concentration detergent solution (e.g., spread-on cleaning or soak-on cleaning). Thus, the present invention also provides a detergent composition containing the mutant protease of the present invention. The detergent composition may be a solid (e.g., powder) detergent composition, but is preferably a liquid detergent composition, and more preferably a concentrated liquid detergent. The detergent composition is also preferably a detergent composition for spread-on cleaning or soak-on cleaning, and more preferably a detergent composition for spread-on cleaning.

[0058] The content of the mutant protease of the present invention in the detergent composition of the present invention is not particularly limited as long as the amount is an amount that allows the protease to exhibit activity, but is preferably 0.1 to 25,000 U, more preferably 0.1 to 5,000 U, and even more preferably 0.1 to 2,500 U per 1 kg of the detergent composition. The activity (U) of the protease in this specification is measured by the following method: 1 / 15 M phosphate buffer (e.g., pH 7.4, Wako Pure Chemical Industries, Ltd. Phosphate Buffer Powder (167-14491) 1 packet (Na per packet) 2 HPO 4 (Anhydrous) 7.6g. KH 2 PO 40.9 mL of a solution containing 1.8 g of (anhydrous) dissolved in 1 L of ion-exchanged water and 0.05 mL of a 40 mM GlT-Ala-Ala-Pro-Leu-p-nitroanilide / dimethyl sulfoxide solution are placed in a test tube and incubated at 30°C for 5 minutes. 0.05 mL of the enzyme solution is added and reacted at 30°C for 10 minutes, after which 2.0 mL of 5% (w / v) citric acid solution is added to stop the reaction, and the absorbance at 420 nm is measured using a spectrophotometer. Here, 1 unit (U) of enzyme is the amount that produces 1 μmol of p-nitroaniline in 1 minute in the above reaction.

[0059] The detergent composition of the present invention contains a surfactant and water in addition to the mutant protease of the present invention. As the surfactant, any surfactant such as a nonionic surfactant, anionic surfactant, cationic surfactant, and amphoteric surfactant can be used alone or in combination of two or more. The content of the surfactant in the detergent composition of the present invention is preferably 10 to 90% by mass, more preferably 10 to 80% by mass, and even more preferably 30 to 75% by mass. When the detergent composition of the present invention is a concentrated liquid detergent, the content of the surfactant in the detergent composition is preferably 40 to 90% by mass, more preferably 45 to 90% by mass, and even more preferably 50 to 75% by mass.

[0060] The nonionic surfactant may be any nonionic surfactant that has a C8 to C22 hydrocarbon group and has several moles or more of C2 oxyalkylene groups added thereto, and is generally used in liquid detergents. Examples of the nonionic surfactant include the following: R 1 O-(AO)mH(R 1 = C8-C22 hydrocarbon, AO = C2-C5 oxyalkylene group, m = 16 to 35) (JP 2010-275468 A); R 1 O-(EO)l-(AO)m-(EO)nH(R 1 = C8-C18 hydrocarbon, EO = C2 oxyalkylene group, AO = C3-C5 oxyalkylene group, l = 3 to 30, m = 1 to 5, l + n = 14 to 50) (JP 2010-265445 A, JP 2011-63784 A); R 1 O-(EO)m / (AO)nH(R 1 = C8-C22 hydrocarbon, EO = C2 oxyalkylene group, AO = C3-C5 oxyalkylene group, m = 10 to 30, n = 0 to 5, EO and AO are random or block bonds) (JP 2010-189551 A); R 1 (CO)lO-(EO)m / (AO)nR 2 (R 1 = C8-C22 hydrocarbon, EO = C2 oxyalkylene group, AO = C3-C5 oxyalkylene group, l = 0-1, m = 14-50, n = 1-5, R 2 = hydrogen (l = 0) or a C1-C3 alkyl group, EO and AO are random or block bonds) (JP 2010-229385 A); R 1 O-(EO)m-(AO)nH(R 1 = C8-C22 hydrocarbon, EO = C2 oxyalkylene group, AO = C3-C5 oxyalkylene group, m = 15 to 30, n = 1 to 5) (JP 2010-229387 A); R 1 O-(AO)m / (Gly)nH and / or R 2 -COO-(AO)p / (Gly)qH(R 1 = C8-C22 hydrocarbon group, R 2 = C7-C21 hydrocarbon group, AO = C2-C3 oxyalkylene group, Gly = glycerol group, m = 0 to 5, n = 2 to 10, p = 0 to 5, q = 2 to 10, AO and Gly are random or block bond) (JP 2010-254881 A); R 1 -COO-(PO)m / (EO)nR 2 (R 1 = C7-C21 hydrocarbon group, COO = carbonyloxy group, R 2 = C1-C3 alkyl group, PO = oxypropylene group, EO = oxyethylene group, m = 0.3 to 5, n = 8 to 25, PO and EO are random or block bond) [JP 2010-265333 A]; R 1 O-(EO)l-(PO)m-(EO)nH(R 1=C8-C20 hydrocarbon, EO = C2 oxyalkylene group, PO = oxypropylene group, l >= 1, n >= 1, 0 < m < l + n, EO and PO are block-bonded) [WO98 / 24865]; R 1 O-(EO)m-(PO)n-H (R 1 =C10-C16 alkyl or alkenyl group, EO = ethylene oxide group, PO = propylene oxide group, m = 5 - 15, n = 1 - 3) [Japanese Patent Laid-Open No. 8-157867]; R 1 (CO)-(EO)m-OR 2 (R 1 =C11-C13 linear or branched alkyl or alkenyl group, R 2 =C1-C3 alkyl group, EO = ethylene oxide group, m = 10 - 20) [Japanese Patent Laid-Open Nos. 2008-7706, 2009-7451, 2009-155594, 2009-155606]; R 1 (CO)-(AO)m-OR 2 (R 1 =C9-C13 linear or branched alkyl or alkenyl group, AO = C2-C4 oxyalkylene group, R 2 =C1-C3 alkyl group, m = 5 - 30) [Japanese Patent Laid-Open Nos. 2009-144002, 2009-173858, 2010-189612]; and, Fatty acid alkanolamide, fatty acid alkanol glucamide, alkyl polyglucoside, etc.

[0061] Examples of the anionic surfactant include carboxylate-type anionic surfactants, sulfonic acid-type or sulfate-type anionic surfactants, non-soap-based anionic surfactants, linear alkylbenzenesulfonic acid, benzenesulfonic acid or a salt thereof, polyoxybenzenesulfonic acid or a salt thereof, polyoxyethylene alkyl sulfate ester salts, polyoxyalkylene alkyl ether sulfate ester salts, α-olefin sulfonates, alkylbenzenesulfonates, α-sulfofatty acid salts, fatty acid soaps, phosphate ester salt-based surfactants, acylalaninates, acyltaurates, alkyl ether carboxylic acids, and alcohol sulfate esters.

[0062] Examples of cationic surfactants include quaternary ammonium salts having a long-chain alkyl group, tertiary amines having one long-chain alkyl group, alkyltrimethylammonium salts, dialkyldimethylammonium salts, alkylpyridinium salts, etc. Preferred are quaternary ammonium surfactants having one long-chain alkyl group with 8 to 22 carbon atoms, and tertiary amines having one long-chain alkyl group with 8 to 22 carbon atoms.

[0063] Examples of the amphoteric ionic surfactant include alkyl acetate betaine, alkanolamidopropyl acetate betaine, alkylimidazoline, alkylalanine, etc., alkyl betaine type, alkylamide betaine type, imidazoline type, alkylamino sulfone type, alkylamino carboxylic acid type, alkylamide carboxylic acid type, amide amino acid type, or phosphoric acid type amphoteric surfactant, etc. Preferred examples include sulfobetaine or carbobetaine having an alkyl group having 10 to 18 carbon atoms.

[0064] The detergent composition of the present invention may further contain components commonly used in detergent compositions, such as a water-soluble polymer, a water-miscible organic solvent, an alkaline agent, an organic acid or a salt thereof, a chelating agent, an enzyme other than the mutant protease of the present invention, an enzyme stabilizer, a fluorescent agent, a redeposition inhibitor, a dispersant, a color transfer inhibitor, a finishing agent, a bleaching agent, an antioxidant, a solubilizing agent, a pH adjuster, a buffer, a preservative, a fragrance, a salt, an alcohol, a sugar, etc.

[0065] Examples of the water-soluble polymer include a polymer compound having a graft structure in which (i) a polyether chain portion comprising a polymerization unit derived from an epoxide having 2 to 5 carbon atoms and (ii) a polymer chain portion comprising a polymerization unit derived from one or more unsaturated carboxylic acid monomers selected from acrylic acid, methacrylic acid, and maleic acid, and in which either (i) or (ii) serves as a backbone and the other serves as a branch chain (JP Patent Publication No. 2010-275468 and JP Patent Publication No. 10-060496); a water-soluble polymer having an alkylene terephthalate unit and / or an alkylene isophthalate unit, and an oxyalkylene unit and / or a polyoxyalkylene unit (JP Patent Publication No. 2009-155606). The content of the water-soluble polymer in the detergent composition of the present invention is preferably 0.2 to 10% by mass, more preferably 0.4 to 5% by mass.

[0066] Examples of the water-miscible organic solvent include alkylene glycols and glycerin, polyalkylene glycols, (poly)alkylene glycol (mono- or di)alkyl ethers, alkyl glyceryl ethers, and aromatic ethers of (poly)alkylene glycol. Preferred are alkylene glycols having 2 to 6 carbon atoms, such as ethylene glycol, propylene glycol, butylene glycol, and hexylene glycol, and glycerin, or polyethylene glycol monophenyl ether, ethylene glycol monobenzyl ether, diethylene glycol monobenzyl ether, and the like. The content of the water-miscible organic solvent in the detergent composition of the present invention is preferably 1 to 40% by mass, more preferably 1 to 35% by mass.

[0067] Examples of the alkaline agent include alkanolamines having 1 to 3 C2-C4 alkanols, such as monoethanolamine, diethanolamine, triethanolamine, polyoxyalkyleneamine, and dimethylaminopropylamine. Monoethanolamine and triethanolamine are preferred. The content of the alkaline agent in the detergent composition of the present invention is preferably 0 to 20% by mass, more preferably 0 to 10% by mass.

[0068] Examples of organic acids or salts thereof include polycarboxylic acids such as saturated fatty acids, succinic acid, maleic acid, fumaric acid, and salts thereof; hydroxycarboxylic acids such as citric acid, malic acid, glycolic acid, p-hydroxybenzoic acid, benzoic acid, and salts thereof; and among these, citric acid or a salt thereof is preferred. The content of the organic acid or a salt thereof in the detergent composition of the present invention is preferably 0 to 5% by mass, more preferably 0 to 3% by mass.

[0069] A chelating agent is a compound capable of forming a coordinate bond with a metal ion. The chelating agent added to a detergent composition has the effect of sequestering metal ions, such as calcium ions and magnesium ions, that adversely affect cleaning in the washing water or dirt. Examples of chelating agents that can be included in the detergent composition of the present invention include aminopolyacetic acids such as nitrilotriacetic acid, iminodiacetic acid, ethylenediamineacetic acid, diethylenetriaminepentaacetic acid, glycoletherdiaminetetraacetic acid, hydroxyethyliminodiacetic acid, triethylenetetraaminehexaacetic acid, and dienecolic acid, or salts thereof, organic acids such as diglycolic acid, oxydisuccinic acid, carboxymethyloxysuccinic acid, citric acid, lactic acid, tartaric acid, oxalic acid, malic acid, oxydisuccinic acid, gluconic acid, carboxymethylsuccinic acid, and carboxymethyltartaric acid, or salts thereof, aminotri(methylenephosphonic acid), 1-hydroxyethylidene-1,1-diphosphonic acid, ethylenediaminetetra(methylenephosphonic acid), diethylenetriaminepenta(methylenephosphonic acid), and alkali metal or lower amine salts thereof. The content of the chelating agent in the detergent composition of the present invention is preferably 0.1 to 5% by mass, more preferably 0.1 to 4% by mass.

[0070] Examples of the soil redeposition inhibitor and dispersant include polyacrylic acid, polymaleic acid, carboxymethyl cellulose, polyethylene glycol having a weight average molecular weight of 5000 or more, maleic anhydride-diisobutylene copolymer, maleic anhydride-methyl vinyl ether copolymer, maleic anhydride-vinyl acetate copolymer, naphthalenesulfonate-formaldehyde condensate, and polymers described in claims 1 to 21 of JP-A-59-62614 (page 1, column 3, line 5 to page 3, column 4, line 14). The contents of the soil redeposition inhibitor and dispersant in the detergent composition of the present invention are preferably 0.01 to 10% by mass, respectively.

[0071] An example of the color transfer inhibitor is polyvinylpyrrolidone, and the content is preferably 0.01 to 10% by mass.

[0072] The bleaching agent is preferably contained in the detergent composition in an amount of 1 to 10% by mass, such as hydrogen peroxide, percarbonate, perborate, etc. When a bleaching agent is used, tetraacetylethylenediamine (TAED) or a bleaching activator such as that described in JP-A-6-316700 can be contained in the detergent composition in an amount of 0.01 to 10% by mass.

[0073] Examples of fluorescent agents include biphenyl-type fluorescent agents (such as Tinopal CBS-X) and stilbene-type fluorescent agents (such as DM-type fluorescent dyes). The content of the fluorescent agent in the detergent composition of the present invention is preferably 0.001 to 2% by mass.

[0074] Examples of enzymes other than the mutant proteases of the present invention include hydrolases such as other proteases, cellulases, β-glucanases, hemicellulases, lipases, peroxidases, laccases, α-amylases, glucoamylases, cutinases, pectinases, reductases, oxidases, phenoloxidases, ligninases, pullulanases, pectate lyases, xyloglucanases, xylanases, pectin acetylesterases, polygalacturonases, rhamnogalacturonases, pectin lyases, mannanases, pectin methylesterases, cellobiohydrolases, and transglutaminase, as well as mixtures of two or more of these.

[0075] Examples of enzyme stabilizers include boron compounds, calcium ion sources (calcium ion supplying compounds), hydroxy compounds, formic acid, etc. Examples of antioxidants include butylhydroxytoluene, distyrenated cresol, sodium sulfite, and sodium hydrogen sulfite, etc. Examples of solubilizers include paratoluenesulfonic acid, cumenesulfonic acid, metaxylenesulfonic acid, benzoates (which also have the effect as a preservative), etc. Furthermore, the detergent composition of the present invention may contain water-immiscible organic solvents such as paraffins such as octane, decane, dodecane, and tridecane, olefins such as decene and dodecene, alkyl halides such as methylene chloride and 1,1,1-trichloroethane, and terpenes such as D-limonene, dyes, fragrances, antibacterial preservatives, and antifoaming agents such as silicone.

[0076] Preferred examples of detergent compositions into which the mutant protease of the present invention can be blended include the liquid detergent composition described in JP 2017-008303 A and the liquid detergent composition described in JP 2013-129729 A. The detergent composition of the present invention can be prepared by blending the mutant protease of the present invention into these detergent compositions.

[0077] The detergent composition of the present invention is not limited, but preferred examples include detergent compositions for washing clothes or fabric products (e.g., sheets, curtains, carpets, wall cloths, etc.) and detergent compositions for kitchen use (e.g., for tableware or cooking utensils). Preferably, the detergent composition of the present invention is a detergent composition for washing with a high concentration detergent solution, more preferably a detergent composition for application washing or soaking washing, and even more preferably a detergent composition for application washing.

[0078] The present invention also includes the following substances, manufacturing methods, uses, methods, etc. as exemplary embodiments, but the present invention is not limited to these embodiments.

[0079] [1] A mutant protease having the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 90% identity thereto, and having at least one amino acid residue selected from the group consisting of the following (a) to (g): (a) Arg or Lys at a position corresponding to position 191 of SEQ ID NO:1; (b) Arg or Lys at a position corresponding to position 17 of SEQ ID NO:1; (c) Ala or Phe at a position corresponding to position 141 of SEQ ID NO:1; (d) Arg or Lys at a position corresponding to position 243 of SEQ ID NO:1; (e) Arg or Lys at a position corresponding to position 300 of SEQ ID NO:1; (f) Arg or Lys at a position corresponding to position 302 of SEQ ID NO:1; (g) Arg or Lys at a position corresponding to position 311 of SEQ ID NO:1. [2] The mutant protease according to [1], preferably having the amino acid residue (a) and at least one amino acid residue selected from the group consisting of (b) to (g). [3] The mutant protease according to [1], preferably having the amino acid residue (e) and the amino acid residue (f). [4] The mutant protease according to [1], which preferably has at least one amino acid residue selected from the group consisting of (a) to (g) above and an amino acid residue of the following (h): (h) Gln at a position corresponding to position 82 of SEQ ID NO:1. [5] The mutant protease according to [4], preferably having the amino acid residue of (a) and the amino acid residue of (h). [6] A polynucleotide encoding the mutant protease according to any one of [1] to [5]. [7] A vector containing the polynucleotide described in [6]. [8] A recombinant Bacillus bacterium comprising the polynucleotide according to [6] or the vector according to [7]. [9] A method for producing a mutant protease using the recombinant Bacillus bacterium described in [8].

[10] The method described in [8], which comprises culturing the recombinant Bacillus bacterium.

[11] A detergent composition comprising the mutant protease according to any one of [1] to [5].

[12] The detergent composition according to

[11] , which is preferably a detergent composition for cleaning with a high-concentration detergent solution, more preferably a detergent composition for application cleaning or soak cleaning, and even more preferably a detergent composition for application cleaning.

[13] A method for producing a mutant protease, comprising introducing at least one mutation selected from the group consisting of the following (A) to (G) into a parent protease having the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 90% identity thereto: (A) substitution of the amino acid residue at position 191 of SEQ ID NO:1 or a position equivalent thereto with Arg or Lys; (B) substitution of the amino acid residue at position 17 of SEQ ID NO:1 or a position equivalent thereto with Arg or Lys; (C) substitution of the amino acid residue at position 141 of SEQ ID NO:1 or a position equivalent thereto with Ala or Phe; (D) substitution of the amino acid residue at position 243 of SEQ ID NO:1 or a position equivalent thereto with Arg or Lys; (E) substitution of the amino acid residue at position 300 of SEQ ID NO:1 or a position equivalent thereto with Arg or Lys; (F) substitution of the amino acid residue at position 302 of SEQ ID NO:1 or a position equivalent thereto with Arg or Lys; (G) Substitution of the amino acid residue at position 311 of SEQ ID NO:1 or a position equivalent thereto with Arg or Lys.

[14] A method for improving the cleaning performance of a protease against protein stains in a high-concentration detergent solution, comprising introducing at least one mutation selected from the group consisting of the following (A) to (G) into a parent protease having the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 90% identity thereto: (A) substitution of the amino acid residue at position 191 of SEQ ID NO:1 or a position equivalent thereto with Arg or Lys; (B) substitution of the amino acid residue at position 17 of SEQ ID NO:1 or a position equivalent thereto with Arg or Lys; (C) substitution of the amino acid residue at position 141 of SEQ ID NO:1 or a position equivalent thereto with Ala or Phe; (D) substitution of the amino acid residue at position 243 of SEQ ID NO:1 or a position equivalent thereto with Arg or Lys; (E) substitution of the amino acid residue at position 300 of SEQ ID NO:1 or a position equivalent thereto with Arg or Lys; (F) substitution of the amino acid residue at position 302 of SEQ ID NO:1 or a position equivalent thereto with Arg or Lys; (G) Substitution of the amino acid residue at position 311 of SEQ ID NO:1 or a position equivalent thereto with Arg or Lys.

[15] The method according to

[13] or

[14] , preferably comprising subjecting the parent protease to the mutation (A) and at least one mutation selected from the group consisting of the mutations (B) to (G).

[16] The method according to

[13] or

[14] , preferably comprising making the mutation (E) and the mutation (F) in the parent protease.

[17] The method according to

[13] or

[14] , preferably comprising subjecting the parent protease to at least one mutation selected from the group consisting of (A) to (G) and the following mutation (H): (H) Substitution of the amino acid residue at position 82 of SEQ ID NO:1 or a position equivalent thereto with Gln. EXAMPLES

[0080] The present invention will be described in more detail below with reference to examples, although the technical scope of the present invention is not limited to these examples.

[0081] Example 1: Construction of mutant proteases The wild-type KP43 protease expression plasmid pHA64TSA described in Reference Example 1 of Patent Document 5 contains a tetracycline resistance gene and the full-length coding sequence of wild-type KP43 protease linked to a promoter region derived from the alkaline cellulase K-64 gene of Bacillus sp. KSM-64 strain. Using pHA64TSA as a template, a site-specific introduction method by PCR using a complementary primer pair was used to repeatedly introduce site-specific mutations into the KP43 protease gene (see Nucleic Acids Research, 2004, 32(14): e115), thereby preparing a plasmid expressing the protease of SEQ ID NO: 1. Using the obtained plasmid expressing the protease of SEQ ID NO:1 as a template, plasmids expressing mutant proteases having the following amino acid substitutions relative to the parent enzyme, the protease of SEQ ID NO:1, were prepared in a similar manner: T311R, N302R, Y300K, N243K, S191R, Y17R, D141A, D141F, Y17R / S191R, S191R / N243K, Y300K / N302R, E82Q / S191R, or S191F. Hereinafter, the mutant proteases expressed from these plasmids are referred to as T311R, N302R, Y300K, N243K, S191R, Y17R, D141A, D141F, Y17R / S191R, S191R / N243K, Y300K / N302R, E82Q / S191R, and S191F, respectively.

[0082] The constructed plasmids expressing the parent enzyme or mutant proteases were used to transform the host bacterium Bacillus sp. KSM9865 (FERM P-18566). Transformation was performed by electroporation, and the transformed cells were smeared on alkaline agar medium containing skim milk [1% skim milk (Difco), 1% bactotryptone (Difco), 0.5% yeast extract (Difco), 1% sodium chloride, 1.5% agar, 0.05% sodium carbonate, 15 ppm tetracycline; % is (w / v)] and cultured at 30°C for several days. Colonies that grew on the agar medium and showed skim milk soluble spots were selected as transformants into which the protease gene had been introduced. The obtained transformant was inoculated into 5 mL of seed medium [6.0% polypeptone S, 0.05% yeast extract, 1.0% maltose, 0.02% magnesium sulfate heptahydrate, 0.1% potassium dihydrogen phosphate, 0.25% sodium carbonate, 30 ppm tetracycline; % is (w / v) %] and cultured with shaking at 30 ° C for 16 hours. Next, the seed culture liquid was inoculated at 1% (v / v) into 20 mL of main medium [8% polypeptone S, 0.3% yeast extract, 10% maltose, 0.04% magnesium sulfate heptahydrate, 0.2% potassium dihydrogen phosphate, 1.5% anhydrous sodium carbonate, 30 ppm tetracycline; % is (w / v) %] and cultured with shaking at 30 ° C for 3 days. The obtained culture liquid was centrifuged to obtain a culture supernatant containing protease. The protein concentration in the culture supernatant was measured using Protein Assay Rapid Kid Wako II (Fujifilm Wako Pure Chemical Industries, Ltd.).

[0083] Example 2 Evaluation of the cleaning power of mutant proteases A commercially available concentrated liquid detergent (Attack ZERO Regular; Kao Corporation) was heated in a microwave oven to inactivate the enzymes in the product. The culture supernatant containing each mutant protease obtained in Example 1 was added to this liquid detergent so that the protein concentration was 0.1 g / L, and the mixture was thoroughly stirred to prepare a liquid detergent composition containing the mutant protease (test composition). As a control, a liquid detergent composition containing the parent enzyme (control composition) was prepared in the same manner. The obtained liquid detergent composition was used to apply and wash artificially soiled cloth DINGY_TN / CP / (CONSUMERTEC), which is a model for collar and sleeve stains. That is, 20 μL of the test composition or control composition was dropped on DINGY_TN / CP / cut into 1.5 cm squares, and left to stand for 5 minutes, after which the soiled cloth was transferred to 600 mL of tap water kept at 20 ° C. in the washing tank of a Tergotometer (Ueshima Seisakusho) and washed for 10 minutes at 60 rpm. After the application and cleaning, the soiled cloth was removed from the cleaning tank, rinsed with tap water, and dried. The lightness (L value) of the soiled cloth before and after the application and cleaning, and the original soiled cloth prepared separately, were measured using a spectrophotometer cm-700d (Konica Minolta), and the cleaning rate of the soiled cloth was calculated using the following formula. Cleaning rate (%) = (L 2 -L 1 ) / (L 0 -L 1 ) × 100 (L 0 : L value of the original soiled fabric, L 1 : L value of the soiled cloth before cleaning, L 2 : L value of soiled cloth after washing). The relative cleaning rate of each test composition to the control composition (cleaning rate with test composition / cleaning rate with control composition) was calculated, and the relative cleaning power of each mutant protease to the parent protease in application cleaning (relative application cleaning power) was evaluated.

[0084] The relative application detergency of each mutant protease (relative cleaning rate of the test composition containing each mutant protease) is shown in Table 2 and Figure 1. All of the mutant proteases except S191F had a relative application detergency of more than 1, and showed a clearly higher application detergency than the parent enzyme.

[0085] [Table 2]

[0086] The effect of mutation at position 191 on the parent protease of SEQ ID NO:1 is summarized in Table 3 and Figure 2. A protease in which serine (S) at position 191 of SEQ ID NO:1 was replaced with arginine (R) (S191R) had improved application and cleaning power compared to the parent protease. On the other hand, substitution of phenylalanine (F) at position 191 (S191F) did not affect application and cleaning power. A mutant with glutamine at position 82 and arginine at position 191 (E82Q / S191R) showed even higher application and cleaning power.

[0087] [Table 3]

[0088] Example 3: Evaluation of stability of mutant proteases A commercially available concentrated liquid detergent (Attack ZERO Regular; Kao Corporation) was heated in a microwave oven to inactivate the enzymes in the product. The culture supernatant containing the mutant protease E82Q / S191R obtained in Example 1 was added to this liquid detergent so that the protein concentration was 0.2 g / L, and the mixture was thoroughly stirred to prepare a liquid detergent composition containing the mutant protease (test sample). As a control, a liquid detergent composition containing the parent enzyme (control sample) was prepared in the same manner. The protease activity of the test sample immediately after the enzyme was added (0h) and after storage at 40°C for 2 weeks (2w) was measured by the following method. One tablet of Protazyme AK Tablets (Megazyme) was suspended in 1 mL of 0.1 M phosphate buffer to prepare a substrate solution. The test samples of 0h and 2w were each diluted 60 times with 0.1 M phosphate buffer. 1 mL of the diluted sample was added to the substrate solution and incubated at 40°C for 30 minutes. The reaction was stopped by adding 10 mL of 2% trisodium phosphate aqueous solution, and after stirring, the reaction solution was divided into 1 mL portions in 1.5 mL tubes. The mixture was centrifuged at 12,000 rpm for 5 minutes, and the supernatant was collected and the absorbance at 590 nm was measured. The absorbance measured in the same manner using a heat-inactivated liquid detergent without enzyme instead of the test sample was used as the blank, and the difference between the absorbance of the test sample and the blank was used as the activity value. The activity values ​​of the 0h and 2w control samples were calculated using the same procedure. The activity value of the 2w sample was divided by the activity value of the 0h sample to determine the remaining activity rate after two weeks, and the value was raised to the power of 1 / 14 to determine the daily activity maintenance rate due to 40°C treatment. The logarithm of the daily activity maintenance rate to the base 0.5 was calculated to determine the half-life (days) of the protease activity. The half-life of the mutant protease was divided by the half-life of the parent protease to determine the relative stability. The relative stability of the E82Q / S191R mutant was 1.89, and the introduction of the E82Q and S191R mutations significantly improved the stability of the protease. Patent Document 3 describes that proteases having 82E or 82Q have high stability in liquid detergents. On the other hand, the E82Q / S191R mutant of this example has improved stability compared to the parent protease having 82E, suggesting that the S191R mutation further improves the stability of the protease.

[0089] Although embodiments of the present invention have been described above, it should be understood that they are not intended to limit the present invention to the specific embodiments described. Various other changes and modifications within the scope of the present invention will be apparent to those skilled in the art. All publications and patent applications cited herein are incorporated by reference as if fully set forth herein.

Claims

1. A mutant protease comprising an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 90% identity thereto, and having at least one amino acid residue selected from the group consisting of the following (a) to (g): (a) Arg or Lys at a position corresponding to position 191 of SEQ ID NO: 1; (b) Arg or Lys at a position corresponding to position 17 of SEQ ID NO: 1; (c) Ala or Phe at a position corresponding to position 141 of SEQ ID NO: 1; (d) Arg or Lys at a position corresponding to position 243 of SEQ ID NO: 1; (e) Arg or Lys at a position corresponding to position 300 of SEQ ID NO: 1; (f) Arg or Lys at a position corresponding to position 302 of SEQ ID NO: 1; (g) Arg or Lys at a position corresponding to position 311 of SEQ ID NO:

1.

2. The mutant protease according to claim 1, having the amino acid residue of (a) and at least one amino acid residue selected from the group consisting of (b) to (g).

3. The mutant protease according to claim 1, having the amino acid residue of (e) and the amino acid residue of (f).

4. The mutant protease according to claim 1, having at least one amino acid residue selected from the group consisting of (a) to (g) and the amino acid residue of the following (h): (h) Gln at a position corresponding to position 82 of SEQ ID NO:

1.

5. The mutant protease according to claim 4, having the amino acid residue of (a) and the amino acid residue of (h).

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

7. A vector containing the polynucleotide according to claim 6.

8. A recombinant Bacillus bacterium comprising the polynucleotide according to claim 6 or a vector containing the polynucleotide.

9. A method for producing a mutant protease using the recombinant Bacillus bacterium according to claim 8.

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

11. The detergent composition according to claim 10, which is a detergent composition for coating and cleaning.

12. A method for producing a mutant protease, comprising subjecting a parent protease comprising an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 90% identity thereto to at least one mutation selected from the group consisting of the following (A) to (G): Substitution of the amino acid residue at position 191 of SEQ ID NO: 1 or a corresponding position thereof with Arg or Lys; Substitution of the amino acid residue at position 17 of SEQ ID NO: 1 or a corresponding position thereof with Arg or Lys; Substitution of the amino acid residue at position 141 of SEQ ID NO: 1 or a corresponding position thereof with Ala or Phe; Substitution of the amino acid residue at position 243 of SEQ ID NO: 1 or a corresponding position thereof with Arg or Lys; Substitution of the amino acid residue at position 300 of SEQ ID NO: 1 or a corresponding position thereof with Arg or Lys; Substitution of the amino acid residue at position 302 of SEQ ID NO: 1 or a corresponding position thereof with Arg or Lys; Substitution of the amino acid residue at position 311 of SEQ ID NO: 1 or a corresponding position thereof with Arg or Lys. **Claim 13** A method for improving the cleaning performance against protein stains in a high-concentration detergent solution of a protease, comprising subjecting a parent protease consisting of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 90% identity therewith to at least one mutation selected from the group consisting of the following (A) to (G): Substitution of the amino acid residue at position 191 of SEQ ID NO: 1 or a corresponding position thereof with Arg or Lys; Substitution of the amino acid residue at position 17 of SEQ ID NO: 1 or a corresponding position thereof with Arg or Lys; Substitution of the amino acid residue at position 141 of SEQ ID NO: 1 or a corresponding position thereof with Ala or Phe; Substitution of the amino acid residue at position 243 of SEQ ID NO: 1 or a corresponding position thereof with Arg or Lys; Substitution of the amino acid residue at position 300 of SEQ ID NO: 1 or a corresponding position thereof with Arg or Lys; Substitution of the amino acid residue at position 302 of SEQ ID NO: 1 or a corresponding position thereof with Arg or Lys; Substitution of the amino acid residue at position 311 of SEQ ID NO: 1 or a corresponding position thereof with Arg or Lys. **Claim 14** The method according to claim 12 or 13, comprising subjecting the parent protease to the mutation of (A) and at least one mutation selected from the group consisting of (B) to (G). **Claim 15** The method according to claim 12 or 13, comprising subjecting the parent protease to the mutation of (E) and the mutation of (F). **Claim 16** The method according to claim 12 or 13, comprising subjecting the parent protease to at least one mutation selected from the group consisting of (A) to (G) and the following mutation (H): (H) substitution of the amino acid residue at position 82 of SEQ ID NO: 1 or a corresponding position thereto with Gln substitution.

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