Lipase variant

Abstract

To provide a lipase variant that mitigates hindrance to activity on a dispersed substrate in the presence of a surfactant.SOLUTION: In one aspect, the lipase variant comprises an amino acid sequence having at least 75% identity to a specific amino acid sequence, and has substitution in a specific amino acid residue. Compared to the parent lipase, hindrance to lipase activity on a dispersed substrate in the presence of a surfactant is mitigated, and the ability to remove triglycerides on a hard surface in the presence of a surfactant is enhanced, so that the lipase variant has superior cleaning performance.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to lipase variants. [Background technology]

[0002] Lipases are useful in a variety of applications, including laundry detergents, dishwashing detergents, oil and fat processing, pulp treatment, feed, pharmaceutical intermediate synthesis, etc. In cleaning, lipases contribute to the removal of oily soils by hydrolyzing ester bonds in lipids to produce fatty acids.

[0003] As a lipase useful for cleaning, lipase derived from Thermomyces lanuginosus (hereinafter referred to as TLL) is sold under the trade name LIPOLASE (registered trademark). Patent Document 1 discloses that lipase Lipr139 derived from Cedecea sp-16640 strain has superior cleaning performance compared to TLL. Patent Document 2 discloses that lipase Lipr138 derived from metagenomics has superior cleaning performance compared to TLL.

[0004] As a method for fully utilizing the cleaning performance of lipase, Patent Document 3 describes, for example, a method for cleaning oily stains by contacting a cleaning agent containing lipase and a sulfosuccinate ester as a surfactant with a hard object on which oily stains are attached and leaving the object without applying external force. In an aqueous solution containing no surfactant, the removal of triglycerides on the hard surface by lipase hardly progresses, but in a cleaning solution containing an alkyl sulfosuccinate ester, the removal of triglycerides on the hard surface by lipase is greatly promoted. On the other hand, even in the absence of a surfactant, lipase can efficiently decompose an ester substrate dispersed in an aqueous solution. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Special Publication No. 2015-523078 [Patent Document 2] Special Publication No. 2015-525248 [Patent Document 3] Patent Publication No. 2021-17508 Summary of the Invention [Problem to be solved by the invention]

[0006] The present inventors have discovered an unexpected phenomenon that a cleaning solution containing sulfosuccinate esters (hereinafter, cleaning solution) significantly promotes the removal of triglycerides from hard surfaces by lipase (Patent Document 3 above), but also significantly inhibits lipase activity against an ester substrate dispersed in the cleaning solution (hereinafter, dispersed substrate). Lipr139 and Lipr138, which are known to be suitable for cleaning, were inhibited in the presence of sulfosuccinate esters to 5% or less in their decomposition activity against dispersed substrates, and almost no activity was detectable. Therefore, a lipase that is less inhibited in lipase activity against dispersed substrates in the presence of surfactants and exhibits high detergency is desired. [Means for solving the problem]

[0007] The present inventors have obtained lipase variants that have improved inhibition of lipase activity against dispersed substrates in the presence of surfactants compared to the parent lipase, and these lipase variants also have significantly improved ability to remove triglycerides from hard surfaces in the presence of surfactants.

[0008] That is, the present invention relates to the following 1) to 7). 1) A lipase variant consisting of an amino acid sequence having at least 75% identity to the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8, and having an amino acid residue other than isoleucine at a position corresponding to position 44 in the numbering of SEQ ID NO: 2. 2) A polynucleotide encoding the lipase variant described in 1). 3) A vector or a DNA fragment comprising the polynucleotide according to 2). 4) A transformed cell containing the vector or DNA fragment described in 3). 5) A cleaning composition comprising the lipase variant described in 1). 6) A method for cleaning stains using the cleaning agent composition according to 5). 7) A method for producing a lipase mutant, comprising a step of replacing an amino acid residue at a position corresponding to position 44 in the numbering of SEQ ID NO: 2 with an amino acid residue other than isoleucine in a polypeptide having lipase activity and consisting of an amino acid sequence having at least 75% identity to the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8. Effect of the Invention

[0009] Compared to the parent lipase, the lipase variant of the present invention has improved inhibition of lipase activity against dispersed substrates in the presence of surfactants, and has improved ability to remove triglycerides from hard surfaces in the presence of surfactants, resulting in excellent cleaning power. [Brief description of the drawings]

[0010] [Figure 1] Enzymatic effect of each lipase on the cleaning power in model cleaning solutions. [Diagram 2] Enzymatic effect of each lipase on the cleaning power in model cleaning solutions. [Diagram 3] Enzymatic effect of each lipase on the cleaning power in model cleaning solutions. [Figure 4] Enzymatic effect of each lipase on the cleaning power in model cleaning solutions. [Diagram 5] The cleaning power of each lipase in a model cleaning solution. [Figure 6] Enzymatic effect of each lipase on the cleaning power in model cleaning solutions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] All patents, non-patent publications, and other publications cited herein are hereby incorporated by reference in their entirety.

[0012] As used herein, the term "lipase" refers to triacylglycerol lipase (EC 3.1.1.3) and means a group of enzymes having the activity of hydrolyzing ester bonds in lipids to produce fatty acids.

[0013] In the present specification, the identity of an amino acid sequence or a nucleotide sequence is calculated by the Lipman-Pearson method (Science, 1985, 227:1435-1441). Specifically, it is calculated by performing an analysis using the search homology program of the genetic information processing software GENETYX Ver. 12 with the unit size to compare (ktup) set to 2.

[0014] As used herein, "at least 75% identity" with respect to an amino acid sequence or a nucleotide sequence means identity of 75% or more, preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, even more preferably 93% or more, even more preferably 94% or more, even more preferably 95% or more, even more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more, and even more preferably 99.5% or more.

[0015] 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 shown in SEQ ID NO: 2) 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]), and the website of the DNA Data Bank of Japan operated by the National Institute of Genetics (DDBJ [www.ddbj.nig.ac.jp / searches-j.html]). 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.

[0016] 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.

[0017] 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).

[0018] In this specification, amino acid positions and variants are described using the accepted IUPAC one-letter amino acid abbreviations as follows: The amino acid at a given position is designated as [amino acid, position], e.g., isoleucine at position 44 is designated "I44." Amino acid "substitutions" are designated as [original amino acid, position, substituted amino acid]. For example, a substitution of an isoleucine at position 44 with an alanine would be designated "I44A".

[0019] 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 so that the gene can be expressed under the control of the control region. The procedure for "operably linked" between a gene and a control region is well known to those skilled in the art.

[0020] 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.

[0021] As used herein, a "parent" polypeptide of a given mutant polypeptide refers to a polypeptide in which a given amino acid residue is mutated to give the mutant polypeptide. In other words, a "parent" polypeptide is a polypeptide before the mutant polypeptide is mutated. Such a parent polypeptide may be a naturally occurring (wild-type) polypeptide or a mutant thereof.

[0022] 1. Lipase variants and methods for producing the same The present invention provides a lipase variant having excellent detergency, and a method for producing the same.

[0023] In one aspect, the invention provides a method for producing a lipase variant, the method comprising the step of substituting an amino acid residue at a predetermined position, numbered based on SEQ ID NO:2, in a parent lipase with another amino acid residue.

[0024] An example of a parent lipase of the lipase variant of the present invention is a polypeptide having lipase activity and consisting of an amino acid sequence having at least 75% identity with the amino acid sequence of SEQ ID NO: 2. Here, the polypeptide having lipase activity and consisting of the amino acid sequence of SEQ ID NO: 2 is lipase CnLip derived from Cedecea neteri (NCBI Accession No. WP_061278013.1). The parent lipase, which is a polypeptide having lipase activity and which consists of an amino acid sequence having at least 75% identity with the amino acid sequence of SEQ ID NO:2, is preferably one which has an I at the position corresponding to position 44 in the numbering of SEQ ID NO:2, an I and an L at the positions corresponding to positions 44 and 120 in the numbering of SEQ ID NO:2, an I and an S at the positions corresponding to positions 44 and 130 in the numbering of SEQ ID NO:2, an I and an A at the positions corresponding to positions 44 and 134 in the numbering of SEQ ID NO:2, an I and an L at the positions corresponding to positions 44 and 136 in the numbering of SEQ ID NO:2, or an I and an A at the positions corresponding to positions 44 and 137 in the numbering of SEQ ID NO:2. More preferred are those having I and S, and even more preferred are those having I, L and S at positions corresponding to positions 44, 120 and 130, respectively, in the numbering of SEQ ID NO:2, those having I, S and L at positions corresponding to positions 44, 130 and 136, respectively, in the numbering of SEQ ID NO:2, those having I, L and L at positions corresponding to positions 44, 120 and 136, respectively, in the numbering of SEQ ID NO:2, those having I, L, S and L at positions corresponding to positions 44, 120, 130 and 136, respectively, in the numbering of SEQ ID NO:2, or those having I, L, S, A, L and S at positions corresponding to positions 44, 120, 130, 134, 136 and 137, respectively, in the numbering of SEQ ID NO:2.

[0025] Another example of the parent lipase of the lipase variant of the present invention is a polypeptide having lipase activity and consisting of an amino acid sequence having at least 75% identity with the amino acid sequence of SEQ ID NO: 4. Here, the polypeptide having lipase activity and consisting of the amino acid sequence of SEQ ID NO: 4 is lipase EbLip (NCBI Accession No. MRT57156.1) derived from Enterobacteriaceae bacterium. The parent lipase, which is a polypeptide having lipase activity and consisting of an amino acid sequence having at least 75% identity with the amino acid sequence of SEQ ID NO: 4, preferably has I at the position corresponding to position 44 in the numbering of SEQ ID NO: 2, has I and L at the positions corresponding to positions 44 and 120 in the numbering of SEQ ID NO: 2, has I and S at the positions corresponding to positions 44 and 130 in the numbering of SEQ ID NO: 2, has I and A at the positions corresponding to positions 44 and 134 in the numbering of SEQ ID NO: 2, more preferred are those having I and L at positions corresponding to positions 44 and 136 in the numbering of SEQ ID NO:2, or those having I and S at positions corresponding to positions 44 and 137 in the numbering of SEQ ID NO:2, and even more preferred are those having I, L and S at positions corresponding to positions 44, 120 and 130 in the numbering of SEQ ID NO:2, or those having I, L, S, A, L and S at positions corresponding to positions 44, 120, 130, 134, 136 and 137 in the numbering of SEQ ID NO:2. In the amino acid sequence of SEQ ID NO:4, the positions corresponding to positions 44, 120, 130, 134, 136 and 137 in the numbering of SEQ ID NO:2 are positions 44, 120, 130, 134, 136 and 137, respectively.

[0026] Another example of the parent lipase of the lipase variant of the present invention is a polypeptide having lipase activity and consisting of an amino acid sequence having at least 75% identity with the amino acid sequence of SEQ ID NO: 6. Here, the polypeptide having lipase activity and consisting of the amino acid sequence of SEQ ID NO: 6 is lipase Ag1Lip (NCBI Accession No. EJF30243.1) derived from Enterobacter sp. The parent lipase, which is a polypeptide having lipase activity and consisting of an amino acid sequence having at least 75% identity with the amino acid sequence of SEQ ID NO: 6, is preferably one having I at the position corresponding to position 44 in the numbering of SEQ ID NO: 2, one having I and L at the positions corresponding to positions 44 and 120 in the numbering of SEQ ID NO: 2, one having I and S at the positions corresponding to positions 44 and 130 in the numbering of SEQ ID NO: 2, one having I and A at the positions corresponding to positions 44 and 134 in the numbering of SEQ ID NO: 2, more preferred are those having I and L at positions corresponding to positions 44 and 136 in the numbering of SEQ ID NO:2, or those having I and S at positions corresponding to positions 44 and 137 in the numbering of SEQ ID NO:2, and even more preferred are those having I, L and S at positions corresponding to positions 44, 120 and 130 in the numbering of SEQ ID NO:2, or those having I, L, S, A, L and S at positions corresponding to positions 44, 120, 130, 134, 136 and 137 in the numbering of SEQ ID NO:2. In the amino acid sequence of SEQ ID NO:6, the positions corresponding to positions 44, 120, 130, 134, 136 and 137 in the numbering of SEQ ID NO:2 are positions 44, 120, 130, 134, 136 and 137, respectively.

[0027] Another example of the parent lipase of the lipase variant of the present invention is a polypeptide having lipase activity and consisting of an amino acid sequence having at least 75% identity with the amino acid sequence of SEQ ID NO: 8. Here, the polypeptide having lipase activity and consisting of the amino acid sequence of SEQ ID NO: 8 is lipase CspLip (NCBI Accession No. WP_016537805.1) derived from Cedecea sp. The parent lipase, which is a polypeptide having lipase activity and consisting of an amino acid sequence having at least 75% identity with the amino acid sequence of SEQ ID NO: 8, is preferably one having I at the position corresponding to position 44 in the numbering of SEQ ID NO: 2, one having I and L at the positions corresponding to positions 44 and 120 in the numbering of SEQ ID NO: 2, one having I and S at the positions corresponding to positions 44 and 130 in the numbering of SEQ ID NO: 2, one having I and A at the positions corresponding to positions 44 and 134 in the numbering of SEQ ID NO: 2, more preferably, those having I and M at positions corresponding to positions 44 and 136 in the numbering of SEQ ID NO:2, or those having I and S at positions corresponding to positions 44 and 137 in the numbering of SEQ ID NO:2, and even more preferably, those having I, L and S at positions corresponding to positions 44, 120 and 130 in the numbering of SEQ ID NO:2, or those having I, L, S, A, M and S at positions corresponding to positions 44, 120, 130, 134, 136 and 137 in the numbering of SEQ ID NO:2. In the amino acid sequence of SEQ ID NO:8, the positions corresponding to positions 44, 120, 130, 134, 136 and 137 in the numbering of SEQ ID NO:2 are positions 45, 121, 131, 135, 137 and 138, respectively.

[0028] When producing a lipase variant of the present invention from a parent lipase, the amino acid residue at the position corresponding to 44 in the numbering of SEQ ID NO:2 in the parent lipase is replaced with an amino acid residue other than I, preferably A, G, S, T, or V.

[0029] When producing the lipase variant of the present invention from the parent lipase, in addition to substituting the amino acid residue at the position corresponding to position 44 in SEQ ID NO: 2 in the parent lipase, preferably one or more, more preferably two or more, even more preferably three or more, even more preferably four or more, and even more preferably all of the steps selected from the group consisting of (i) to (v) below are carried out, from the viewpoint of improving cleaning power. (i) substituting an amino acid residue at a position corresponding to position 120 in the numbering of SEQ ID NO:2 with an amino acid residue other than L; (ii) substituting an amino acid residue at a position corresponding to position 130 in the numbering of SEQ ID NO:2 with an amino acid residue other than S; (iii) substituting the amino acid residue at the position corresponding to position 134 in the numbering of SEQ ID NO:2 with an amino acid residue other than A; (iv) substituting an amino acid residue at a position corresponding to position 136 in the numbering of SEQ ID NO:2 with an amino acid residue other than L; and (v) substituting an amino acid residue at a position corresponding to position 137 in the numbering of SEQ ID NO:2 with an amino acid residue other than S. In the case where only step (iv) among the above steps (i) to (v) is carried out and where the substitution is made with M, the parent lipase preferably consists of an amino acid sequence having at least 75% identity with the amino acid sequence of SEQ ID NO: 2, 4 or 6. The above (i) to (v) are more preferably the following (i') to (v'), respectively. (i') substituting the amino acid residue at the position corresponding to position 120 in the numbering of SEQ ID NO:2 with A, F, G, H, I, K, M, N, Q, R, S, T, V, W, or Y; (ii') substituting the amino acid residue at the position corresponding to position 130 in the numbering of SEQ ID NO:2 with A, C, E, F, G, H, I, K, L, M, Q, R, T, V, W, or Y; (iii') substituting the amino acid residue at the position corresponding to position 134 in SEQ ID NO:2 with C, D, E, G, I, T, or V; (iv') substituting the amino acid residue at the position corresponding to position 136 in the numbering of SEQ ID NO:2 with A, C, E, F, G, H, I, M, S, T, V, or W; and (v') substituting the amino acid residue at the position corresponding to position 137 in the numbering of SEQ ID NO:2 with C, N or T. In the case where only step (iv') among the above steps (i') to (v') is performed and where the substitution is made with M, it is preferable that the parent lipase consists of an amino acid sequence having at least 75% identity with the amino acid sequence of SEQ ID NO: 2, 4 or 6.

[0030] The present invention also provides a lipase variant, which is a polypeptide having lipase activity and consisting of an amino acid sequence in which an amino acid residue at a predetermined position, as numbered based on SEQ ID NO: 2, in the amino acid sequence of a parent lipase is substituted with another amino acid residue.

[0031] The lipase variant of the present invention consists of an amino acid sequence having at least 75% identity to the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8, and has an amino acid residue other than I, preferably A, G, S, T or V, at the position corresponding to position 44 in the numbering of SEQ ID NO: 2.

[0032] From the viewpoint of improving detergency, the lipase variant of the present invention further contains preferably one or more, more preferably two or more, even more preferably three or more, even more preferably four or more, and even more preferably all of the amino acid residues selected from the group consisting of the following (a) to (e). (a) an amino acid residue other than L at a position corresponding to 120 in the numbering of SEQ ID NO:2; (b) an amino acid residue other than S at a position corresponding to 130 in the numbering of SEQ ID NO:2; (c) an amino acid residue other than A at a position corresponding to 134 in the numbering of SEQ ID NO:2; (d) an amino acid residue other than L at a position corresponding to position 136 in the numbering of SEQ ID NO:2; and (e) an amino acid residue other than S at a position corresponding to position 137 in the numbering of SEQ ID NO:2. When the lipase mutant has only the amino acid residue (d) among the above (a) to (e) and the amino acid residue is M, it is preferable that the lipase mutant has an amino acid sequence having at least 75% identity with the amino acid sequence of SEQ ID NO: 2, 4 or 6. The above (a) to (e) are more preferably the following (a') to (e'), respectively. (a') A, F, G, H, I, K, M, N, Q, R, S, T, V, W or Y at a position corresponding to 120 in the numbering of SEQ ID NO:2; (b') A, C, E, F, G, H, I, K, L, M, Q, R, T, V, W, or Y at a position corresponding to 130 in the numbering of SEQ ID NO:2; (c') C, D, E, G, I, T, or V at a position corresponding to position 134 in the numbering of SEQ ID NO:2; (d') A, C, E, F, G, H, I, M, S, T, V, or W at a position corresponding to position 136 in the numbering of SEQ ID NO:2; and (e') C, N, or T at a position corresponding to position 137 in the numbering of SEQ ID NO:2. When the lipase variant has only M (d') among the above (a') to (e'), it preferably has an amino acid sequence having at least 75% identity with the amino acid sequence of SEQ ID NO: 2, 4 or 6.

[0033] In a preferred embodiment, the lipase variant of the present invention is a lipase variant consisting of an amino acid sequence having at least 75% identity with the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8 and having any of the amino acid residues No. 1 to 57 in Table 1 below. Among these, lipase variants having an amino acid sequence having at least 75% identity with the amino acid sequence of SEQ ID NO: 2 and having any of the amino acid residues No. 1 to 23, 25 to 47, 49 and 50 in Table 1 below, lipase variants having an amino acid sequence having at least 75% identity with the amino acid sequence of SEQ ID NO: 4 and having any of the amino acid residues No. 4, 24, 44, 49 and 52 to 57 in Table 1 below, lipase variants having an amino acid sequence having at least 75% identity with the amino acid sequence of SEQ ID NO: 6 and having any of the amino acid residues No. 4, 44 and 50 in Table 1 below, and lipase variants having an amino acid sequence having at least 75% identity with the amino acid sequence of SEQ ID NO: 8 and having any of the amino acid residues No. 1 to 5, 24, 44, 48 and 51 in Table 1 below are more preferred.

[0034] [Table 1]

[0035] The substitution of the amino acid residues at the above-mentioned predetermined positions is for improving the inhibition of the lipase activity of the lipase against a dispersed substrate in the presence of a surfactant, and for improving the ability of the lipase to remove triglycerides on a hard surface in the presence of a surfactant. In other words, the substitution of the amino acid residues at the above-mentioned predetermined positions is for improving the detergency of the lipase. Therefore, the lipase variant of the present invention has improved detergency compared to the parent lipase, i.e., the lipase before the substitution of the amino acid residues at the above-mentioned predetermined positions. Note that positions 120, 130, 134, 136, and 137 in the numbering of SEQ ID NO: 2 are all present in the lid domain of the lipase (the lid of the active center located at positions 108 to 161 in the numbering of SEQ ID NO: 2).

[0036] In addition to the mutations at the above-mentioned predetermined positions, the lipase variant of the present invention may have a mutation (e.g., deletion, substitution, addition, insertion) at any other position relative to the parent lipase, so long as the mutation does not interfere with its detergency. The mutation may be naturally occurring or artificially introduced.

[0037] <2. Polynucleotide encoding the lipase variant of the present invention> The lipase variant of the present invention can be produced by using various mutagenesis techniques known in the art, for example, by mutating a polynucleotide encoding an amino acid residue to be substituted in a parent lipase gene (reference lipase gene) encoding the reference amino acid sequence to a polynucleotide encoding the substituted amino acid residue, and then expressing the variant from the mutant gene.

[0038] The polynucleotide encoding the lipase variant of the present invention may be in the form of single- or double-stranded DNA, RNA, or an artificial nucleic acid, or may be cDNA, or chemically synthesized DNA containing no introns.

[0039] In the present invention, various mutation introduction techniques known in the art can be used as a means for mutating amino acid residues of a parent lipase. For example, in a polynucleotide encoding the amino acid sequence of a parent lipase (hereinafter also referred to as a parent gene), a nucleotide sequence encoding an amino acid residue to be mutated is mutated to a nucleotide sequence encoding the mutated amino acid residue, thereby obtaining a polynucleotide encoding a lipase variant of the present invention.

[0040] The introduction of a desired mutation into a parent gene can basically be carried out using various site-directed mutagenesis methods well known to those skilled in the art. Site-directed mutagenesis can be carried out by any method, such as inverse PCR or annealing. Commercially available site-directed mutagenesis kits (e.g., Stratagene's QuickChange II Site-Directed Mutagenesis Kit, QuickChange Multi Site-Directed Mutagenesis Kit, etc.) can also be used.

[0041] Site-specific mutagenesis of a parent gene 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 gene, 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 (Gene, 1989, 77(1): p61-68).

[0042] The template DNA containing the parent gene can be prepared by extracting genomic DNA from the microorganism that produces the above-mentioned parent lipase in a conventional manner, or by extracting RNA and synthesizing cDNA by reverse transcription. Alternatively, a corresponding nucleotide sequence may be chemically synthesized based on the amino acid sequence of the parent lipase and used as the template DNA. DNA sequences containing a base sequence encoding lipases consisting of the amino acid sequences shown in SEQ ID NOs: 2, 4, 6 and 8 are shown in SEQ ID NOs: 1, 3, 5 and 7, respectively.

[0043] The mutation primer can be prepared by a well-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 that manufactured by ABI). A primer set including the mutation primer is used to perform site-specific mutagenesis as described above using a parent gene as a template DNA, thereby obtaining a polynucleotide encoding the lipase mutant of the present invention having the desired mutation.

[0044] The polynucleotide encoding the lipase variant of the present invention may comprise single-stranded or double-stranded DNA, cDNA, RNA or other artificial nucleic acid. The DNA, cDNA and RNA may be chemically synthesized. The polynucleotide may comprise a nucleotide sequence of an untranslated region (UTR) in addition to an open reading frame (ORF). The polynucleotide may be codon-optimized according to the species of the transformant used to produce the mutant polypeptide of the present invention. Information on codons used by various organisms is available from the Codon Usage Database ([www.kazusa.or.jp / codon / ]).

[0045] <3. Vector or DNA fragment> The obtained polynucleotide encoding the lipase variant of the present invention can be incorporated into a vector. The type of vector containing the polynucleotide is not particularly limited, and may be any vector, such as a plasmid, 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, preferably in Bacillus bacteria (e.g., Bacillus subtilis or a mutant thereof), and more preferably an expression vector that can induce expression of an introduced gene in Bacillus bacteria. Among them, a shuttle vector, which is a vector that can be replicated in both Bacillus bacteria and other organisms, can be suitably used for recombinantly producing the lipase variant of the present invention. Preferred examples of the vector include, but are not limited to, shuttle vectors such as pHA3040SP64, pHSP64R or pASP64 (Patent No. 3492935), pHY300PLK (an expression vector capable of transforming both Escherichia coli and Bacillus subtilis; Jpn J Genet, 1985, 60:235-243), and pAC3 (Nucleic Acids Res, 1988, 16:8732); and plasmid vectors that can be used for transformation of bacteria of the genus Bacillus, such as pUB110 (J Bacteriol, 1978, 134:318-329) and pTA10607 (Plasmid, 1987, 18:8-15). Plasmid vectors derived from E. coli (e.g., pET22b(+), pBR322, pBR325, pUC57, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.) can also be used.

[0046] The vector may contain a DNA replication origin region or a DNA region containing a replication origin. Alternatively, the vector may have a control sequence, such as a promoter region for initiating transcription of the gene, a terminator region, or a secretion signal region for secreting the expressed protein outside the cell, operably linked upstream of a polynucleotide encoding the lipase variant of the present invention (i.e., the lipase variant gene).

[0047] The types of the control sequences such as the promoter region, terminator region, and secretion signal region are not particularly limited, and promoters and secretion signal sequences that are commonly used can be appropriately selected and used depending on the host to be introduced. For example, suitable examples of the control sequences that can be incorporated into the vector include the promoter and secretion signal sequence of the cellulase gene of Bacillus sp. KSM-S237 strain.

[0048] Alternatively, the vector of the present invention may further incorporate a marker gene (e.g., a resistance gene to a drug such as ampicillin, neomycin, kanamycin, or chloramphenicol) for selecting a host into which the vector has been appropriately introduced. Alternatively, when an auxotrophic strain is used as a host, a gene encoding an enzyme for synthesizing the required nutrient may be incorporated as a marker gene into the vector. Furthermore, when a selective medium requiring a specific metabolism for growth is used, a gene related to the metabolism may be incorporated as a marker gene into the vector. An example of such a metabolism-related gene is the acetamidase gene for utilizing acetamide as a nitrogen source.

[0049] The polynucleotide encoding the lipase variant of the present invention can be ligated to a control sequence and a marker gene by a method known in the art, such as splicing by overlap extension (SOE)-PCR (Gene, 1989, 77:61-68). The procedure for introducing the ligated fragment into a vector is well known in the art.

[0050] 4. Transformed cells The transformed cell of the present invention can be obtained by introducing a vector containing a polynucleotide encoding the lipase variant of the present invention into a host, or by introducing a DNA fragment containing a polynucleotide encoding the lipase variant of the present invention into the genome of the host.

[0051] Examples of host cells include microorganisms such as bacteria and filamentous fungi. Examples of bacteria include bacteria belonging to the genera Escherichia coli, Staphylococcus, Enterococcus, Listeria, and Bacillus, among which Escherichia coli and Bacillus are preferred, Bacillus are more preferred, and Bacillus subtilis (e.g., Bacillus subtilis Marburg No. 168 (Bacillus subtilis 168 strain) or a mutant thereof) are even more preferred. Examples of Bacillus subtilis mutants include the KA8AX, a nine-fold protease deletion strain described in J. Biosci. Bioeng., 2007, 104(2):135-143, and the D8PA strain, an eight-fold protease deletion strain with improved protein folding efficiency described in Biotechnol. Lett., 2011, 33(9):1847-1852. Examples of filamentous fungi include the genera Trichoderma, Aspergillus, and Rhizopus.

[0052] The vector can be introduced into the host by a method commonly used in the art, such as the protoplast method, electroporation, etc. A strain into which the vector has been appropriately introduced can be selected based on the expression of a marker gene, nutritional requirements, etc., to obtain a desired transformant into which the vector has been introduced.

[0053] Alternatively, a fragment in which a polynucleotide encoding the lipase variant of the present invention, a control sequence, and a marker gene are linked can be directly introduced into the genome of a host. For example, a DNA fragment in which sequences complementary to the host genome are added to both ends of the linked fragment is constructed by SOE-PCR or the like, and this is introduced into a host to cause homologous recombination between the host genome and the DNA fragment, thereby introducing the polynucleotide encoding the lipase variant of the present invention into the genome of the host.

[0054] When the thus obtained transformant into which a polynucleotide encoding the lipase variant of the present invention or a vector containing the same has been introduced is cultured in an appropriate medium, the gene encoding the protein on the vector is expressed to produce the lipase variant of the present invention. The medium used for culturing the transformant can be appropriately selected by those skilled in the art depending on the type of microorganism used as the transformant.

[0055] Alternatively, the lipase variant of the present invention may be expressed from a polynucleotide encoding the lipase variant of the present invention or a transcription product thereof using a cell-free translation system. The "cell-free translation system" refers to an in vitro transcription / translation system or an in vitro translation system that is prepared by adding reagents such as amino acids required for protein translation to a suspension obtained by mechanically disrupting host cells.

[0056] The lipase variant of the present invention produced in the above culture or cell-free translation system can be isolated or purified by a general method used for protein purification, such as centrifugation, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc., either alone or in appropriate combination. The protein recovered from the culture may be further purified by known means.

[0057] <5. Cleaning composition> The thus obtained lipase variant of the present invention has a significantly improved inhibition of lipase activity against a dispersed substrate in the presence of a surfactant, compared to the parent lipase. The lipase variant of the present invention also has a significantly improved ability to remove triglycerides on a hard surface in the presence of a surfactant, compared to the parent lipase. The lipase variant of the present invention can remove triglycerides on a hard surface in the presence of a surfactant without the application of an external force, and therefore can remove triglycerides not only on hard surfaces but also on soft surfaces. Thus, the lipase variant of the present invention has excellent detergency and exhibits good detergency even in the presence of a surfactant. Here, "detergency" refers to the ability to remove dirt, particularly oily dirt, in a washing or cleaning process.

[0058] The surfactant may be one or a combination of anionic surfactants, nonionic surfactants, amphoteric surfactants, and cationic surfactants, preferably one or a combination of anionic surfactants and nonionic surfactants, more preferably one or a combination of sulfosuccinate esters or salts thereof, SDS, and Triton X-100, which will be described in detail below, and even more preferably a sulfosuccinate ester or a salt thereof.

[0059] The term "dispersed substrate" refers to an ester substrate that contains an ester bond that can be hydrolyzed by lipase and is solubilized, emulsified or dispersed in an aqueous solution. Examples of dispersed substrates include 4-nitrophenol fatty acid esters, triglycerides, methyl resorufin esters, arachidonic acid-1-thioglycerol, fluorescein diacetate, fluorescein isothiocyanate diacetate, EnzChek TM Lipase Substrate (Invitrogen TM ) can be used. When the substrate is insoluble, it can be emulsified by adding an emulsion stabilizer or emulsifier. Among these, from the viewpoint of convenience of measuring lipase activity, 4-nitrophenol fatty acid esters are preferred, 4-nitrophenol fatty acid esters having a fatty acid chain length of 2 to 16 are more preferred, and 4-nitrophenyl octanoate is even more preferred.

[0060] "Inhibition of lipase activity on a dispersed substrate in the presence of a surfactant" means that the lipase activity on a dispersed substrate in the presence of a surfactant is lower than the lipase activity on a dispersed substrate in the absence of a surfactant. The degree of inhibition of lipase activity on a dispersed substrate in the presence of a surfactant can be measured using a method well known in the art. For example, lipase and a surfactant solution are mixed, a 4-nitrophenyl octanoate solution as a dispersed substrate is added to the mixture, and the change in absorbance at 405 nm (OD / min) associated with the liberation of 4-nitrophenol is measured, and the difference ΔOD / min (lipase activity value in the surfactant solution) from the blank (sample without lipase added) is taken to determine the lipase activity on a dispersed substrate in the presence of a surfactant. Separately, ΔOD / min (lipase activity value in the buffer) is determined when a buffer is used instead of the surfactant solution. Next, the lipase activity value in the surfactant solution is divided by the lipase activity value in the buffer to determine the ΔOD / min ratio. The ΔOD / min ratio is a relative measure of the lipase activity retention rate in a surfactant solution compared to that in a buffer, and the smaller the value, the more the lipase activity is inhibited in the presence of a surfactant. Therefore, the degree of inhibition of lipase activity against a dispersed substrate in the presence of a surfactant can be evaluated using this value as an index. In addition, by dividing the ΔOD / min ratio of the lipase variant by the ΔOD / min ratio of the parent lipase, the degree of improvement in the inhibition of lipase activity in the presence of a surfactant in the lipase variant compared to the parent lipase can be obtained. If the degree of improvement is greater than 1, the inhibition of lipase activity in the presence of a surfactant in the lipase variant is improved compared to the parent lipase. The lipase variant of the present invention is a lipase having a degree of improvement ((ΔOD / min ratio of lipase variant) / (ΔOD / min ratio of parent lipase)) of preferably 1.3 or more, more preferably 1.7 or more, even more preferably 2.4 or more, and even more preferably 3.5 or more under the conditions of (3) in the Examples described below.

[0061] "Hard surface" refers to a hard solid surface of an inanimate object. Inanimate objects having such hard surfaces include tableware, kitchen items, and / or living environment items such as bathrooms, toilets, and floors. Specific examples of tableware include so-called tableware such as plates and bowls; storage containers such as Tupperware and bottles; cooking utensils such as knives, cutting boards, pots, frying pans, and fish grills; and cooking appliances such as food processors and mixers, and other components and utensils that come into contact with food. Kitchen items are items used around the kitchen, and specific examples include storage locations for food, tableware, and cooking utensils such as refrigerators and cupboards; cooking locations for food such as drains, counters, range hoods, sinks, gas ranges, and microwave ovens; and floors and walls around the storage locations and cooking locations. The lipase variant of the present invention is preferably used for removing triglycerides on the hard surfaces of items selected from tableware, storage containers, cooking utensils, and cooking appliances.

[0062] "Soft surface" refers to the soft solid surface of an inanimate object. Examples of inanimate objects having such soft surfaces include textile products. Here, textile products include fabrics in which a large number of fibers are processed into a thin, wide sheet shape by weaving or knitting thread-like natural fibers, regenerated fibers, or synthetic fibers, and processed fabrics made by sewing, pressing, or the like using such fabrics. Examples of processed fabrics include clothing (e.g., underwear, shirts, sweaters, skirts, sweatshirts, etc.), towels, accessories (e.g., slippers, scarves, stoles, gloves, socks, etc.), orthotics (e.g., supporters, masks, gauze, bandages, etc.), bedding (e.g., futons, mats, cushions, pillows, blankets, etc.), covers (e.g., futon covers, cushion covers, pillow covers, sheets, cushion covers, toilet seat covers, etc.), toys (e.g., stuffed toys, etc.), etc.

[0063] "Triglyceride" includes ester bonds and can be hydrolyzed by lipase. Triglycerides are not particularly limited, but preferably include triglycerides that are attached or may be attached to hard surfaces, such as triglycerides having a fatty acid selected from palmitic acid, stearic acid, and oleic acid as a constituent fatty acid. In addition, oils containing triglycerides include oils derived from animals such as cows and pigs, and oils derived from plants such as rapeseed oil and olive oil.

[0064] The "ability to remove triglycerides from a hard surface" of lipase means the ability of lipase to remove triglycerides from a hard surface by hydrolyzing the triglycerides attached to the hard surface, and can be an index of cleaning power. The ability to remove triglycerides from a hard surface in the presence of a surfactant can be evaluated using a method well known in the art. For example, a triglyceride-containing model oil stain containing a predetermined indicator substance (e.g., a staining agent with high solubility in fat such as Sudan III) is attached to a hard surface, and a cleaning solution containing lipase and a surfactant is added to perform a cleaning treatment under predetermined conditions. A part of the cleaning solution is separated, and the concentration of the indicator substance in the model oil stain solubilized in the cleaning solution by the cleaning treatment is measured, for example, by absorbance measurement, and the difference from before cleaning can be obtained as an index of the ability to remove triglycerides from a hard surface in the presence of a surfactant.

[0065] The lipase variant of the present invention is useful as an enzyme to be incorporated in various detergent compositions, and is particularly useful as an enzyme to be incorporated in detergent compositions suitable for low-temperature washing. Here, "low temperature" includes 40°C or lower, 35°C or lower, 30°C or lower, and 25°C or lower, as well as 5°C or higher, 10°C or higher, and 15°C or higher. Further examples include 5 to 40°C, 10 to 35°C, 15 to 30°C, and 15 to 25°C.

[0066] The amount of the lipase variant of the present invention to be incorporated in the detergent composition is not particularly limited as long as the lipase variant exhibits activity, but is, for example, preferably 0.1 mg or more, more preferably 1 mg or more, even more preferably 5 mg or more, and preferably 5000 mg or less, more preferably 1000 mg or less, even more preferably 500 mg or less, per 1 kg of the detergent composition. The amount is preferably 0.1 to 5000 mg, more preferably 1 to 1000 mg, and even more preferably 5 to 500 mg.

[0067] The detergent composition preferably contains a sulfosuccinate ester or a salt thereof in addition to the lipase variant of the present invention. Sulfosuccinate esters or salts thereof are known as components to be incorporated into detergent compositions (for example, JP 2019-182911 A). The sulfosuccinate ester or salt thereof is preferably a sulfosuccinate branched alkyl ester having a branched alkyl group having 9 to 12 carbon atoms or a salt thereof, more preferably a sulfosuccinate branched alkyl ester having a branched alkyl group having 9 or 10 carbon atoms or a salt thereof, and even more preferably a sulfosuccinate branched alkyl ester having a branched alkyl group having 10 carbon atoms or a salt thereof. Furthermore, the sulfosuccinic acid ester or a salt thereof is a dibranched alkyl sulfosuccinate or a salt thereof, and is preferably a dibranched alkyl sulfosuccinate or a salt thereof in which the two branched alkyl groups are each a branched alkyl group having from 9 to 12 carbon atoms, more preferably a dibranched alkyl sulfosuccinate or a salt thereof in which the two branched alkyl groups are each a branched alkyl group having 9 or 10 carbon atoms, even more preferably a dibranched alkyl sulfosuccinate or a salt thereof in which the two branched alkyl groups are each a branched alkyl group having 10 carbon atoms, and still more preferably bis-(2-propylheptyl)sulfosuccinic acid or a salt thereof.

[0068] Examples of the salt include alkali metal salts and alkanolamine salts. An alkali metal salt or an alkanolamine salt is preferred, a salt selected from sodium salt, potassium salt, triethanolamine salt, diethanolamine salt, and monoethanolamine salt is more preferred, and a sodium salt is even more preferred.

[0069] Examples of sulfosuccinate esters or salts thereof include compounds represented by the following formula 1.

[0070] [ka]

[0071] [In formula 1, R 1 , R 2 are each a branched alkyl group having 9 to 12 carbon atoms, 1 O, A 2 Each O is an alkyleneoxy group having 2 to 4 carbon atoms, x1 and x2 are the average number of moles added, each being a number of 0 to 10, and M is a cation.

[0072] In formula 1, R 1 , R 2 are each preferably a branched alkyl group selected from a branched nonyl group, a branched decyl group, and a branched dodecyl group, and more preferably a branched decyl group. The branched decyl group is preferably a 2-propylheptyl group.

[0073] In formula 1, A 1 O, A 2 Each O is an alkyleneoxy group having 2 to 4 carbon atoms, preferably 2 or 3 carbon atoms in terms of lubricity to water. 1 O, A 2 Each of these represents the average number of moles of O added, and is from 0 to 10, and from the viewpoint of lubricity in water, is preferably 6 or less, more preferably 4 or less, even more preferably 2 or less, with 0 being even more preferable.

[0074] In formula 1, M is a cation. M is preferably a cation other than a hydrogen ion. Examples of M include alkali metal ions such as lithium ion, sodium ion, and potassium ion, alkaline earth metal ions such as calcium ion and barium ion, and organic ammonium ions such as triethanolammonium ion, diethanolammonium ion, monoethanolammonium ion, trimethylammonium ion, and monomethylammonium ion. From the viewpoint of dispersibility in water, M is preferably an alkali metal ion or an alkanol ammonium ion, more preferably a sodium ion, a potassium ion, a triethanol ammonium ion, a diethanol ammonium ion or a monoethanol ammonium ion, and further preferably a sodium ion.

[0075] The sulfosuccinate or a salt thereof is preferably a compound represented by the following formula 1-1: The compound of formula 1-1 is a compound in which x1 and x2 in formula 1 are each 0.

[0076] [ka]

[0077] [In formula 1-1, R 1 , R 2 are each a branched alkyl group having 9 to 12 carbon atoms, and M is a cation. R in Formula 1-1 1 , R 2 Specific and preferred examples of M are the same as those of formula 1. In a preferred embodiment, the sulfosuccinic acid ester or its salt is bis-(2-propylheptyl)sulfosuccinic acid or its salt.

[0078] The amount of the sulfosuccinate or its salt in the detergent composition is preferably 0.01% by mass or more, more preferably 0.1% by mass or more, and preferably 30.0% by mass or less, more preferably 10.0% by mass or less, and even more preferably 2.0% by mass or less. The amount is preferably 0.01 to 30.0% by mass, more preferably 0.1 to 10.0% by mass, and even more preferably 0.1 to 2.0% by mass.

[0079] In addition to the lipase variant of the present invention, various enzymes can also be used in combination with the detergent composition. For example, hydrolases, oxidases, reductases, transferases, lyases, isomerases, ligases, synthetases, etc. Among these, lipases other than the lipase variant of the present invention, amylases, proteases, cellulases, keratinases, esterases, cutinases, pullulanases, pectinases, mannanases, glucosidases, glucanases, cholesterol oxidases, peroxidases, laccases, etc. are preferred, and proteases, cellulases, amylases, and lipases are particularly preferred. Examples of commercially available proteases include Alcalase, Esperase, Everlase, Savinase, Kannase, Progress Uno (registered trademark; Novozymes), PREFERENZ, EFFECTENZ, EXCELLENZ (registered trademark; DuPont), Lavergy (registered trademark; BASF), and KAP (Kao). Examples of cellulases include Celluclean, Carezyme (registered trademark; Novozymes), KAC, alkaline cellulase produced by Bacillus sp. KSM-S237 strain described in JP-A-10-313859, and mutant alkaline cellulase described in JP-A-2003-313592 (all of which are Kao products). Examples of amylase include Termamyl, Duramyl, Stainzyme, Stainzyme Plus, Amplify Prime (registered trademark; Novozymes), PREFERENZ, EFFECTENZ (registered trademark; DuPont), and KAM (Kao). Examples of lipase include Lipolase and Lipex (registered trademark; Novozymes).

[0080] The detergent composition may contain known detergent components. Examples of the known detergent components include the following:

[0081] (1) Surfactants The surfactant is blended in the detergent composition in an amount of 0.5 to 60% by mass, preferably 10 to 45% by mass for powder detergent compositions and 20 to 90% by mass for liquid detergent compositions. When the detergent composition is a laundry detergent or an automatic dishwashing machine detergent, the surfactant is generally blended in an amount of 1 to 10% by mass, preferably 1 to 5% by mass.

[0082] The surfactant used in the detergent composition may be one or a combination of anionic surfactants, nonionic surfactants, amphoteric surfactants, and cationic surfactants other than the above-mentioned sulfosuccinic acid esters or salts thereof, with amphoteric surfactants being preferred.

[0083] As the amphoteric surfactant, an amine oxide type surfactant or a betaine type surfactant is preferred, and a tertiary amine oxide type surfactant, a sulfobetaine type surfactant or a carbobetaine type surfactant is more preferred. As the tertiary amine oxide type surfactant, there can be mentioned a tertiary amine oxide type surfactant in which one of the groups bonded to the nitrogen atom is an alkyl group having 8 to 18 carbon atoms, preferably an alkyl group having 8 to 16 carbon atoms, more preferably an alkyl group having 8 to 14 carbon atoms, which may be interrupted by an amide group or an ester group, and the remaining is an alkyl group having 1 to 3 carbon atoms, preferably a methyl group. As the sulfobetaine type surfactant, a compound having one alkyl group having 10 to 18 carbon atoms, preferably 16 to 14 carbon atoms, two alkyl groups having 1 to 3 carbon atoms, preferably two methyl groups, and a 3-sulfopropyl group or a 2-hydroxy-3-sulfopropyl group is preferred. In addition, the carbobetaine type surfactant is preferably a carbobetaine type surfactant having one alkyl group having 10 or more and 18 or less, preferably 16 or less, and more preferably 14 or less, carbon atoms, which may be interrupted by an amide group or an ester group, two alkyl groups having 1 to 3 carbon atoms, preferably two methyl groups, and one carboxyalkyl group, preferably a carboxymethyl group.

[0084] (2) Divalent metal ion scavengers The divalent metal ion trapping agent is blended in an amount of 0.01 to 50 mass%, preferably 5 to 40 mass%. Examples of the divalent metal ion trapping agent used in the detergent composition include condensed phosphates such as tripolyphosphates, pyrophosphates, and orthophosphates, aluminosilicates such as zeolites, synthetic layered crystalline silicates, nitrilotriacetates, ethylenediaminetetraacetates, citrates, isocitrates, and polyacetal carboxylates. Among these, crystalline aluminosilicates (synthetic zeolites) are particularly preferred, and among A-, X-, and P-type zeolites, A-type is particularly preferred. As for the synthetic zeolites, those having an average primary particle size of 0.1 to 10 μm, particularly 0.1 to 5 μm, are preferably used.

[0085] (3) Alkaline agents The alkaline agent is blended in an amount of 0.01 to 80% by mass, preferably 1 to 40% by mass. In the case of powder detergents, examples of the alkaline agent include alkali metal carbonates such as sodium carbonate, which are generally called dense ash or light ash, and amorphous alkali metal silicates such as JIS No. 1, No. 2, and No. 3. These inorganic alkaline agents are effective in forming the skeleton of particles when the detergent is dried, and a detergent that is relatively hard and has excellent fluidity can be obtained. Other examples of alkalis include sodium sesquicarbonate and sodium hydrogencarbonate, and phosphates such as tripolyphosphates also act as alkaline agents. In addition to the above alkaline agents, sodium hydroxide and mono-, di-, or triethanolamine can be used as alkaline agents used in liquid detergents, and they can also be used as counterions of the activators.

[0086] (4) Anti-redeposition agent The redeposition inhibitor is blended in an amount of 0.001 to 10% by mass, preferably 1 to 5% by mass. Examples of redeposition inhibitors used in the detergent composition include polyethylene glycol, carboxylic acid polymers, polyvinyl alcohol, and polyvinylpyrrolidone. Among these, the carboxylic acid polymers have the function of capturing metal ions and dispersing solid particle soils from clothing into the wash bath in addition to the ability to prevent redeposition. The carboxylic acid polymers are homopolymers or copolymers of acrylic acid, methacrylic acid, itaconic acid, and the like, and the copolymers are preferably copolymers of the above monomers and maleic acid, and preferably have a molecular weight of several thousand to 100,000. In addition to the above carboxylic acid polymers, polymers such as polyglycidyl acid salts, cellulose derivatives such as carboxymethylcellulose, and aminocarboxylic acid polymers such as polyaspartic acid are also preferred because they have the ability to capture metal ions, dispersant, and prevent redeposition.

[0087] (5) Bleach For example, a bleaching agent such as hydrogen peroxide or percarbonate is preferably blended at 1 to 10% by mass. When a bleaching agent is used, tetraacetylethylenediamine (TAED) or a bleaching activator such as that described in JP-A-6-316700 can be blended at 0.01 to 10% by mass.

[0088] (6) Fluorescent agent The fluorescent agent used in the cleaning composition may be a biphenyl type fluorescent agent (such as Tinopal CBS-X) or a stilbene type fluorescent agent (such as DM type fluorescent dye). The fluorescent agent is preferably blended in an amount of 0.001 to 2% by mass.

[0089] (7) Other ingredients The detergent composition may contain solvents, builders, softeners, reducing agents (such as sulfites), foam inhibitors (such as silicones), fragrances, antibacterial and antifungal agents (such as Proxel [trade name] and benzoic acid), and other additives known in the field of laundry detergents.

[0090] Examples of the solvent include monohydric alcohols having 1 to 3 carbon atoms; polyhydric alcohols having 2 to 4 carbon atoms; di- or trialkylene glycols having 2 to 4 carbon atoms in the alkylene glycol unit; and monoalkoxy (methoxy, ethoxy, propoxy, butoxy), phenoxy, or benzooxy ethers of di- or tetraalkylene glycols having 2 to 4 carbon atoms in the alkylene glycol unit. The solvent is preferably a water-soluble organic solvent having 2 or more carbon atoms, preferably 3 or more carbon atoms, and 10 or less carbon atoms, preferably 8 or less carbon atoms. Here, the water-soluble organic solvent refers to a solvent having an octanol / water partition coefficient (LogPow) of 3.5 or less. Specifically, ethanol, isopropyl alcohol, ethylene glycol, propylene glycol, glycerin, isoprene glycol, diethylene glycol, dipropylene glycol, propylene glycol monomethyl ether, propylene glycol monoethyl ether, diethylene glycol monobutyl ether (also called butyl diglycol, etc.), phenoxyethanol, phenoxytriethylene glycol, and phenoxyisopropanol are listed. The solvent is preferably selected from ethanol, propylene glycol, dipropylene glycol, diethylene glycol monobutyl ether, phenoxyethanol, phenyl glycol, and phenoxyisopropanol. The solvent is preferably one having an alkoxy group, and further preferably contains one or more selected from monoalkoxy, phenoxy, or benzooxy ether of di- or tetraalkylene glycol having 2 to 4 carbon atoms in the alkylene glycol unit, and more preferably contains diethylene glycol monobutyl ether.

[0091] The detergent composition can be produced by combining the lipase variant of the present invention obtained by the above-mentioned method with the above-mentioned known detergent components in a conventional manner. The form of the detergent can be selected according to the application, and can be, for example, liquid, powder, granule, paste, solid, etc.

[0092] The detergent composition thus obtained can be used as a clothing detergent, a dishwashing detergent, a bleaching agent, a detergent for cleaning hard surfaces, a drain cleaner, a denture cleaner, a germicidal cleaner for medical instruments, etc., but is preferably used as a clothing detergent or a dishwashing detergent, and more preferably used as a laundry detergent (laundry laundry detergent), a dishwashing agent for hand washing, or a detergent for automatic dishwashers. The detergent composition is suitable for use at temperatures of 40° C. or lower, 35° C. or lower, 30° C. or lower, or 25° C. or lower, and 5° C. or higher, 10° C. or higher, or 15° C. or higher. The detergent composition is also suitable for use at temperatures of 5 to 40° C., 10 to 35° C., 15 to 30° C., or 15 to 25° C. Preferred modes of use include use in low-temperature (15 to 30° C.) washing in a laundry and low-temperature (15 to 30° C.) washing in an automatic dishwashing machine.

[0093] By using the detergent composition of the present invention, it is possible to clean objects requiring stain removal (e.g., clothes, tableware, hard surfaces, drainage pipes, dentures, medical instruments, etc.), i.e., to remove stains. Such a cleaning method includes a step of contacting the object requiring stain removal with the detergent composition of the present invention. Preferably, the stain is an oil stain.

[0094] In the cleaning method of the present invention, the object to be cleaned may be brought into contact with the detergent composition by immersing the object in water in which the detergent composition has been dissolved, or by directly applying the detergent composition to the object to be cleaned. In the method of the present invention, the object to be cleaned after the immersion or application of the detergent composition may be further washed by hand washing, scrubbing with a sponge, or in a washing machine, but this is not necessarily required.

[0095] In relation to the above-described embodiment, the present invention further discloses the following aspects. <1> A lipase variant consisting of an amino acid sequence having at least 75% identity to the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8, and having an amino acid residue other than I at the position corresponding to position 44 in the numbering of SEQ ID NO: 2. <2> having A, G, S, T or V at the position corresponding to position 44 in SEQ ID NO:2; <1> The lipase variant described in <3> Preferably, the amino acid sequence contains one or more amino acid residues selected from the group consisting of the following (a) to (e), more preferably two or more amino acid residues, even more preferably three or more amino acid residues, even more preferably four or more amino acid residues, and even more preferably all amino acid residues selected from the group consisting of the following (a) to (e): <1> or <2> Lipase variants according to (a) an amino acid residue other than L at a position corresponding to 120 in the numbering of SEQ ID NO:2; (b) an amino acid residue other than S at a position corresponding to 130 in the numbering of SEQ ID NO:2; (c) an amino acid residue other than A at a position corresponding to 134 in the numbering of SEQ ID NO:2; (d) an amino acid residue other than L at a position corresponding to position 136 in the numbering of SEQ ID NO:2; and (e) an amino acid residue other than S at a position corresponding to position 137 in the numbering of SEQ ID NO:2. <4> Among the amino acid residues (a) to (e), the amino acid sequence has only the amino acid residue (d), and when the amino acid residue is M, the amino acid sequence has at least 75% identity with the amino acid sequence of SEQ ID NO: 2, 4 or 6. <3> The lipase variant described in <5> The (a) to (e) are the following (a') to (e'), respectively: <3> or <4> Lipase variants according to (a') A, F, G, H, I, K, M, N, Q, R, S, T, V, W or Y at a position corresponding to 120 in the numbering of SEQ ID NO:2; (b') A, C, E, F, G, H, I, K, L, M, Q, R, T, V, W, or Y at a position corresponding to 130 in the numbering of SEQ ID NO:2; (c') C, D, E, G, I, T, or V at a position corresponding to position 134 in the numbering of SEQ ID NO:2; (d') A, C, E, F, G, H, I, M, S, T, V, or W at a position corresponding to position 136 in the numbering of SEQ ID NO:2; and (e') C, N, or T at a position corresponding to position 137 in the numbering of SEQ ID NO:2. <6> When only M of (d') among (a') to (e') is present, the amino acid sequence has at least 75% identity with the amino acid sequence of SEQ ID NO: 2, 4 or 6. <5> The lipase variant described in <7> Having any of the amino acid residues No. 1 to 57 in Table 2 below. <1> ~ <6> 2. The lipase variant according to claim 1 .

[0096] [Table 2]

[0097] <8> a lipase variant consisting of an amino acid sequence having at least 75% identity with the amino acid sequence of SEQ ID NO: 2 and having any of the amino acid residues No. 1 to 23, 25 to 47, 49 and 50 in Table 2; a lipase variant consisting of an amino acid sequence having at least 75% identity with the amino acid sequence of SEQ ID NO: 4 and having any of the amino acid residues No. 4, 24, 44, 49 and 52 to 57 in Table 2; a lipase variant consisting of an amino acid sequence having at least 75% identity with the amino acid sequence of SEQ ID NO: 6 and having any of the amino acid residues No. 4, 44 and 50 in Table 2; or a lipase variant consisting of an amino acid sequence having at least 75% identity with the amino acid sequence of SEQ ID NO: 8 and having any of the amino acid residues No. 1 to 5, 24, 44, 48 and 51 in Table 2. <7> The lipase variant described in

[0098] <9> <1> ~ <8> A polynucleotide encoding the lipase variant according to any one of claims 1 to 5. <10> <9> A vector or DNA fragment comprising the polynucleotide described in . <11> <10> A transformed cell containing the vector or DNA fragment described in . <12> It is a microorganism, <11> A transformed cell according to claim 1. <13> Escherichia coli or Bacillus bacteria, preferably Bacillus bacteria, more preferably Bacillus subtilis; <12> A transformed cell according to claim 1. <14> <11> ~ <13> A method for producing a lipase variant, comprising a step of culturing the transformed cell described in any one of the above.

[0099] <15> <1> ~ <8> 13. A cleaning composition comprising the lipase variant according to any one of claims 1 to 12. <16> The composition further contains a sulfosuccinate ester or a salt thereof, preferably a branched alkyl sulfosuccinate ester having a branched alkyl group having from 9 to 12 carbon atoms or a salt thereof, more preferably a branched alkyl sulfosuccinate ester having a branched alkyl group having 9 or 10 carbon atoms or a salt thereof, and even more preferably a branched alkyl sulfosuccinate ester having a branched alkyl group having 10 carbon atoms or a salt thereof. <15> The cleaning composition according to claim 1. <17> The sulfosuccinic acid diester or a salt thereof, preferably a sulfosuccinic acid dibranched alkyl ester or a salt thereof, each of which has 9 to 12 carbon atoms as its branched alkyl group, more preferably a sulfosuccinic acid dibranched alkyl ester or a salt thereof, each of which has 9 or 10 carbon atoms as its branched alkyl group, even more preferably a sulfosuccinic acid dibranched alkyl ester or a salt thereof, each of which has 10 carbon atoms as its branched alkyl group, and even more preferably a sulfosuccinic acid dibranched alkyl ester or a salt thereof, each of which has 10 carbon atoms as its branched alkyl group, and even more preferably bis-(2-propylheptyl)sulfosuccinic acid or a salt thereof, <15> The cleaning composition according to claim 1. <18> A laundry detergent or dishwashing detergent, <15> ~ <17> The cleaning composition according to any one of the above. <19> It is a powder or liquid. <15> ~ <18> The cleaning composition according to any one of the above. <20> Used at low temperatures, <15> ~ <19> The cleaning composition according to any one of the above. <21> Used at 40°C or less, 35°C or less, 30°C or less, 25°C or less, and 5°C or more, 10°C or more, 15°C or more, or used at 5 to 40°C, 10 to 35°C, 15 to 30°C, 15 to 25°C, <20> The cleaning composition according to claim 1.

[0100] <22> <15> ~ <21> 2. A method for cleaning stains using the cleaning agent composition according to claim 1. <23> The item to be cleaned <15> ~ <21> The cleaning composition according to any one of the preceding claims, <22> The method according to <24> For the preparation of cleaning compositions <1> ~ <8> 2. Use of a lipase variant according to any one of claims 1 to 11. <25> For cleaning dirt <1> ~ <8> 2. Use of a lipase variant according to any one of claims 1 to 11.

[0101] <26> A method for producing a lipase mutant, comprising a step of replacing an amino acid residue at a position corresponding to position 44 in the numbering of SEQ ID NO: 2 with an amino acid residue other than I in a polypeptide having lipase activity and consisting of an amino acid sequence having at least 75% identity to the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8. <27> The substitution of the amino acid residue at the position corresponding to position 44 in the numbering of SEQ ID NO:2 is a substitution of A, G, S, T or V. <26> The method according to <28> The method further includes preferably one or more steps, more preferably two or more steps, even more preferably three or more steps, even more preferably four or more steps, and even more preferably all steps selected from the group consisting of the following (i) to (v): <26> or <27> Method to do this: (i) substituting an amino acid residue at a position corresponding to position 120 in the numbering of SEQ ID NO:2 with an amino acid residue other than L; (ii) substituting an amino acid residue at a position corresponding to position 130 in the numbering of SEQ ID NO:2 with an amino acid residue other than S; (iii) substituting the amino acid residue at the position corresponding to position 134 in the numbering of SEQ ID NO:2 with an amino acid residue other than A; (iv) substituting an amino acid residue at a position corresponding to position 136 in the numbering of SEQ ID NO:2 with an amino acid residue other than L; and (v) substituting an amino acid residue at a position corresponding to position 137 in the numbering of SEQ ID NO:2 with an amino acid residue other than S. <29> The method includes carrying out only step (iv) among steps (i) to (v), and when the substitution is made with M, the polypeptide has an amino acid sequence having at least 75% identity with the amino acid sequence of SEQ ID NO: 2, 4 or 6. <28> The method according to <30> The (i) to (v) are the following (i') to (v'), respectively: <28> or <29> Method to do this: (i') substituting the amino acid residue at the position corresponding to position 120 in the numbering of SEQ ID NO:2 with A, F, G, H, I, K, M, N, Q, R, S, T, V, W, or Y; (ii') substituting the amino acid residue at the position corresponding to position 130 in the numbering of SEQ ID NO:2 with A, C, E, F, G, H, I, K, L, M, Q, R, T, V, W, or Y; (iii') substituting the amino acid residue at the position corresponding to position 134 in SEQ ID NO:2 with C, D, E, G, I, T, or V; (iv') substituting the amino acid residue at the position corresponding to position 136 in the numbering of SEQ ID NO:2 with A, C, E, F, G, H, I, M, S, T, V, or W; and (v') substituting the amino acid residue at the position corresponding to position 137 in the numbering of SEQ ID NO:2 with C, N or T. <31> The method includes carrying out only step (iv') among steps (i') to (v'), and when the step is replaced with M, the polypeptide has an amino acid sequence having at least 75% identity with the amino acid sequence of SEQ ID NO: 2, 4 or 6. <30> The method according to <32> The substitution is a substitution with any of the amino acid residues No. 1 to 57 in Table 2. <26> ~ <31> 2. The method according to claim 1 . <33> the substitution is a substitution of any one of the amino acid residues No. 1 to 23, 25 to 47, 49, and 50 in Table 2 in a polypeptide having an amino acid sequence having at least 75% identity with the amino acid sequence of SEQ ID NO: 2 and having lipase activity, or a substitution of any one of the amino acid residues No. 4, 24, 44, 49, and 52 to 57 in Table 2 in a polypeptide having an amino acid sequence having at least 75% identity with the amino acid sequence of SEQ ID NO: 4 and having lipase activity, or a substitution of any one of the amino acid residues No. 4, 44, and 50 in Table 2 in a polypeptide having an amino acid sequence having at least 75% identity with the amino acid sequence of SEQ ID NO: 6 and having lipase activity, or a substitution of any one of the amino acid residues No. 1 to 5, 24, 44, 48, and 51 in Table 2 in a polypeptide having an amino acid sequence having at least 75% identity with the amino acid sequence of SEQ ID NO: 8 and having lipase activity. <32> The method according to EXAMPLES

[0102] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples.

[0103] (1) Construction of lipase expression plasmid Using the VHH expression plasmid of SEQ ID NO: 26 containing the Bacillus subtilis spoVG gene-derived promoter described in WO2021 / 153129 as a template, the following lipase expression plasmid was constructed by replacing each lipase gene by In-Fusion reaction on the full-length ORF containing the VHH gene. Plasmids pHY-CnLip, pHY-EbLip, pHY-Ag1Lip, pHY-Lipr138, and pHY-Lipr139 were constructed from artificially synthesized lipase genes CnLip, EbLip, Ag1Lip, Lipr138, and Lipr139 (encoding the polynucleotides of SEQ ID NOs: 1, 3, 5, 9, and 11, respectively, and the amino acid sequences of SEQ ID NOs: 2, 4, 6, 10, and 12, respectively). Plasmid pHY-amyEsig-TLL was constructed by replacing the full-length ORF containing the Lipr139 gene of plasmid pHY-Lipr139 with an artificially synthesized gene for lipase TLL (polynucleotide of SEQ ID NO: 13, encoding the amino acid sequence of SEQ ID NO: 14) to which a signal sequence derived from Bacillus subtilis amyE (polynucleotide of SEQ ID NO: 15, encoding the amino acid sequence of SEQ ID NO: 16) was linked at the N-terminus by an In-Fusion reaction. Plasmid pHY-S237 described in Example 7 of WO2006 / 068148 was used as a template to construct plasmid pHY-CspLip by replacing the entire ORF of the alkaline cellulase gene with an artificially synthesized CspLip gene (encoding the polynucleotide sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 8) by an In-Fusion reaction. Mutations were introduced into each lipase by site-directed mutagenesis using PCR with complementary primer pairs (Zheng, Lei, Ulrich Baumann, and Jean-Louis Reymond. Nucleic Acids Research 32.14(2004):e115-e115.).

[0104] (2) Preparation of lipase solution The lipase expression plasmid was introduced into a Bacillus subtilis strain by the protoplast method, and the strain was cultured in 2xL-maltose medium (2% tryptone, 1% yeast extract, 1% NaCl, 7.5% maltose, 7.5 ppm manganese sulfate pentahydrate, 0.04% calcium chloride dihydrate, 15 ppm tetracycline; % is (w / v)%) at 30°C for 3 days, and the culture supernatant containing lipase was recovered by centrifugation. The culture supernatant was mixed with an equal volume of 2×Laemmli Sample buffer (Bio-Rad) containing 100 mM DTT and incubated at 100° C. for 5 minutes. 6 μL of each sample was applied to each lane of Any kDTM Mini-PROTEAN® TGX Stain-FreeTM Protein Gel (Bio-Rad), and electrophoresis was performed at a constant voltage of 200 V. The gel after electrophoresis was photographed using a Chemi Doc MP Imaging system (Bio-Rad), and the band intensity of the band estimated to be lipase was quantified. A calibration curve was created using the band intensity of BSA, and the concentration of each lipase in the culture supernatant was calculated.

[0105] (3) Activity measurement for dispersed substrates in model cleaning solutions 4-Nitrophenyl octanoate (SIGMA) was used as a substrate. Lipase activity can be determined by measuring the rate of increase in absorbance associated with the release of 4-nitrophenol by the action of lipase. Model cleaning solution (Table 3) or 20 mM Tris-HCl (pH 7.0) was used as a test solution, and 4-nitrophenyl octanoate was added to each test solution at a final concentration of 2 mM and mixed to use as a substrate solution. 2 μL of culture supernatant containing lipase diluted 200-fold with 20 mM Tris-HCl (pH 7.0) and 100 μL of each substrate solution were mixed in each well of a 96-well assay plate, and the change in absorbance (OD / min) at 405 nm at 30°C was measured. The difference ΔOD / min from the blank (sample without enzyme addition) was calculated. The degree of improvement of the mutants was calculated as follows. For each lipase, the ΔOD / min when the model cleaning solution was used as the test solution was divided by the ΔOD / min when 20 mM Tris-HCl (pH 7.0) was used as the test solution to calculate the value (ΔOD / min ratio). The ΔOD / min ratio is a relative measure of the activity retention rate in the model cleaning solution compared to in buffer, and the smaller the value, the more the activity is inhibited in the model cleaning solution. The value of the ΔOD / min ratio of the mutant divided by the ΔOD / min ratio of the wild-type lipase was calculated as the degree of improvement of the mutant. A mutant with an improvement degree of more than 1 has improved activity inhibition in the model cleaning solution compared to the wild-type lipase. The relative activity (%) was calculated as follows. 2 μL of 20 mM Tris-HCl (pH 7.0) containing 47 to 1500 μM of 4-nitrophenol was mixed with the model cleaning solution or 100 μL of 20 mM Tris-HCl (pH 7.0), and the absorbance at 405 nm was measured to create a calibration curve. The release rate of 4-nitrophenol per minute (μM / min) was calculated from the calibration curve and ΔOD / min value of each test solution. For each lipase, the release rate (μM / min) when the model cleaning solution was used as the test solution was divided by the release rate (μM / min) when 20 mM Tris-HCl (pH 7.0) was used as the test solution, and the value multiplied by 100 was used as the relative activity (%).

[0106] [Table 3]

[0107] (4) Cleaning evaluation in model cleaning solutions The ability to remove triglycerides from hard surfaces was evaluated as cleaning power. Beef tallow (SIGMA, 03-0660) and rapeseed oil (SIGMA, 23-0450) were mixed in a weight ratio of 9:1, dissolved in three times the amount of chloroform, and colored with 0.2 wt% Sudan III to prepare a model soil. 10 μL of the model soil was dropped onto the bottom of each well of a 96-well polypropylene deep well plate, and the chloroform was evaporated and dried to prepare a soiled plate. The model cleaning solution shown in Table 3 was added with lipase solution to prepare the cleaning solution. 300 μL of the cleaning solution was slowly added to the soiled plate and left to stand at room temperature (about 22 °C) for 10 to 30 minutes to perform immersion cleaning. 100 μL of the cleaning solution was taken and transferred to a new 96-well plate without touching the dirt at the bottom. The absorbance at 500 nm (A500) was measured to quantify the amount of Sudan III in the model dirt solubilized in the cleaning solution by immersion cleaning. A500 corresponds to the amount of oil released into the cleaning solution and can be used as an index of cleaning power. The enzyme effect (ΔA500) of cleaning power was calculated by subtracting the A500 of the cleaning solution containing each lipase from the A500 of the cleaning solution to which 20 mM Tris-HCl (pH 7.0) was added instead of lipase.

[0108] (5) Activity measurement of wild-type lipase in model washing solution The relative activity of each wild-type lipase in a model washing solution relative to that in a buffer was measured using the method described in (3). The activities of Lipr138, whose suitability for washing is disclosed in Patent Document 2, Lipr139, whose suitability for washing is disclosed in Patent Document 1, and CnLip against a dispersed substrate were all significantly inhibited in the model washing solution (Table 4).

[0109] [Table 4]

[0110] (6) Activity measurement of CnLip mutants with substitution at position 44 in model washing solution The relative activity of each CnLip mutant in a model washing solution relative to that in a buffer was measured using the method described in (3). Substitution of position 44 with A, G, S, T, or V significantly improved the inhibition of activity in the model washing solution (Table 5).

[0111] [Table 5]

[0112] (7) Activity measurement of CnLip multiple mutants in model washing solution For the CnLip mutants with a substitution at position 44, mutations at other positions were searched for to further improve the activity inhibition in the model washing solution. The relative activity in the model washing solution to that in the buffer was measured for each CnLip mutant by the method described in (3). As a result, the activity inhibition in the model washing solution was improved by substitutions at positions 120, 130, 134, 136, or 137 (Table 6). Therefore, the relative activity in the model washing solution to that in the buffer was measured for multiple CnLip mutants containing a substitution at position 44 and substitutions of one or more residues selected from positions 120, 130, 134, 136, and 137 by the method described in (3). The activity inhibition in the model washing solution was further improved by combining the substitution at position 44 with substitutions of one or more residues selected from positions 120, 130, 134, 136, and 137 (Table 7).

[0113] [Table 6]

[0114] [Table 7]

[0115] (8) Evaluation of the cleaning ability of CnLip mutants The cleaning power of each wild-type lipase and CnLip mutant in a model cleaning solution was evaluated using the method described in (4). The lipase concentration of the culture supernatant was diluted with 20 mM Tris-HCl (pH 7.0) to 200 mg / L and used as a lipase solution. 1 / 50 of the lipase solution was added to the model cleaning solution and used for immersion cleaning for 10 or 30 minutes. The results of the 30-minute cleaning are shown in Figures 1 to 3, and the results of the 10-minute cleaning are shown in Figure 4. The mutants, which showed reduced inhibition of activity against dispersed substrates in the model cleaning solution and surfactant solution, showed significantly improved removal performance of triglycerides from hard surfaces in the model cleaning solution. In a similar 30-minute cleaning test, the cleaning power of the CnLip mutants was compared with that of Lipr138, Lipr139, and TLL. As a result, the CnLip mutants with improved cleaning power showed significantly higher cleaning power than Lipr138, Lipr139, and TLL (Figure 5).

[0116] (9) Activity measurement of EbLip, Ag1Lip, and CspLip mutants in model washing solutions (3) The relative activity in model washing solution was measured for each mutant of EbLip, Ag1Lip, and CspLip in buffer by the method described above (Table 8). As with CnLip, the inhibition of activity in model washing solution was significantly improved by substitution at position 44 for EbLip and Ag1Lip, or by combining substitution at position 44 with substitution at one or more residues selected from positions 120, 130, 134, 136, and 137, and by substitution at position 45 (corresponding to position 44 of CnLip) for CspLip, or by combining substitution at position 45 with substitution at one or more residues selected from positions 121, 131, 135, 137, and 138 (corresponding to positions 120, 130, 134, 136, and 137 of CnLip, respectively).

[0117] [Table 8]

[0118] (10) Evaluation of the cleaning ability of CspLip mutants The cleaning power of each CspLip mutant in a model cleaning solution was evaluated using the method described in (4). The culture supernatant containing lipase was subjected to SDS-PAGE, and the lipase concentration was quantified by comparing the band intensity with that of BSA of known concentration. The lipase concentration of the culture supernatant was diluted to 200 mg / L and used as a lipase solution. 1 / 50 of the lipase was added to the model cleaning solution and subjected to immersion cleaning for 10 minutes. The results are shown in Figure 6. As with CnLip, the mutants with reduced activity inhibition against dispersed substrates in the model cleaning solution and surfactant solution showed significantly improved removal performance of triglycerides from hard surfaces in the model cleaning solution.

Claims

1. A lipase variant consisting of an amino acid sequence having at least 75% identity with the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8, and having an amino acid residue other than isoleucine at a position corresponding to position 44 in the numbering of SEQ ID NO:

2.

2. The lipase variant according to claim 1 , which has alanine, glycine, serine, threonine or valine at the position corresponding to position 44 in the numbering of SEQ ID NO:

2.

3. The lipase variant of claim 1, having one or more amino acid residues selected from the group consisting of (a) to (e) below: (a) an amino acid residue other than leucine at a position corresponding to position 120 in the numbering of SEQ ID NO:2; (b) an amino acid residue other than serine at a position corresponding to position 130 in the numbering of SEQ ID NO:2; (c) an amino acid residue other than alanine at a position corresponding to position 134 in the numbering of SEQ ID NO:2; (d) an amino acid residue other than leucine at a position corresponding to position 136 in the numbering of SEQ ID NO:2; and (e) an amino acid residue other than serine at a position corresponding to position 137 in the numbering of SEQ ID NO:

2.

4. The lipase variant of claim 2, having one or more amino acid residues selected from the group consisting of the following (a) to (e): (a) an amino acid residue other than leucine at a position corresponding to position 120 in the numbering of SEQ ID NO:2; (b) an amino acid residue other than serine at a position corresponding to position 130 in the numbering of SEQ ID NO:2; (c) an amino acid residue other than alanine at a position corresponding to position 134 in the numbering of SEQ ID NO:2; (d) an amino acid residue other than leucine at a position corresponding to position 136 in the numbering of SEQ ID NO:2; and (e) an amino acid residue other than serine at a position corresponding to position 137 in the numbering of SEQ ID NO:

2.

5. The lipase variant according to claim 3, wherein (a) to (e) are the following (a') to (e'), respectively: (a') alanine, phenylalanine, glycine, histidine, isoleucine, lysine, methionine, asparagine, glutamine, arginine, serine, threonine, valine, tryptophan, or tyrosine at a position corresponding to position 120 in the numbering of SEQ ID NO:2; (b') alanine, cysteine, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, glutamine, arginine, threonine, valine, tryptophan, or tyrosine at a position corresponding to position 130 in the numbering of SEQ ID NO:2; (c') a cysteine, aspartic acid, glutamic acid, glycine, isoleucine, threonine, or valine at a position corresponding to position 134 in the numbering of SEQ ID NO:2; (d') an alanine, cysteine, glutamic acid, phenylalanine, glycine, histidine, isoleucine, methionine, serine, threonine, valine, or tryptophan at a position corresponding to position 136 in the numbering of SEQ ID NO:2; and (e') a cysteine, asparagine, or threonine at a position corresponding to position 137 in the numbering of SEQ ID NO:

2.

6. The lipase variant according to claim 4, wherein (a) to (e) are the following (a') to (e'), respectively: (a') alanine, phenylalanine, glycine, histidine, isoleucine, lysine, methionine, asparagine, glutamine, arginine, serine, threonine, valine, tryptophan, or tyrosine at a position corresponding to position 120 in the numbering of SEQ ID NO:2; (b') alanine, cysteine, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, glutamine, arginine, threonine, valine, tryptophan, or tyrosine at a position corresponding to position 130 in the numbering of SEQ ID NO:2; (c') a cysteine, aspartic acid, glutamic acid, glycine, isoleucine, threonine, or valine at a position corresponding to position 134 in the numbering of SEQ ID NO:2; (d') an alanine, cysteine, glutamic acid, phenylalanine, glycine, histidine, isoleucine, methionine, serine, threonine, valine, or tryptophan at a position corresponding to position 136 in the numbering of SEQ ID NO:2; and (e') a cysteine, asparagine, or threonine at a position corresponding to position 137 in the numbering of SEQ ID NO:

2.

7. A polynucleotide encoding the lipase variant according to any one of claims 1 to 6.

8. A vector or DNA fragment comprising the polynucleotide of claim 7.

9. A transformed cell containing the vector or DNA fragment according to claim 8.

10. The transformed cell of claim 9, which is a microorganism.

11. A detergent composition comprising the lipase variant according to any one of claims 1 to 6.

12. The detergent composition according to claim 11, which is a laundry detergent or a dish detergent.

13. A method for cleaning stains, comprising using the cleaning agent composition according to claim 11.

14. A method for producing a lipase variant, comprising a step of substituting an amino acid residue at a position corresponding to position 44 in the numbering of SEQ ID NO: 2 with an amino acid residue other than isoleucine in a polypeptide having lipase activity and consisting of an amino acid sequence having at least 75% identity to the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8.

15. The method of claim 14, further comprising one or more steps selected from the group consisting of: (i) to (v) (i) substituting the amino acid residue at the position corresponding to position 120 in the numbering of SEQ ID NO:2 with an amino acid residue other than leucine; (ii) substituting the amino acid residue at the position corresponding to position 130 in the numbering of SEQ ID NO:2 with an amino acid residue other than serine; (iii) substituting the amino acid residue at the position corresponding to position 134 in the numbering of SEQ ID NO:2 with an amino acid residue other than alanine; (iv) substituting the amino acid residue at the position corresponding to position 136 in the numbering of SEQ ID NO:2 with an amino acid residue other than leucine; and (v) substituting the amino acid residue at the position corresponding to position 137 in the numbering of SEQ ID NO:2 with an amino acid residue other than serine.

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