Mutant proteases

Abstract

Provided is a mutant protease having improved cleaning performance against protein stains in a high-concentration detergent solution. A mutant protease comprising an amino acid sequence represented by 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 the 17-position of SEQ ID NO: 1; (c) Ala or Phe at a position corresponding to the 141 bit of SEQ ID NO: 1; (d) Arg or Lys at a position corresponding to the 243 position of SEQ ID NO: 1; (e) Arg or Lys at a position corresponding to the 300-bit 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 311 bit in SEQ ID NO: 1.

Description

Technical Field

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

[0002] The purpose of adding protease to detergents is to decompose protein stains from food, dirt, sweat, etc. attached to clothing, tableware, etc. to promote the removal of stains. The pH of detergents is generally on the alkaline side, so proteases with an optimal pH on the alkaline side have been developed as proteases for detergents. Patent document 1 discloses a protease with a molecular weight of about 43,000 that has a cleaning effect on complex stains mixed with proteins and lipids. Patent documents 2 to 5 disclose mutant proteases obtained by introducing mutations that improve the cleaning performance of the protease, stability in liquid detergents, solubility in liquid detergents, or stability under acidic conditions.

[0003] (Patent Document 1) International Publication No. 99 / 18218

[0004] (Patent Document 2) Japanese Patent Application Publication No. 2008-212084

[0005] (Patent Document 3) Japanese Patent Application Publication No. 2010-273672

[0006] (Patent Document 4) Japanese Patent Application Publication No. 2013-233141

[0007] (Patent Document 5) Japanese Patent Application Publication No. 2020-145938 Summary of the invention

[0008] The present invention provides a mutant protease, which consists of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 90% identity thereto, and has at least one amino acid residue selected from the following (a) to (g):

[0009] (a) Arg or Lys in the position corresponding to position 191 of SEQ ID NO: 1;

[0010] (b) Arg or Lys in the position corresponding to position 17 of SEQ ID NO: 1;

[0011] (c) Ala or Phe in the position corresponding to position 141 of SEQ ID NO: 1;

[0012] (d) Arg or Lys in the position corresponding to position 243 of SEQ ID NO: 1;

[0013] (e) Arg or Lys in the position corresponding to position 300 of SEQ ID NO: 1;

[0014] (f) Arg or Lys in the position corresponding to position 302 of SEQ ID NO: 1;

[0015] (g) Arg or Lys at the position corresponding to position 311 of SEQ ID NO:1.

[0016] In addition, the present invention provides a polynucleotide encoding the above-mentioned mutant protease.

[0017] In addition, the present invention provides a vector comprising the above-mentioned polynucleotide.

[0018] In addition, the present invention provides a recombinant Bacillus bacterium comprising the above polynucleotide or the above vector.

[0019] In addition, the present invention provides a method for producing a mutant protease using the above-mentioned recombinant Bacillus bacteria.

[0020] In addition, the present invention provides a detergent composition comprising the above-mentioned mutant protease.

[0021] In addition, the present invention provides a method for producing a mutant protease, comprising the step of 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 thereto to at least one mutation selected from the following (A) to (G):

[0022] (A) Substitution of the amino acid residue at position 191 of SEQ ID NO:1 or a position equivalent thereto with Arg or Lys;

[0023] (B) Substitution of the amino acid residue at position 17 of SEQ ID NO: 1 or a position equivalent thereto with Arg or Lys;

[0024] (C) substitution of the amino acid residue at position 141 of SEQ ID NO:1 or a position equivalent thereto with Ala or Phe;

[0025] (D) Substitution of the amino acid residue at position 243 of SEQ ID NO:1 or a position equivalent thereto with Arg or Lys;

[0026] (E) Substitution of the amino acid residue at position 300 of SEQ ID NO:1 or a position equivalent thereto with Arg or Lys;

[0027] (F) Substitution of the amino acid residue at position 302 of SEQ ID NO:1 or a position equivalent thereto with Arg or Lys;

[0028] (G) Substitution of the amino acid residue at position 311 of SEQ ID NO: 1 or a position equivalent thereto with Arg or Lys.

[0029] In addition, the present invention provides a method for improving the cleaning performance of protease on protein stains in a high concentration detergent solution, which comprises the step of subjecting a parent protease composed of the amino acid sequence of sequence number 1 or an amino acid sequence having at least 90% identity thereto to at least one mutation selected from the above (A) to (G). BRIEF DESCRIPTION OF THE DRAWINGS

[0030] [ Figure 1 ] Relative coating cleaning power of mutant proteases.

[0031] [ Figure 2 ] Improvement of coating cleaning power by utilizing mutation at position 191. DETAILED DESCRIPTION

[0032] In the present specification, "amino acid residue" refers to the 20 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).

[0033] In the present specification, "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 96% or more, further preferably 97% or more, further preferably 98% or more, further preferably 99% or more.

[0034] 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, the identity is calculated by using the Search homology program of the genetic information processing software Genetyx-Win with a unit size to compare (ktup) of 2.

[0035] In the present specification, as "an amino acid sequence in which one or several amino acids are deleted, inserted, substituted or added", an amino acid sequence in which preferably 1 to 10, more preferably 1 to 5, and further preferably 1 to 3 amino acids are deleted, inserted, substituted or added. In addition, in the present specification, as "a nucleotide sequence in which one or several nucleotides are deleted, inserted, substituted or added", a nucleotide sequence in which preferably 1 to 30, more preferably 1 to 15, and further preferably 1 to 10 nucleotides are deleted, inserted, substituted or added can be listed. In addition, in the present specification, "addition" of amino acids or nucleotides includes adding one or several amino acids or nucleotides to one end and both ends of the sequence.

[0036] In the present specification, the "equivalent position" on an amino acid sequence or a nucleotide sequence is determined by arranging (aligning) the target sequence and the reference sequence (e.g., the amino acid sequence of SEQ ID NO: 1) in a manner that gives the greatest identity. The alignment of amino acid sequences or nucleotide sequences can be performed using a known algorithm, and its steps are well known to those skilled in the art. For example, the alignment can be performed using the Clustal W multiple alignment program (Thompson, JD et al, 1994, Nucleic Acids Res. 22: 4673-4680) with default settings. Alternatively, a revised version of Clustal W, Clustal W2 or Clustal omega, can also be used. Clustal W, Clustal W2 and Clustal omega can be used, for example, on the website of Clustal operated by University College Dublin [www.clustal.org], the European Bioinformatics Institute (European Bioinformatics Institute: EBI [www.ebi.ac.uk / index.html]), and the website of the DNA Data Bank of Japan (DDBJ [www.ddbj.nig.ac.jp / searches-j.html]) operated by the National Institute of Genetics of Japan. The position of the target sequence located at an arbitrary position of the reference sequence by the above-mentioned alignment is regarded as the "equivalent position" to the arbitrary position.

[0037] As long as one is skilled in the art, the comparison of the amino acid sequences obtained above can be optimized by further fine-tuning. Such an optimal comparison is preferably determined by considering the similarity of the amino acid sequences, the frequency of the inserted intervals, etc. Here, the similarity of the amino acid sequence refers to the ratio (%) of the number of positions where the same or similar amino acid residues exist in the sequences of both parties when two amino acid sequences are compared to the number of amino acid residues in the whole length. Similar amino acid residues refer to the 20 kinds of amino acids constituting proteins that have similar properties in terms of polarity and charge, that is, amino acid residues that undergo so-called conservative substitutions. Grouping composed of such similar amino acid residues is well known to those skilled in the art, for example, arginine and lysine or glutamine; glutamic acid and aspartic acid or glutamine; serine and threonine or alanine; glutamine and asparagine or arginine; leucine and isoleucine, etc., but are not limited thereto.

[0038] The position of the amino acid residue of the target sequence located at a position corresponding to an arbitrary position of the reference sequence by the above alignment is regarded as the "corresponding position" to the arbitrary position, and the amino acid residue becomes the "amino acid residue at the corresponding position".

[0039] In this specification, the "parent" polypeptide of a mutant polypeptide refers to a polypeptide that becomes the mutant polypeptide by forming a specified mutation in its amino acid residues. In other words, the "parent" polypeptide refers to the polypeptide before the polypeptide mutant mutates. Similarly, the "parent" polynucleotide of a mutant polynucleotide refers to a polynucleotide that becomes the mutant polynucleotide by forming a specified mutation in its nucleotides. In other words, the "parent" polynucleotide refers to the polynucleotide before the mutant polynucleotide mutates.

[0040] In this specification, "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. The steps of "operably linking" a gene and a control region are well known to those skilled in the art.

[0041] 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 arranged downstream of a promoter" means that the gene is present on the 3' side of the promoter in the positive strand of DNA, and the upstream of a gene refers to the region on the 5' side of the gene in the positive strand of DNA.

[0042] In this specification, the term "originally" used for the function, property, and trait of a cell is used to indicate that the function, property, and trait originally exist in the cell. In contrast, the term "exogenous" is used to indicate that the function, property, and trait are not originally present in the cell but are introduced from the outside. For example, a "exogenous" gene or polynucleotide refers to a gene or polynucleotide introduced into a cell from the outside. A foreign gene or polynucleotide can come from an organism of the same species as the cell into which it is introduced, or it can come from an organism of a different species (i.e., a heterologous gene or polynucleotide).

[0043] The present invention is directed to mutant proteases which provide improved cleaning performance on proteinaceous stains in highly concentrated detergent liquors.

[0044] In order to remove stains that are difficult to remove, such as dyeing, the original liquid of a liquid detergent is sometimes directly applied to the stain without diluting it with water. For stains containing protein, such as food spills and collar and cuff stains, it is believed that the application of a liquid detergent containing protease is effective. However, the original liquid of a liquid detergent has a high concentration of cleaning components such as surfactants, which is a harsh condition for proteases. It is desired to further improve the cleaning performance of proteases in solutions containing high concentrations of cleaning components, such as the original liquid of a liquid detergent.

[0045] The inventors of the present invention have found that a mutant protease derived from protease KP43 (see Patent Document 1) having a molecular weight of 43,000 has improved cleaning performance (hereinafter also referred to as simply cleaning performance) for protein stains even in a stock solution of a liquid detergent.

[0046] The mutant protease of the present invention has improved cleaning performance for protein stains in a high-concentration detergent solution (e.g., a stock solution of a liquid detergent). The mutant protease of the present invention is effective as a protease used in cleaning using a high-concentration detergent solution, such as coating cleaning or immersion cleaning. For example, the mutant protease of the present invention is effective as a protease formulated in a detergent for cleaning a stain by bringing a stock solution of a liquid detergent or a high-concentration detergent solution into direct contact with the stain.

[0047] The mutant protease of the present invention is a mutant protease having at least one mutation selected from the following (A) to (G) relative to a parent protease consisting of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 90% identity thereto:

[0048] (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;

[0049] (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;

[0050] (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;

[0051] (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;

[0052] (E) Substitution of the amino acid residue at position 300 of SEQ ID NO: 1 or a position equivalent thereto with Arg or Lys, preferably Lys;

[0053] (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;

[0054] (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.

[0055] Preferably, the mutant protease of the present invention has the mutation (A) above relative to the parent protease. In a preferred embodiment, the mutant protease of the present invention has the mutation (A) above relative to the parent protease and at least one mutation selected from the above (B) to (G).

[0056] In one embodiment, the mutant protease of the present invention has the mutation of (A) and at least one mutation selected from the mutations of (B) and (D) relative to the parent protease. In another embodiment, the mutant protease of the present invention has the mutation of (A) and any one mutation selected from the mutations of (B) and (D) relative to the parent protease. Preferably, the mutation of (A) is a substitution of an amino acid residue at position 191 or a position equivalent thereto to Arg, the mutation of (B) is a substitution of an amino acid residue at position 17 or a position equivalent thereto to Arg, and the mutation of (D) is a substitution of an amino acid residue at position 243 or a position equivalent thereto to Lys.

[0057] In one embodiment, the mutant protease of the present invention has, in addition to at least one mutation selected from the above (A) to (G), the following mutation (H):

[0058] (H) Substitution of the amino acid residue at position 82 of SEQ ID NO: 1 or a position equivalent thereto with Gln.

[0059] By having the mutation of (H) in addition to any of the mutations of (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 of (A) and the mutation of (H). Preferably, the mutation of (A) is a substitution of an amino acid residue at position 191 or a position equivalent thereto to Arg.

[0060] In one embodiment, the mutant protease of the present invention may have at least one mutation selected from the above (B) to (G). In a preferred embodiment, the mutant protease of the present invention has the above (E) mutation and the above (F) mutation relative to the above parent protease. By having the above (E) and (F) mutations, the cleaning performance of the mutant protease of the present invention in a high concentration detergent solution is further improved. Preferably, the mutation of (E) is a substitution of an amino acid residue at position 300 or a position equivalent thereto to Lys, and the mutation of (F) is a substitution of an amino acid residue at position 302 or a position equivalent thereto to Arg.

[0061] Examples of the parent protease of the mutant protease of the present invention include a protease consisting of the amino acid sequence of SEQ ID NO: 1 and a protease consisting of an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1.

[0062] As a preferred example of a protease consisting of an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1, there can be listed proteases having 95% or more identity, more preferably 96% or more identity, further preferably 97% or more identity, further preferably 98% or more identity, further preferably 99% or more identity with the amino acid sequence of SEQ ID NO: 1. As another example of a protease consisting of an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1, there can be listed proteases consisting of an amino acid sequence in which one or more amino acids are deleted, inserted, substituted or added to the amino acid sequence of SEQ ID NO: 1. The protease consisting of an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1 used as the parent protease in the present invention may be a mutant protease from the protease consisting of the amino acid sequence of SEQ ID NO: 1.

[0063] Preferably, in the amino acid sequence of the parent protease, the amino acid residue at the position corresponding to position 30 of 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 listed in Table 1(ii) at the positions corresponding to the respective positions listed in the following Table 1(i) in SEQ ID NO. 1. The amino acid residues listed in Table 1 are highly conserved amino acid residues between protease KP43 (Patent Document 1), which is the origin of the parent protease, and mutant proteases derived therefrom (Saeki et al., Journal of bioscience and Bioengineering, 2007, 103: 501-508).

[0064] [Table 1]

[0065] (i) Location (ii) Amino acid residues (i) Location (ii) Amino acid residues 10 Alanine 159 Alanine 18 Glycine 161 Glycine 21 Glycine 162 Asparagine 26 Valine 173 Proline 27 Alanine 182 Valine 28 Valine 183 Glycine 30 Aspartic acid 184 Alanine 32 Glycine 203 Alanine 43 Histidine 205 Phenylalanine 52 Alanine 206 Serine 64 Aspartic acid 209 Glycine 66 Asparagine 222 Alanine 68 Histidine 223 Proline 69 Glycine 224 Glycine 70 Threonine 225 Threonine 71 Histidine 229 Serine 72 Valine 253 Glycine 73 Alanine 254 Threonine 74 Glycine 255 Serine 85 Glycine 256 Methionine 87 Alanine 257 Alanine 88 Proline 259 Proline 92 Leucine 261 Valine 106 Glycine 262 Alanine 118 Alanine 263 Glycine 129 Serine 266 Alanine 131 Glycine 289 Leucine 145 Valine 297 Glycine

[0066] The parent protease of the mutant of the present invention is a polypeptide having proteolytic activity on the alkaline side (preferably at pH 8 or above). Preferably, the parent protease is a polypeptide having an optimum pH on the alkaline side (preferably at pH 8 or above). More preferably, the parent protease has any of the following enzymatic properties possessed by the protease consisting of the amino acid sequence of SEQ ID NO: 1 (see Patent Document 1): 1) having antioxidant properties, acting on the alkaline side (pH 8 or above), and being stable. Among them, having antioxidant properties means that the residual activity (synthetic substrate method) of the protease after being placed in a 50mM hydrogen peroxide solution (containing 5mM calcium chloride) (20mM Britton-Robinson buffer, pH10) at 20°C for 20 minutes is at least 50%; 2) the residual activity is 80% or more when treated at 50°C, pH10 for 10 minutes; 3) the activity is inhibited by diisopropyl fluorophosphate (DFP) and phenylmethylsulfonyl fluoride (PMSF); 4) the molecular weight measured by SDS-PAGE is 43,000±2,000. More preferably, the parent protease has all the enzymatic properties of 1) to 4) above.

[0067] The mutant protease of the present invention can be produced by subjecting the amino acid sequence of the parent protease to at least one mutation selected from the above (A) to (G) of the amino acid sequence of SEQ ID NO: 1. Preferably, the mutant protease of the present invention can be produced by subjecting the amino acid sequence of the parent protease to at least one mutation selected from the above (A) to (G) and the above mutation (H).

[0068] Therefore, the mutant protease of the present invention has at least one amino acid residue selected from the following amino acid residues (a) to (g):

[0069] (a) Arg or Lys, preferably Arg, at the position corresponding to position 191 of SEQ ID NO: 1;

[0070] (b) Arg or Lys, preferably Arg, at the position corresponding to position 17 of SEQ ID NO: 1;

[0071] (c) Ala or Phe, preferably Ala, at the position corresponding to position 141 of SEQ ID NO: 1;

[0072] (d) Arg or Lys, preferably Lys, at the position corresponding to position 243 of SEQ ID NO: 1;

[0073] (e) Arg or Lys, preferably Lys, at the position corresponding to position 300 of SEQ ID NO: 1;

[0074] (f) Arg or Lys, preferably Arg, at the position corresponding to position 302 of SEQ ID NO: 1;

[0075] (g) Arg or Lys, preferably Arg, at the position corresponding to position 311 of SEQ ID NO: 1.

[0076] Preferably, the mutant protease of the present invention has at least 90% sequence identity with the amino acid sequence of SEQ ID NO:1.

[0077] 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 (b) to (g) above.

[0078] In one embodiment, the mutant protease of the present invention has the amino acid residue of the above-mentioned (a) and at least one amino acid residue selected from the above-mentioned (b) and (d). In another embodiment, the mutant protease of the present invention has the amino acid residue of the above-mentioned (a) and the amino acid residue of any one of the above-mentioned (b) and (d). Preferably, the amino acid residue of the (a) is Arg, the amino acid residue of the (b) is Arg, and the amino acid residue of the (d) is Lys.

[0079] In one embodiment, the mutant protease of the present invention has at least one amino acid residue selected from the above (a) to (g), and the following amino acid residue (h):

[0080] (h) Gln in the position corresponding to position 82 of sequence number 1.

[0081] In a preferred embodiment, the mutant protease of the present invention has the amino acid residues of (a) and (h) above. Preferably, the amino acid residue of (a) is Arg.

[0082] In one embodiment, the mutant protease of the present invention may have at least one amino acid residue selected from the above (b) to (g). In a preferred embodiment, the mutant protease of the present invention has the above (e) and (f) amino acid residues. Preferably, the amino acid residue of (e) is Lys, and the amino acid residue of (f) is Arg.

[0083] The mutant protease of the present invention is a mutant protease having improved cleaning performance for protein stains in a detergent solution containing a surfactant at a high concentration (so-called high-concentration detergent solution). In more detail, the mutant protease of the present invention can exert a higher cleaning performance than the protease consisting of the amino acid sequence of sequence number 1 in a high-concentration detergent solution. Preferably, the mutant protease of the present invention can exert a higher cleaning performance than the protease consisting of the amino acid sequence of sequence number 1 in the stock solution of a concentrated liquid detergent. Preferably, the mutant protease of the present invention can exert a cleaning performance at least equal to or higher than that of the protease consisting of the amino acid sequence of sequence number 1 in a detergent solution diluted with water to the extent usually used for 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 cleaning (such as coating cleaning or immersion cleaning) using a high-concentration detergent solution. For example, the mutant protease of the present invention is effective as a protease in a concentrated liquid detergent for cleaning a stain by directly applying its stock solution or high-concentration detergent solution to the stain. Furthermore, 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 immersing the stains in the original solution or a highly concentrated detergent solution.

[0084] As the "protein stain" in this specification, for example, stains caused by protein-containing foods, protein-containing body components (such as blood, urine, dirt, sweat, sebum, etc.), etc. As more specific examples of "protein stains", food spilled on clothing, collar and sleeve stains, etc. can be listed.

[0085] The "detergent liquid" in this specification includes the original liquid of the liquid detergent and the detergent aqueous solution obtained by diluting the liquid detergent or the solid detergent (such as the powder detergent) with water. In this specification, the "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 of the surfactant, more preferably 20% by mass or more, for example, 10 to 90% by mass, preferably 20 to 90% by mass, and more preferably 20 to 75% by mass. As an example of a high-concentration detergent liquid, the original liquid of a concentrated liquid detergent or an aqueous solution containing it at a high concentration can be cited. In this specification, the "concentrated liquid detergent" refers to a liquid detergent containing 40% by mass or more of the surfactant, preferably 40 to 90% by mass, more preferably 45 to 90% by mass, and further preferably 50 to 75% by mass. As an example of the "concentrated liquid detergent", as long as it is a liquid detergent for clothes, a liquid detergent expressed as 7 to 13g or less in a standard usage amount relative to 30L of water in a water tank washing machine can be cited.

[0086] In this specification, "cleaning with a high-concentration detergent liquid" refers to a cleaning method in which a high-concentration detergent liquid is brought into direct contact with stains on a cleaning object (e.g., cloth, hard surfaces of tableware, cooking utensils, etc.), and after standing or cleaning for a prescribed time as needed, the cleaning object is washed with water. Examples of "cleaning with a high-concentration detergent liquid" include coating cleaning and immersion cleaning. "Coating cleaning" refers to a cleaning method in which a high-concentration detergent liquid is directly applied to the stains on the cleaning object, and after standing or cleaning for a prescribed time as needed, the cleaning object is washed with water. "Immersion cleaning" refers to a cleaning method in which the stains on the cleaning object are immersed in a high-concentration detergent liquid, and after standing for a prescribed time as needed or cleaning in the high-concentration detergent liquid, the cleaning object is washed with water. Therefore, in the above-mentioned cleaning using a high-concentration detergent liquid, methods for bringing the high-concentration detergent liquid into direct contact with the stain on the cleaning object include applying the high-concentration detergent liquid to the stain (for example, using a roll-on, spray, dripping, etc.), immersing the stain in the high-concentration detergent liquid, etc.

[0087] In the mutant protease of the present invention, in addition to at least one mutation selected from the above (A) to (G), as long as it can maintain a cleaning performance higher than that of the parent protease in the above-mentioned high-concentration detergent solution, it can also have a mutation (for example, deletion, substitution, addition, insertion) in any other position relative to the parent protease. The mutation can be naturally occurring or artificially introduced. Among them, it is preferred that the amino acid residues at least at positions 30, 68, and 255 of sequence number 1 of the mutant protease of the present invention are the same as those of the parent protease. More preferably, the amino acid residues at positions corresponding to the positions recorded in the above-mentioned Table 1 (i) in sequence number 1 of the mutant protease of the present invention are the same as those of the parent protease.

[0088] In the present invention, various mutation introduction techniques known in the art can be used as methods for mutating the amino acid residues of the parent protease. For example, in a polynucleotide encoding the amino acid sequence of the parent protease (hereinafter, also referred to as the parent polynucleotide), the nucleotide sequence encoding the amino acid residue to be mutated is mutated into a nucleotide sequence encoding the mutated amino acid residue, and then the protein is expressed from the mutant polynucleotide, thereby obtaining the target mutant protease.

[0089] About introducing the target mutation to the parental polynucleotide, for example, basically can be based on the PCR amplification using the parental polynucleotide as the template DNA and the replication reaction using various DNA polymerases, using various site-specific mutation introduction methods known to those skilled in the art to carry out. Site-specific mutation introduction method can be carried out using any method such as inverse PCR method or annealing method (Muramatsu et al., "Revised 4th Edition New Gene Engineering Handbook", Yangtu Company, p82-88). Various commercially available site-specific mutation introduction kits such as Stratagene's QuickChange II site-specific mutation kit (Site-Directed Mutagenesis Kit), QuickChange multi-site-specific mutation kit (Multi Site-Directed Mutagenesis Kit) can also be used.

[0090] Site-specific mutation introduction to a parent polynucleotide can most commonly be performed using a mutation primer containing the nucleotide mutation to be introduced. The mutation primer can be designed as follows, that is, it can anneal to the region of the parent polynucleotide containing the nucleotide sequence encoding the amino acid residue to be mutated, and contains a nucleotide sequence having a nucleotide sequence (codon) encoding the amino acid residue after the mutation to replace the nucleotide sequence (codon) encoding the amino acid residue to be mutated. As long as it is a person skilled in the art, based on common textbooks, etc., it is possible to appropriately identify and select the nucleotide sequence (codon) encoding the amino acid residue before and after the mutation. Alternatively, site-specific mutation introduction can also be performed using the following method: using two complementary primers containing the nucleotide mutation to be introduced, respectively, the upstream and downstream sides of the mutation site are amplified, and the amplified DNA fragments are connected into one using SOE (splicing by overlap extension)-PCR (Horton et al, Gene, 1989, 77 (1): p61-68).

[0091] 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 the microbial strain that maintains it by a known method. The primer for mutation can be made by a known oligonucleotide synthesis method such as the phosphoramidite method (Nucleic Acids Research, 1989, 17: 7059-7071). The primer synthesis can also be implemented using a commercially available oligonucleotide synthesizer (manufactured by ABI) or the like. Using a primer set containing the primer for mutation, the parent polynucleotide is used as a template DNA to carry out the site-specific mutation introduction as described above, thereby obtaining a polynucleotide that has been introduced with the target mutation and encodes a mutant protease.

[0092] Therefore, the present invention also provides 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 can include single-stranded or double-stranded DNA, cDNA, RNA or other artificial nucleic acid. The DNA, cDNA and RNA can be chemically synthesized. In addition, the polynucleotide of the present invention can also include the nucleotide sequence of the untranslated region (UTR) in addition to the open reading frame (ORF).

[0093] The present invention also provides a vector containing a polynucleotide encoding the mutant protease of the present invention. The vector can be made by inserting the polynucleotide of the present invention into any vector and linking it using a common method. The type of the vector is not particularly limited, and can be any vector of a plasmid, a phage, a phasmid, a clay, a virus, a YAC vector, a shuttle vector, etc. In addition, the vector is not limited, and is preferably a vector that can be amplified in bacteria, particularly in Bacillus bacteria, and more preferably an expression vector that can induce the expression of the introduced gene in Bacillus bacteria. Among them, the shuttle vector as a vector that can replicate in Bacillus bacteria and other organisms can be suitable for recombinant production of the mutant protease of the present invention. Examples of preferred vectors include, but are not particularly limited to, shuttle vectors such as pHA3040SP64 (Japanese Patent Application Publication No. 2013-233141), pHSP64R or pASP64 (Japanese Patent No. 3492935), pHA64, pHY300PLK (an expression vector capable of transforming both Escherichia 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), and pTA10607 (Bron et al., 1989, 135: 319-321). al, Plasmid, 1987, 18: 8-15) and the like that can be used for transformation of Bacillus bacteria; secretion vectors that can impart secretion signals to recombinant proteins (Yamane et al., Fusion proteins using Bacillus subtilis secretion vectors, Starch Science, 34. (1987), 163-170), etc. In addition, plasmids derived from Escherichia coli (e.g., pET22b(+), pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.) can also be used.

[0094] When the mutant protease of the present invention is produced by recombination, the above-mentioned vector is preferably an expression vector. The expression vector may contain various elements necessary for expression in the host organism such as a transcription promoter, a terminator, a ribosome binding site, etc.; cis elements such as a polylinker and an enhancer; a PolyA tailing signal; a ribosome binding sequence (SD sequence); a selection marker gene such as a drug resistance gene (e.g., ampicillin, neomycin, kanamycin, tetracycline, chloramphenicol, etc.), etc., as required.

[0095] The present invention also provides a transformant comprising a polynucleotide encoding a mutant protease of the present invention or a vector containing the polynucleotide. The transformant can be produced by introducing a polynucleotide encoding a mutant protease of the present invention or a vector containing the polynucleotide (preferably a recombinant expression vector) into a host. Therefore, the transformant comprises an external polynucleotide encoding a mutant protease of the present invention.

[0096] As the host of the above-mentioned transformant, bacteria such as Escherichia coli and Bacillus subtilis, microorganisms represented by yeast cells, and any cells such as insect cells, animal cells (e.g., mammalian cells), and plant cells can be cited. The host is preferably a bacterium of the genus Bacillus, more preferably Bacillus subtilis or a mutant thereof. Therefore, the transformant of the present invention is preferably a recombinant Bacillus bacterium, more preferably a recombinant of Bacillus subtilis or a mutant thereof.

[0097] For transformation of the host, for example, known transformation techniques such as the calcium phosphate method, electroporation, lipofection, gene gun method, and PEG method can be applied. For example, transformation methods applicable to Bacillus bacteria include competent cell transformation method (J Bacteriol, 1967, 93: 1925-1937), electroporation method (FEMS Microbiol Lett, 1990, 55: 135-138), protoplast transformation method (Mol Gen Genet, 1979, 168: 111-115), Tris-PEG method (J Bacteriol, 1983, 156: 1130-1134), and the like.

[0098] By culturing the transformant of the present invention, the mutant protease of the present invention can be produced. Therefore, the present invention also provides a method for producing a mutant protease using the transformant of the present invention. Those skilled in the art can culture the transformant for producing the mutant protease according to the usual method. For example, as a culture medium for culturing a transformant based on a microbial host such as Escherichia coli, yeast cells, etc., as long as it contains a carbon source, nitrogen source, inorganic salts, etc. that can be used by the microbial host, and can efficiently culture the transformant. The culture medium can use any kind of natural culture medium and synthetic culture medium. For example, in the culture of Bacillus subtilis transformants for producing recombinant proteins, LB culture medium, 2×YT culture medium, 2×L-maltose culture medium, or CSL fermentation culture medium can be used. In the culture medium, a drug corresponding to the type of drug resistance gene (selection marker gene) introduced into the transformant can be added. In addition, when a microorganism transformed with an expression vector using an inducible promoter is cultured, an inducer can be added to the culture medium as needed. For example, when culturing a microorganism transformed with an expression vector using a Lac promoter, isopropyl-1-thio-β-D-galactoside (IPTG) or the like can be added to the culture medium, and when culturing a microorganism transformed with an expression vector using a trp promoter, indoleacetic acid (IAA) or the like can be added to the culture medium.

[0099] Alternatively, the mutant protease of the present invention can be expressed by a polynucleotide encoding the mutant protease of the present invention or its transcription product using a cell-free translation system. A "cell-free translation system" is a reagent such as amino acids necessary for protein translation added to a suspension obtained by mechanically disrupting cells that serve as a host, thereby constituting an in vitro transcription-translation system or an in vitro translation system.

[0100] The mutant protease of the present invention obtained by utilizing the transformant or the cell-free translation system can be separated or purified from the culture fluid, cell-disrupted liquid, the reaction solution of the cell-free translation system, etc. by using the 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, the culture supernatant or lysate supernatant solution after separation or concentration using centrifugation or ultrafiltration filters can be used directly as a crude enzyme solution. When the expressed mutant protease is not secreted from the cell, the cell can be broken and the protein can be separated and purified.

[0101] The DNA extraction, mRNA preparation, cDNA preparation, PCR, RT-PCR, library preparation, connection to a vector, cell transformation, determination of DNA base sequence, nucleic acid chemical synthesis, determination of the amino acid sequence on the N-terminal side of the protein, mutation induction, protein extraction and other operations used in the present invention can be performed using the methods described in common experimental books. As such experimental books, for example, Sambrook et al. Molecular Cloning, A laboratory manual, 2001, 3rd Ed., Sambrook, J. & Russell, DW. Cold Spring Harbor Laboratory Press can be cited. In addition, for example, with respect to the gene recombination experiment of Bacillus subtilis, reference can be made to "7.2 Bacillus subtilis system" by Yoshikawa Hirofumi, "Continued Biochemistry Experiment Lecture 1. Gene Research Method II", 1986, Tokyo Chemical Co., Ltd. (Tokyo), p150-169 and other general experimental books on the gene manipulation of Bacillus subtilis.

[0102] The mutant protease obtained by the production method of the present invention has improved cleaning performance in a high-concentration detergent solution compared to the parent protease, and is useful as a protease used in a detergent for cleaning (e.g., coating cleaning or immersion cleaning) using a high-concentration detergent solution. Therefore, another embodiment of the present invention can be a method for improving the cleaning performance of a protease on protein stains in a high-concentration detergent solution, comprising the step of subjecting the amino acid sequence of the parent protease to at least one mutation selected from the above (A) to (G).

[0103] The mutant protease of the present invention is useful as a detergent enzyme, and is more suitable as an enzyme used for cleaning (e.g., coating cleaning or immersion cleaning) using a high concentration detergent solution. Therefore, the present invention also provides a detergent composition containing the mutant protease of the present invention. The detergent composition can be a solid (e.g., powder) detergent composition, preferably a liquid detergent composition, more preferably a concentrated liquid detergent. In addition, the detergent composition is preferably a detergent composition for coating cleaning or immersion cleaning, more preferably a detergent composition for coating cleaning.

[0104] 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 it is an amount in which the protease exhibits activity, but is preferably 0.1 to 25000 U per 1 kg of the detergent composition, more preferably 0.1 to 5000 U, and even more preferably 0.1 to 2500 U. The activity (U) of the protease in the present specification is measured by the following method: 0.9 mL of 1 / 15 M phosphate buffer (e.g., pH 7.4, a solution obtained by dissolving 1 packet of Wako Pure Chemical Industries, Ltd. phosphate buffer powder (167-14491) (1 packet contains 7.6 g of Na2HPO4 (anhydrous) and 1.8 g of KH2PO4 (anhydrous)) in 1 L of ion-exchanged water) and 0.05 mL of 40 mM Glt-Ala-Ala-Pro-Leu-p-nitroaniline / dimethyl sulfoxide solution are placed in a test tube and incubated at 30° C. for 5 minutes. 0.05 mL of enzyme solution was added thereto, and the mixture was reacted at 30°C for 10 minutes, then 2.0 mL of 5% (w / v) citric acid aqueous solution was added to stop the reaction, and the absorbance at 420 nm was measured using a spectrophotometer. 1 unit (U) of enzyme was defined as the amount that generates 1 μmol of p-nitroaniline in 1 minute in the above reaction.

[0105] The detergent composition of the present invention contains a surfactant and water in addition to the mutant protease of the present invention. As a surfactant, any surfactant such as a nonionic surfactant, anionic surfactant, cationic surfactant, and amphoteric surfactant can be used as one or a 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 further 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 further preferably 50 to 75% by mass.

[0106] The nonionic surfactant may be any nonionic surfactant having a C8 to C22 hydrocarbon group and having several moles or more of C2 oxyalkylene added thereto, which is usually contained in liquid detergents. Examples thereof include the following:

[0107] R1O-(AO)mH (R1 = C8-C22 hydrocarbon, AO = C2-C5 oxyalkylene, m = 16 to 35) [Japanese Patent Application Publication No. 2010-275468];

[0108] R1O-(EO)l-(AO)m-(EO)nH (R1=C8-C18 hydrocarbon, EO=C2 oxide

[0109] Alkylene, AO = C3-C5 oxyalkylene, l = 3 to 30, m = 1 to 5, l + n = 14 to 50) [Japanese Patent Application Publication No. 2010-265445, Japanese Patent Application Publication No. 2011-63784];

[0110] R1O-(EO)m / (AO)nH (R1=C8-C22 hydrocarbon, EO=C2 oxyalkylene, AO=C3-C5 oxyalkylene, m=10-30, n=0-5, EO and AO are randomly or block-bonded) [Japanese Patent Application Publication No. 2010-189551];

[0111] R1(CO)1O-(EO)m / (AO)n-R2(R1=C8-C22 hydrocarbon, EO=C2 oxidation

[0112] Alkylene, AO = C3-C5 oxyalkylene, l = 0-1, m = 14-50, n = 1-5, R2 = hydrogen (l = 0) or C1-C3 alkyl, EO and AO are randomly or block-bonded) [Japanese Patent Application Publication No. 2010-229385];

[0113] R1O-(EO)m-(AO)nH (R1=C8-C22 hydrocarbon, EO=C2 oxyalkylene, AO=C3-C5 oxyalkylene, m=15-30, n=1-5) [Japanese Patent Application Publication No. 2010-229387];

[0114] R1O-(AO)m / (Gly)nH and / or R2-COO-(AO)p / (Gly)qH (R1=C8-C22 hydrocarbon group, R2=C7-C21 hydrocarbon group, AO=C2-C3 oxyalkylene group, Gly=glyceryl group, m=0-5, n=2-10, p=0-5, q=2-10, AO and Gly are randomly or block-bonded) [Japanese Patent Application Publication No. 2010-254881];

[0115] R1-COO-(PO)m / (EO)n-R2 (R1=C7-C21 hydrocarbon group, COO=carbonyloxy group, R2=C1-C3 alkyl group, PO=propylene oxide, EO=ethylene oxide, m=0.3-5, n=8-25, PO and EO are randomly or block-bonded) [Japanese Patent Publication No. 2010-265333];

[0116] R1O-(EO)l-(PO)m-(EO)nH(R1=C8-C20 hydrocarbon, EO=C2 oxidized

[0117] Alkylene, PO = oxypropylene, l>=1, n>=1, 0<m<l+n, EO and PO block combination)〔WO98 / 24865〕;

[0118] R1O-(EO)m-(PO)nH (R1 = C10-C16 alkyl or alkenyl, EO = ethylene oxide, PO = propylene oxide, m = 5 to 15, n = 1 to 3) [Japanese Patent Publication No. 8-157867];

[0119] R1(CO)-(EO)m-OR2(R1=C11-C13 linear or branched alkyl or alkenyl, R2=C1-C3 alkyl, EO=ethylene oxide, m=10~20)〔Japanese Patent Publication No. 2008-7706, Japanese Patent Publication No. 2009-7451, Japanese Patent Publication No. 2009-155594, Japanese Patent Publication No. 2009-155606〕;

[0120] R1(CO)-(AO)m-OR2 (R1=C9-C13 straight chain or branched alkyl or alkenyl, AO=C2-C4 oxyalkylene, R2=C1-C3 alkyl, m=5 to 30) [Japanese Patent Application Publication No. 2009-144002, Japanese Patent Application Publication No. 2009-173858, Japanese Patent Application Publication No. 2010-189612]; and

[0121] Fatty acid alkanolamides, fatty acid alkanol glucamides, alkyl polyglucosides, etc.

[0122] Examples of the anionic surfactant include carboxylate anionic surfactants, sulfonic acid or sulfate anionic surfactants, non-soap anionic surfactants, linear alkylbenzenesulfonic acid, benzenesulfonic acid or its salts, polyoxybenzenesulfonic acid or its salts, polyoxyethylene alkyl sulfate ester salts, polyoxyalkylene alkyl ether sulfate ester salts, α-olefin sulfonates, alkylbenzenesulfonates, α-sulfofatty acid salts, fatty acid soaps, phosphate ester surfactants, acyl alanine esters, acyl taurates, alkyl ether carboxylic acids, alcohol sulfates, and the like.

[0123] Examples of the cationic surfactant include quaternary ammonium salts having a long-chain alkyl group, tertiary amines having one long-chain alkyl group, alkyl trimethyl ammonium salts, dialkyl dimethyl ammonium salts, alkyl pyridinium salts, etc. Preferred examples include quaternary ammonium surfactants having one long-chain alkyl group having 8 to 22 carbon atoms, and tertiary amines having one long-chain alkyl group having 8 to 22 carbon atoms.

[0124] Examples of the zwitterionic surfactant include alkyl acetate betaine, alkanolamidopropyl acetate betaine, alkyl imidazoline, alkyl alanine, alkyl betaine type, alkyl amide betaine type, imidazoline type, alkyl amino sulfone type, alkyl amino carboxylic acid type, alkyl amide carboxylic acid type, amide amino acid type or phosphoric acid type amphoteric surfactants, etc. Preferably, sulfobetaine or carbobetaine having an alkyl group with 10 to 18 carbon atoms can be used.

[0125] The detergent composition of the present invention may also contain ingredients commonly used in detergent compositions, such as water-soluble polymers, water-miscible organic solvents, alkali agents, organic acids or their salts, chelating agents, enzymes other than the mutant protease of the present invention, enzyme stabilizers, fluorescent agents, re-pollution inhibitors, dispersants, anti-color transfer agents, modifiers, bleaching agents, antioxidants, solubilizers, pH preparation agents, buffers, preservatives, fragrances, salts, alcohols, sugars, etc.

[0126] Examples of the water-soluble polymer include: a polymer compound having (i) a polyether chain portion composed of a polymerization unit derived from an epoxide having 2 to 5 carbon atoms and (ii) a polymer chain portion composed of a polymerization unit derived from one or more unsaturated carboxylic acid monomers selected from acrylic acid, methacrylic acid and maleic acid, and having a graft structure in which one of (i) or (ii) is the main chain and the other is a branched chain (Japanese Patent Publication No. 2010-275468, Japanese 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 (Japanese Patent Publication No. 2009-155606), etc. 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.

[0127] As water-miscible organic solvents, for example, alkylene glycols and glycerol of alkanols, polyalkylene glycols, (poly)alkylene glycol (mono or di) alkyl ethers, alkyl glycerol ethers, and aromatic ethers of (poly)alkylene glycols can be cited. Alkylene glycols having 2 to 6 carbon atoms such as ethylene glycol, propylene glycol, butylene glycol, or hexylene glycol, or glycerol, or polyethylene glycol monophenyl ether, ethylene glycol monobenzyl ether, diethylene glycol monobenzyl ether, etc. are preferred. 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.

[0128] As the alkali agent, for example, as an alkanolamine having 1 to 3 C2-C4 alkanols, monoethanolamine, diethanolamine, triethanolamine, polyoxyalkyleneamine, dimethylaminopropylamine, etc. can be mentioned. Monoethanolamine and triethanolamine are preferred. The content of the alkali agent in the detergent composition of the present invention is preferably 0 to 20% by mass, more preferably 0 to 10% by mass.

[0129] Examples of the organic acid or its salt include polycarboxylic acids such as saturated fatty acids, succinic acid, maleic acid, fumaric acid, or their salts; hydroxycarboxylic acids such as citric acid, malic acid, glycolic acid, p-hydroxybenzoic acid, benzoic acid, or their salts, and the like, among which citric acid or its salt is preferred. The content of the organic acid or its salt in the detergent composition of the present invention is preferably 0 to 5% by mass, more preferably 0 to 3% by mass.

[0130] Chelating agents refer to compounds that can coordinate with metal ions. The chelating agents added to the detergent composition have the effect of blocking metal ions such as calcium ions and magnesium ions in the washing water or stains that have an adverse effect on cleaning. Examples of chelating agents that can be contained in the detergent composition of the present invention include amino polyacetic acids such as nitrilotriacetic acid, iminodiacetic acid, ethylenediamineacetic acid, diethylenetriaminepentaacetic acid, ethylene glycol ether diaminetetraacetic acid, hydroxyethyliminodiacetic acid, triethylenetetraaminehexaacetic acid, and leucine or their salts, diglycolic acid, oxydisuccinic acid, carboxymethyloxysuccinic acid, citric acid, lactic acid, tartaric acid, oxalic acid, malic acid, oxydisuccinic acid, gluconic acid, carboxymethylsuccinic acid, carboxymethyltartaric acid or their salts, aminotris(methylenephosphonic acid), 1-hydroxyethyl-1,1-diphosphonic acid, ethylenediaminetetra(methylenephosphonic acid), diethylenetriaminepenta(methylenephosphonic acid) and their alkali metal or lower amine salts, etc. 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.

[0131] Examples of the anti-recontamination agent and dispersant include polyacrylic acid, polymaleic acid, carboxymethyl cellulose, polyethylene glycol having a weight average molecular weight of 5,000 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 (Page 1, Column 3, Line 5 to Page 3, Column 4, Line 14) of Japanese Patent Application Laid-Open No. 59-62614. The content of the anti-recontamination agent and dispersant in the detergent composition of the present invention is preferably 0.01 to 10% by mass, respectively.

[0132] As an anti-color transfer agent, polyvinyl pyrrolidone is mentioned, for example, and the content is preferably 0.01 to 10% by mass.

[0133] As the bleaching agent, for example, it is preferred that the detergent composition contain 1 to 10% by mass of hydrogen peroxide, percarbonate, perborate or the like. When a bleaching agent is used, the detergent composition may contain 0.01 to 10% by mass of a bleaching activator such as tetraacetylethylenediamine (TAED) or the like described in Japanese Patent Application Laid-Open No. 6-316700.

[0134] Examples of the fluorescent agent include biphenyl fluorescent agents (Tinopal CBS-X, etc.) and stilbene fluorescent agents (DM fluorescent dyes, etc.) The content of the fluorescent agent in the detergent composition of the present invention is preferably 0.001 to 2% by mass.

[0135] Examples of other enzymes other than the mutant protease of the present invention include 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, polygalacturons, rhamnogalacturons, pectin lyases, mannanases, pectin methylesterases, cellobiohydrolases, transglutaminases and other hydrolases, and mixtures of two or more thereof.

[0136] As enzyme stabilizers, for example, boron compounds, calcium ion sources (calcium ion supplying compounds), hydroxy compounds, formic acid, etc. can be cited. As antioxidants, butylated hydroxytoluene, distyrenated cresol, sodium sulfite and sodium bisulfite, etc. can be cited. As solubilizing agents, p-toluenesulfonic acid, isopropylbenzenesulfonic acid, meta-xylenesulfonic acid, benzoates (also having the effect of being a preservative), etc. can be cited. In addition, the detergent composition of the present invention can also contain alkanes such as octane, decane, dodecane, and tridecane, olefins such as decene and dodecene, alkyl halides such as dichloromethane and 1,1,1-trichloroethane, terpenes such as D-limonene, etc., non-aqueous miscible organic solvents, pigments, fragrances, antimicrobial preservatives, defoamers such as silicone, etc.

[0137] Preferred examples of detergent compositions that can be formulated with the mutant protease of the present invention include the liquid detergent compositions described in Japanese Patent Application Laid-Open No. 2017-008303 and the liquid detergent compositions described in Japanese Patent Application Laid-Open No. 2013-129729. By formulating the mutant protease of the present invention in these detergent compositions, the detergent composition of the present invention can be prepared.

[0138] The detergent composition of the present invention is not limited, and preferred examples include detergent compositions for cleaning clothing or cloth products (e.g., bed sheets, curtains, carpets, wall coverings, etc.), and detergent compositions for kitchen use (e.g., tableware or cooking utensils). Preferably, the detergent composition of the present invention is a detergent composition for cleaning using a high-concentration detergent solution, more preferably a detergent composition for coating cleaning or immersion cleaning, and even more preferably a detergent composition for coating cleaning.

[0139] As exemplary embodiments, the present invention also includes the following substances, production methods, uses, methods, etc. However, the present invention is not limited to these embodiments.

[0140] [1] A mutant protease, which consists of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 90% identity thereto, and has at least one amino acid residue selected from the group consisting of (a) to (g) below:

[0141] (a) Arg or Lys in the position corresponding to position 191 of SEQ ID NO: 1;

[0142] (b) Arg or Lys in the position corresponding to position 17 of SEQ ID NO: 1;

[0143] (c) Ala or Phe in the position corresponding to position 141 of SEQ ID NO: 1;

[0144] (d) Arg or Lys in the position corresponding to position 243 of SEQ ID NO: 1;

[0145] (e) Arg or Lys in the position corresponding to position 300 of SEQ ID NO: 1;

[0146] (f) Arg or Lys in the position corresponding to position 302 of SEQ ID NO: 1;

[0147] (g) Arg or Lys at the position corresponding to position 311 of SEQ ID NO:1.

[0148] [2] The mutant protease according to [1], which preferably has the amino acid residue of (a) above and at least one amino acid residue selected from (b) to (g) above.

[0149] [3] The mutant protease according to [1], which preferably has the amino acid residue of (e) and the amino acid residue of (f).

[0150] [4] The mutant protease according to [1], preferably having a member selected from the group consisting of (a) to

[0151] at least one amino acid residue of (g) and the following amino acid residue of (h):

[0152] (h) Gln in the position corresponding to position 82 of sequence number 1.

[0153] [5] The mutant protease according to [4], which preferably has the amino acid residue of (a) and the amino acid residue of (h).

[0154] [6] A polynucleotide encoding the mutant protease according to any one of [1] to [5].

[0155] [7] A vector comprising the polynucleotide described in [6].

[0156] [8] A recombinant Bacillus bacterium, comprising the polynucleotide described in [6] or the vector described in [7].

[0157] [9] A method for producing a mutant protease, comprising using the recombinant Bacillus bacterium described in [8].

[0158]

[10] The method as described in [9], which includes the step of culturing the above-mentioned recombinant Bacillus bacteria.

[0159]

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

[0160]

[12] The detergent composition according to

[11] , which is preferably a detergent composition for cleaning using a high-concentration detergent solution, more preferably a detergent composition for coating cleaning or immersion cleaning, and still more preferably a detergent composition for coating cleaning.

[0161]

[13] A method for producing a mutant protease, comprising the step of 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 thereto to at least one mutation selected from the group consisting of (A) to (G) below:

[0162] (A) Substitution of the amino acid residue at position 191 of SEQ ID NO:1 or a position equivalent thereto with Arg or Lys;

[0163] (B) Substitution of the amino acid residue at position 17 of SEQ ID NO: 1 or a position equivalent thereto with Arg or Lys;

[0164] (C) substitution of the amino acid residue at position 141 of SEQ ID NO:1 or a position equivalent thereto with Ala or Phe;

[0165] (D) Substitution of the amino acid residue at position 243 of SEQ ID NO:1 or a position equivalent thereto with Arg or Lys;

[0166] (E) Substitution of the amino acid residue at position 300 of SEQ ID NO:1 or a position equivalent thereto with Arg or Lys;

[0167] (F) Substitution of the amino acid residue at position 302 of SEQ ID NO:1 or a position equivalent thereto with Arg or Lys;

[0168] (G) Substitution of the amino acid residue at position 311 of SEQ ID NO: 1 or a position equivalent thereto with Arg or Lys.

[0169]

[14] A method for improving the cleaning performance of a protease on protein stains in a high concentration detergent solution, comprising the step of 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 thereto to at least one mutation selected from the group consisting of (A) to (G) below:

[0170] (A) Substitution of the amino acid residue at position 191 of SEQ ID NO:1 or a position equivalent thereto with Arg or Lys;

[0171] (B) Substitution of the amino acid residue at position 17 of SEQ ID NO: 1 or a position equivalent thereto with Arg or Lys;

[0172] (C) substitution of the amino acid residue at position 141 of SEQ ID NO:1 or a position equivalent thereto with Ala or Phe;

[0173] (D) Substitution of the amino acid residue at position 243 of SEQ ID NO:1 or a position equivalent thereto with Arg or Lys;

[0174] (E) Substitution of the amino acid residue at position 300 of SEQ ID NO:1 or a position equivalent thereto with Arg or Lys;

[0175] (F) Substitution of the amino acid residue at position 302 of SEQ ID NO:1 or a position equivalent thereto with Arg or Lys;

[0176] (G) Substitution of the amino acid residue at position 311 of SEQ ID NO: 1 or a position equivalent thereto with Arg or Lys.

[0177]

[15] The method as described in

[13] or

[14] , which preferably includes the step of subjecting the parent protease to the mutation (A) and at least one mutation selected from the above (B) to (G).

[0178]

[16] The method as described in

[13] or

[14] , which preferably includes the steps of subjecting the parent protease to the mutations (E) and (F).

[0179]

[17] The method according to

[13] or

[14] , wherein the parent protease is preferably subjected to the step of subjecting the parent protease to at least one mutation selected from the above (A) to (G) and the following mutation (H):

[0180] (H) Substitution of the amino acid residue at position 82 of SEQ ID NO: 1 or a position equivalent thereto with Gln.

[0181] Example

[0182] Hereinafter, the present invention will be described in more detail using examples, but the technical scope of the present invention is not limited to these examples.

[0183] Example 1 Preparation of mutant protease

[0184] The wild-type KP43 protease expression plasmid pHA64TSA described in Reference Example 1 of Patent Document 5 comprises a tetracycline resistance gene and the full length coding sequence of the wild-type KP43 protease linked to the promoter region of the alkaline cellulase K-64 gene from the Bacillus sp. KSM-64 strain. Using pHA64TSA as a template, site-specific mutations were repeatedly introduced into the KP43 protease gene by a site-specific introduction method (see Nucleic Acids Research, 2004, 32(14): e115) using PCR with a complementary primer pair, thereby preparing a plasmid expressing the protease of SEQ ID NO: 1. Using the plasmid expressing the protease of sequence number 1 obtained as a template, a plasmid expressing a mutant protease having the following amino acid substitutions with respect to the protease of sequence number 1 as the parent enzyme: T311R, N302R, Y300K, N243K, S191R, Y17R, D141A, D141F, Y17R / S191R, S191R / N243K, Y300K / N302R, E82Q / S191R, or S191F is prepared by the same method. Hereinafter, the mutant proteases expressed by 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.

[0185] The prepared plasmid expressing the parent enzyme or mutant protease was used to transform the host bacillus KSM9865 strain (FERM P-18566). The transformation was performed by electroporation, and the transformed cells were spread on an alkaline agar medium containing skim milk [1% skim milk (Difco), 1% Bacto Tryptone (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. Among the colonies grown on the agar medium, the ones that showed skim milk lysis spots were selected as transformants into which the protease gene was 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% sodium dihydrogen phosphate, 0.25% sodium carbonate, 30 ppm tetracycline; % is (w / v) %], and cultured with shaking at 30°C for 16 hours. Next, 1% (v / v) of the seed culture solution was inoculated into 20 mL of the main medium [8% polypeptone S, 0.3% yeast extract, 10% maltose, 0.04% magnesium sulfate heptahydrate, 0.2% sodium 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 solution was centrifuged to obtain a culture supernatant containing protease. The protein concentration contained in the culture supernatant was measured using Protein Assay Rapid Kit Wako II (Fujifilm Wako Pure Chemical Industries, Ltd.).

[0186] Example 2 Evaluation of coating cleaning power of mutant proteases

[0187] A commercially available concentrated liquid detergent (Attack ZERO Regular; Kao Corporation) was heated in a microwave oven to inactivate the enzyme in the product. The culture supernatant containing each mutant protease obtained in Example 1 was added to the liquid detergent so that the protein concentration became 0.1 g / L, and the mixture was stirred thoroughly to prepare a liquid detergent composition (test composition) containing the mutant protease. As a control, a liquid detergent composition (control composition) containing the parent enzyme was prepared in the same manner. The obtained liquid detergent composition was used to apply and clean the artificial contaminated cloth DINGY_TN / CP / (CONSUMERTEC) as a collar and sleeve stain model. That is, 20 μL of the test composition or the control composition was dripped onto DINGY_TN / CP / cut into 1.5 cm squares, and after standing for 5 minutes, the contaminated cloth was transferred to 600 mL of tap water kept at 20°C in the cleaning tank of a stirring cleaning power tester (Tergotometer, Ueshima Seisakusho), and cleaned at 60 rpm for 10 minutes. The contaminated cloth after coating and cleaning was taken out of the cleaning tank, rinsed with tap water, and dried. The lightness (L value) of the contaminated cloth before and after coating and cleaning and the original contaminated cloth prepared separately was measured using a spectrophotometer cm-700d (Konica Minolta), and the cleaning rate of the contaminated cloth was calculated using the following formula.

[0188] Cleaning rate (%) = (L2-L1) / (L0-L1)×100

[0189] (L0: L value of the original contaminated cloth; L1: L value of the contaminated cloth before cleaning; L2: L value of the contaminated cloth after cleaning).

[0190] The relative cleaning rate of each test composition to the control composition (cleaning rate in the test composition / cleaning rate in the control composition) was calculated, and the relative cleaning power of each mutant protease to the parent protease in coating cleaning (relative coating cleaning power) was evaluated.

[0191] The relative coating cleaning power of each mutant protease (relative cleaning rate of the test composition containing each mutant protease) is shown in Tables 2 and Figure 1 The relative coating cleaning power of each mutant protease except S191F was greater than 1, showing a coating cleaning power significantly higher than that of the parent enzyme.

[0192] [Table 2]

[0193] Enzymes Relative coating cleaning power Parent enzyme (SEQ ID NO: 1) 1 T311R 1.35 N302R 1.30 Y300K 1.46 N243K 1.15 S191R 1.27 Y17R 1.41 D141A 1.49 D141F 1.36 Y17R / S191R 1.61 S191R / N243K 1.58 Y300K / N302R 2.21 E82Q / S191R 2.01 S191F 1.03

[0194] In Table 3 and Figure 2The effect of mutation at position 191 of the parent protease of sequence number 1 is summarized in the figure. The protease (S191R) in which the serine (S) at position 191 of sequence number 1 is replaced with arginine (R) has improved coating cleaning power compared to the parent protease. On the other hand, the substitution of phenylalanine (F) at position 191 (S191F) does not have an effect on coating cleaning power. The mutant with glutamine at position 82 and arginine at position 191 (E82Q / S191R) shows higher coating cleaning power.

[0195] [Table 3]

[0196] Enzymes 191 82 bits Relative coating cleaning power Parent enzyme (SEQ ID NO: 1) S E 1 S191R R E 1.27 S191F F E 1.03 E82Q / S191R R Q 2.01

[0197] Example 3 Stability Evaluation of Mutant Proteases

[0198] A commercially available concentrated liquid detergent (Attack ZERO Regular; Kao Corporation) was heated in a microwave oven to inactivate the enzyme in the product. The culture supernatant containing the mutant protease E82Q / S191R obtained in Example 1 was added to the liquid detergent so that the protein concentration was 0.2 g / L, and the mixture was stirred thoroughly to prepare a liquid detergent composition (test sample) containing the mutant protease. As a control, a liquid detergent composition (control sample) containing the parent enzyme was prepared in the same manner. The protease activity of the test sample just after the enzyme was added (0h) and after being stored 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 0.1M phosphate buffer as a substrate solution. The test samples at 0h and 2w were diluted 60 times with 0.1M phosphate buffer, respectively. 1 mL of the diluted sample was added to the substrate solution and incubated at 40°C for 30 minutes. Add 10mL of 2% trisodium phosphate aqueous solution to stop the reaction. After stirring, take 1mL of the reaction solution in each 1.5mL tube. Centrifuge at 12000rpm for 5 minutes, take the supernatant, and measure the absorbance at 590nm. Instead of the test sample, use a heat-inactivated liquid detergent without adding enzyme, and measure the absorbance in the same way as a blank control. The difference between the absorbance of the test sample and the blank control is taken as the activity value. Use the same steps to calculate the activity value of the control sample at 0h and 2w. By dividing the activity value of the sample at 2w by the activity value of the sample at 0h, calculate the residual activity rate after two weeks, and multiply its value by 1 / 14 to calculate the daily activity maintenance rate obtained by 40℃ treatment. Take the daily activity maintenance rate as the base, and calculate the logarithm of 0.5 as the half-life (days) of the protease activity. Divide the half-life of the mutant protease by the half-life of the parent protease to calculate the relative stability. The relative stability of the mutant E82Q / S191R is 1.89, and the introduction of mutations of E82Q and S191R greatly improves the stability of the protease. Patent Document 3 states that the protease with 82E or 82Q has high stability in liquid detergents. The mutant E82Q / S191R of this example has improved stability compared to the parent protease with 82E, suggesting that the mutation of S191R further improves the stability of the protease.

[0199] Although the 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 made within the scope of the present invention will be apparent to those skilled in the art.

[0200] The documents and patent applications cited in this specification are incorporated by reference as if fully described in this specification.

Claims

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

1.

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

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

4. The mutant protease according to claim 1, wherein The mutant protease has at least one amino acid residue selected from (a) to (g) and the following amino acid residue (h): (h) Gln in the position corresponding to position 82 of sequence number 1.

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

6. A polynucleotide, wherein The polynucleotide encodes the mutant protease according to any one of claims 1 to 5.

7. A vector, wherein The vector contains the polynucleotide according to claim 6.

8. A recombinant Bacillus bacterium, wherein: The recombinant Bacillus bacterium comprises the polynucleotide of claim 6 or the vector of claim 7.

9. A method for producing a mutant protease, wherein: The method uses the recombinant Bacillus bacterium according to claim 8.

10. A detergent composition, wherein The detergent composition contains the mutant protease according to any one of claims 1 to 5.

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

12. A method for producing a mutant protease, wherein: The method comprises the step of 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 thereto to at least one mutation selected from the following (A) to (G): (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.

13. A method for improving the cleaning performance of protease on protein stains in a high concentration detergent solution, wherein: The method comprises the step of 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 thereto to at least one mutation selected from the following (A) to (G): (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. The method according to claim 12 or 13, wherein: The method comprises the steps of subjecting the parent protease to the mutation (A) and at least one mutation selected from the group consisting of (B) to (G).

15. The method according to claim 12 or 13, wherein: The method comprises the steps of subjecting the parent protease to the mutation of (E) and the mutation of (F).

16. The method according to claim 12 or 13, wherein: The method comprises the step of subjecting the parent protease to at least one mutation selected from (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.

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