Lipase variant

Abstract

To provide a lipase variant exhibiting excellent stability.SOLUTION: The present invention provides a lipase variant, which is a variant of a parent lipase, comprising substitution of amino acid residues at one or more positions selected from positions corresponding to L16, G22, Y29, Y43, A45, T46, L50, S120, K123, R126, T134, L142, A145, G156, K174, A181, Q186, K188, L190, D191, L208, H209, M212, L215, V220, A221, L223, A224, F225, L228, R233, F234, S256, V260, L266, V269, N272, T274, K278, and R286 in numbering according to SEQ ID NO: 4, wherein the parent lipase or the lipase variant has at least 80% sequence identity to an amino acid sequence represented by SEQ ID NO: 4 or 6.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to lipase mutants.

Background Art

[0002] Lipases are useful in various applications such as laundry detergents, dishwashing detergents, oil processing, pulp treatment, feeds, and synthesis of pharmaceutical intermediates. In cleaning, lipases contribute to the removal of oil-containing stains by hydrolyzing ester bonds in lipids to produce fatty acids.

[0003] Lipases for cleaning have been employed in detergent compositions for removing oily stains. Patent Document 1 describes a method of washing oil stains by bringing a detergent containing an alkyl sulfosuccinate into contact with stains together with lipase and then leaving it without applying an external force. Also, as described in Patent Document 2, a detergent composition for dishwashing may also be used for cleaning around sinks made of stainless steel or resin. To impart a function of cleaning scale adhering to the sink area in the kitchen, it is conceivable to blend a chelating agent such as citric acid at a high concentration. Such detergent compositions are characterized by having a larger amount of water and containing more solvent than surfactant compared to general detergents.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] The stability of the incorporated lipase was unknown in an environment harsh for enzymes in a detergent composition having a large amount of water and containing more solvent than surfactant as compared with a general detergent, but as a result of investigations by the present inventors, it became clear that the stability was remarkably reduced as compared with when incorporated into a general detergent. Therefore, the present invention relates to providing a lipase showing excellent stability.

Means for Solving the Problems

[0006] The present inventors obtained a lipase mutant having improved stability in the presence of a large amount of water and / or more solvent than surfactant as compared with the parent lipase.

[0007] That is, the present invention relates to the following 1) to 6). 1) A mutant of a parent lipase, comprising substitution of an amino acid residue at one or more positions selected from the positions corresponding to positions L16, G22, Y29, Y43, A45, T46, L50, S120, K123, R126, T134, L142, A145, G156, K174, A181, Q186, K188, L190, D191, L208, H209, M212, L215, V220, A221, L223, A224, F225, L228, R233, F234, S256, V260, L266, V269, N272, T274, K278 and R286 in the numbering of SEQ ID NO: 4, wherein the parent lipase or lipase mutant has at least 80% sequence identity to the amino acid sequence represented by SEQ ID NO: 4 or 6. 2) A polynucleotide encoding the lipase mutant according to 1). 3) A vector or DNA fragment containing the polynucleotide according to 2). 4) A transformed cell containing the vector or DNA fragment according to 3). 5) A detergent composition containing the lipase mutant according to 1). 6) A method for improving the stability of lipase, comprising a step of substituting amino acid residues at one or more positions selected from positions corresponding to positions L16, G22, Y29, Y43, A45, T46, L50, S120, K123, R126, T134, L142, A145, G156, K174, A181, Q186, K188, L190, D191, L208, H209, M212, L215, V220, A221, L223, A224, F225, L228, R233, F234, S256, V260, L266, V269, N272, T274, K278 and R286 in the numbering of SEQ ID NO: 4, wherein the lipase or the lipase after amino acid residue substitution has at least 80% sequence identity to the amino acid sequence represented by SEQ ID NO: 4 or 6.

Advantages of the Invention

[0008] The lipase variant of the present invention has improved stability compared to the parent lipase. Such a lipase variant can also be suitably formulated in a detergent composition containing a larger amount of solvent than a large amount of water and / or surfactant.

Modes for Carrying Out the Invention

[0009] All patent documents, non-patent documents, and other publications cited in this specification are hereby incorporated by reference in their entirety.

[0010] In this specification, "lipase" refers to triacylglycerol lipase (EC 3.1.1.3) and means a group of enzymes having an activity of hydrolyzing an ester bond in a lipid to produce a fatty acid. Lipase activity can be determined by measuring the rate of increase in absorbance accompanying the release of 4-nitrophenol by hydrolysis of 4-nitrophenyl butyrate. The specific procedure for measuring lipase activity is described in detail in the examples below.

[0011] In this 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 homology analysis (Search homology) program of the genetic information processing software GENETYX Ver. 12 with Unit size to compare (ktup) set to 2.

[0012] In this specification, "at least 80% identity" with respect to an amino acid sequence or a nucleotide sequence means an identity of 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 93% or more, still more preferably 94% or more, still more preferably 95% or more, still more preferably 96% or more, still more preferably 97% or more, still more preferably 98% or more, still more preferably 99% or more, and still more preferably 99.5% or more.

[0013] As used herein, the "corresponding position" on an amino acid sequence or nucleotide sequence can be determined by aligning the target sequence and a reference sequence (for example, the amino acid sequence shown in SEQ ID NO: 4) to give the 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, the alignment can be carried out by 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 is a revised version of Clustal W, can also be used. Clustal W, Clustal W2, and Clustal omega can be used, for example, on the Clustal website [www.clustal.org] operated by University College Dublin, the website of the European Bioinformatics Institute (EBI [www.ebi.ac.uk / index.html]), or the website of the DNA Data Bank of Japan (DDBJ [www.ddbj.nig.ac.jp / searches-j.html]) operated by the National Institute of Genetics. The position of the target sequence aligned with any position of the reference sequence by the above alignment is regarded as the "corresponding position" to the any position.

[0014] A person skilled in the art can further finely adjust the alignment of the amino acid sequences obtained above so as to optimize it. Such optimal alignment is preferably determined in consideration of the similarity of the amino acid sequences, the frequency of inserted gaps, etc. Here, the similarity of amino acid sequences refers to the ratio (%) of the number of positions where identical or similar amino acid residues exist in both sequences when two amino acid sequences are aligned to the total number of amino acid residues in the full length. Similar amino acid residues mean amino acid residues that have similar properties to each other in terms of polarity and charge among the 20 amino acids constituting proteins and cause so-called conservative substitutions. Such groups of similar amino acid residues are well known to those skilled in the art, and examples include, but are not limited to, arginine and lysine or glutamine; glutamic acid and aspartic acid or glutamine; serine and threonine or alanine; glutamine and asparagine or arginine; leucine and isoleucine, etc.

[0015] In this specification, "amino acid residue" means the 20 amino acid residues constituting proteins, alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0016] In this specification, the description of the position of amino acids and variants is represented as follows using the recognized one-letter amino acid abbreviations of IUPAC. The amino acid at a predetermined position is represented as [amino acid, position]. For example, valine at position 220 is shown as "V220". Regarding the "substitution" of an amino acid, it is represented as [original amino acid, position, substituted amino acid]. For example, the substitution of valine at position 220 with histidine is denoted as "V220H". Variants containing multiple modifications are represented by the plus sign ("+"). For example, "V220H+N272G" represents the substitution of valine at position 220 with histidine and the substitution of asparagine at position 272 with glycine, respectively. When different modifications can be introduced at one position, the different modifications are separated by a slash (" / "). For example, "G22C / M" represents the substitution of glycine at position 22 with cysteine or methionine.

[0017] As used herein, the "operable linkage" of a control region such as a promoter and a gene means that the gene and the control region are linked such that the gene can be expressed under the control of the control region. The procedure for "operable linkage" of a gene and a control region is well known to those skilled in the art.

[0018] As used herein, "upstream" and "downstream" with respect to a gene refer to upstream and downstream in the transcription direction of the gene. For example, a "gene located downstream of a promoter" means that the gene is present on the 3'-side of the promoter in the DNA sense strand, and upstream of the gene means the region on the 5'-side of the gene in the DNA sense strand.

[0019] <1. Lipase Variant> The lipase variant of the present invention is a variant of a parent lipase, which contains substitution(s) of amino acid residue(s) at one or more positions selected from the positions corresponding to positions L16, G22, Y29, Y43, A45, T46, L50, S120, K123, R126, T134, L142, A145, G156, K174, A181, Q186, K188, L190, D191, L208, H209, M212, L215, V220, A221, L223, A224, F225, L228, R233, F234, S256, V260, L266, V269, N272, T274, K278 and R286 in SEQ ID NO: 4 numbering, and the parent lipase or the lipase variant has at least 80% sequence identity to the amino acid sequence represented by SEQ ID NO: 4 or 6.

[0020] That is, the "variant" means a polypeptide having lipase activity in which one or more amino acid residues at positions selected from the positions corresponding to positions L16, G22, Y29, Y43, A45, T46, L50, S120, K123, R126, T134, L142, A145, G156, K174, A181, Q186, K188, L190, D191, L208, H209, M212, L215, V220, A221, L223, A224, F225, L228, R233, F234, S256, V260, L266, V269, N272, T274, K278 and R286 in SEQ ID NO: 4 numbering are substituted in the parent lipase. Substitution of amino acid residues at such predetermined positions is a modification for improving stability. Therefore, the variant has improved stability compared to the parent lipase. The stability here more specifically refers to stability in the presence of a larger amount of solvent than a large amount of water and / or surfactant.

[0021] The "parent lipase" means a reference lipase that is modified to produce the variant of the present invention. In the present invention, it is a lipase having at least 80% sequence identity to the amino acid sequence represented by SEQ ID NO: 4 or 6. The parent lipase can be a natural (wild-type) polypeptide or a variant thereof.

[0022] As an example of the parent lipase, there is a polypeptide comprising an amino acid sequence having at least 80% identity with the amino acid sequence represented by SEQ ID NO: 4 and having lipase activity. Here, the lipase consisting of the amino acid sequence represented by SEQ ID NO: 4 is a lipase CnLip (NCBI Accession No. WP_061278013.1) derived from Cedecea neteri, in which amino acid residues at positions I44T, L120S, S130A, A134T, A135V, L136M, and S137N are substituted (CnLipA having excellent detergency described in Japanese Patent Application No. 2023-025141), and further, amino acid residues at positions F16L, I47L, A91V, E97D, and A149E are substituted, and it is CnLipAm shown in the following examples. The above CnLip and CnLipA can also be the parent lipases of the variants of the present invention.

[0023] As another example of the parent lipase, there is a polypeptide comprising an amino acid sequence having at least 80% identity with the amino acid sequence represented by SEQ ID NO: 6 and having lipase activity. Here, the lipase consisting of the amino acid sequence represented by SEQ ID NO: 6 is a lipase in which amino acid residues at positions I45T, L121S, S131A, A135T, A136V, and S138N are substituted in the amino acid sequence constituting lipase CspLip (NCBI Accession No. WP_016537805.1) derived from Cedecea sp. (CspLipA having excellent detergency described in Japanese Patent Application No. 2023-025141), and it is CspLipA shown in the following examples.

[0024] The "substitution" of amino acid residues performed at the above-mentioned predetermined positions means replacing an amino acid at a certain position with a different amino acid. In the present invention, the number of substitution sites of amino acid residues can be two or more, but from the viewpoint of stability, it is preferably two or more, more preferably 2 to 20 sites, and even more preferably 3 to 10 sites. In addition, from the viewpoints of stability and detergency, the variant is preferably a lipase having at least 80% identity to the amino acid sequence represented by SEQ ID NO: 4 or 6. Also, the variant may contain any number of conservative amino acid substitutions as long as it retains the properties as the above-mentioned variant.

[0025] Preferred embodiments of substitutions of amino acid residues at positions corresponding to the positions of L16, G22, Y29, Y43, A45, T46, L50, S120, K123, R126, T134, L142, A145, G156, K174, A181, Q186, K188, L190, D191, L208, H209, M212, L215, V220, A221, L223, A224, F225, L228, R233, F234, S256, V260, L266, V269, N272, T274, K278 and R286 numbered with SEQ ID NO: 4 in the lipase variant of the present invention are shown below. That is, the substitution of L16 with F (L16F) is preferred; The substitution of G22 with C or M (G22C / M) is preferred; The substitution of Y29 with F (Y29F) is preferred; The substitution of Y43 with W (Y43W) is preferred; The substitution of A45 with V (A45V) is preferred; The substitution of T46 with Q (T46Q) is preferred; The substitution of L50 with I, M or V (L50I / M / V) is preferred; The substitution of S120 with A or Q (S120A / Q) is preferred; The substitution of K123 with I or T (K123I / T) is preferred; The substitution of R126 with E (R126E) is preferred; The substitution of T134 with A (T134A) is preferred; The substitution of L142 with F (L142F) is preferred; The substitution of A145 with C or Q (A145C / Q) is preferred; The substitution of G156 with A (G156A) is preferred; K174 is preferably substituted with D (K174D); A181 is preferably substituted with K (A181K); Q186 is preferably substituted with E (Q186E); K188 is preferably substituted with Q (K188Q); L190 is preferably substituted with F (L190F), D191 is preferably substituted with A, E or V (D191A / E / V); L208 is preferably substituted with C (L208C); H209 is preferably substituted with K (H209K); M212 is preferably substituted with L (M212L); L215 is preferably substituted with C (L215C); V220 is preferably substituted with H (V220H); A221 is preferably substituted with H (A221H); L223 is preferably substituted with A, E, S or V (L223A / E / S / V); A224 is preferably substituted with F, Q or Y (A224F / Q / Y); F225 is preferably substituted with T (F225T); L228 is preferably substituted with W (L228W); R233 is preferably substituted with K, Q or T (R233K / Q / T); F234 is preferably substituted with W (F234W); S256 is preferably substituted with N (S256N); V260 is preferably substituted with A, C, F, G, L, M or T (V260A / C / F / G / L / M / T); L266 is preferably substituted with P (L266P); V269 is preferably substituted with I, L or M (V269I / L / M); N272 is preferably substituted with C, G or T (N272C / G / T); T274 is preferably substituted with I or Q (T274I / Q); K278 is preferably substituted with A (K278A); Substitution of R286 with M (R286M) is preferred.

[0026] Next, suitable combinations of mutations that contribute to improved stability are shown in (A) to (D) below. Therefore, a variant having at least any one of the combinations of mutations in (A) to (D) below is a variant that contributes to improved stability. (A) One or more substitutions selected from N272C / G / T substitution at the numbering of SEQ ID NO: 4 and L16F, G22C / M, Y29F, Y43W, A45V, T46Q, L50I / M / V, S120A / Q, K123I / T, R126E, T134A, L142F, A145C / Q, G156A, K174D, A181K, Q186E, K188Q, L190F, D191A / E / V, L208C, H209K, M212L, L215C, V220H, A221H, L223A / E / S / V, A224F / Q / Y, F225T, L228W, R233K / Q / T, F234W, S256N, V260A / C / F / G / L / M / T, L266P, V269I / L / M, T274I / Q, K278A, and R286M (B) One or more substitutions selected from V220H substitution at the numbering of SEQ ID NO: 4 and L16F, G22C / M, Y29F, Y43W, A45V, T46Q, L50I / M / V, S120A / Q, K123I / T, R126E, T134A, L142F, A145C / Q, G156A, K174D, A181K, Q186E, K188Q, L190F, D191A / E / V, L208C, H209K, M212L, L215C, A221H, L223A / E / S / V, A224F / Q / Y, F225T, L228W, R233K / Q / T, F234W, S256N, V260A / C / F / G / L / M / T, L266P, V269I / L / M, N272C / G / T, T274I / Q, K278A, and R286M (C) One or more substitutions selected from V260A / C / F / G / L / M / T substitution with the numbering of SEQ ID NO: 4 and L16F, G22C / M, Y29F, Y43W, A45V, T46Q, L50I / M / V, S120A / Q, K123I / T, R126E, T134A, L142F, A145C / Q, G156A, K174D, A181K, Q186E, K188Q, L190F, D191A / E / V, L208C, H209K, M212L, L215C, V220H, A221H, L223A / E / S / V, A224F / Q / Y, F225T, L228W, R233K / Q / T, F234W, S256N, L266P, V269I / L / M, N272C / G / T, T274I / Q, K278A and R286M (D) One or more substitutions selected from L208C substitution with the numbering of SEQ ID NO: 4 and L16F, G22C / M, Y29F, Y43W, A45V, T46Q, L50I / M / V, S120A / Q, K123I / T, R126E, T134A, L142F, A145C / Q, G156A, K174D, A181K, Q186E, K188Q, L190F, D191A / E / V, H209K, M212L, L215C, V220H, A221H, L223A / E / S / V, A224F / Q / Y, F225T, L228W, R233K / Q / T, F234W, S256N, V260A / C / F / G / L / M / T, L266P, V269I / L / M, N272C / G / T, T274I / Q, K278A and R286M

[0027] More preferred combinations of mutations that contribute to improved stability are shown in (A') to (D') below. Therefore, a variant having at least any one of the combinations of mutations in (A') to (D') below is a variant that contributes to improved stability. (A’) One or more substitutions selected from the substitution of N272C / G with the numbering of SEQ ID NO: 4 and L16F, G22M, Y29F, Y43W, L50I / V, S120A / Q, K123I / T, R126E, L142F, G156A, A181K, Q186E, K188Q, L190F, D191V, L208C, M212L, V220H, A221H, L223V, A224Q, F225T, L228W, R233K / T, F234W, S256N, V260A / C / L, V269I / L, T274Q and K278A (B’) One or more substitutions selected from the substitution of V220H with the numbering of SEQ ID NO: 4 and L16F, G22C / M, Y43W, L50I / V, S120Q, K123I / T, R126E, L142F, G156A, A181K, Q186E, K188Q, L190F, D191A / E / V, L208C, M212L, A221H, L223V, A224Q, F225T, L228W, R233K / Q, F234W, S256N, V260A / C / L, V269I / L / M, N272C / G, T274Q and K278A (C’) One or more substitutions selected from the substitution of V260A / C / L with the numbering of SEQ ID NO: 4 and L16F, G22M, T46Q, L50I / V, K123I, R126E, L142F, G156A, L190F, D191A / E, L208C, M212L, V220H, A224F / Q / Y, F225T, L228W, R233K / Q, R234W, V269I / M, N272C / G and K278A (D’) One or more substitutions selected from the substitution of L208C with the numbering of SEQ ID NO: 4 and L16F, G22C, L50I / V, K123I, R126E, K188Q, L190F, D191A, M212L, V220H, L223V, A224Q / Y, F225T, L228W, R233K, F234W, S256N, V260A / C / L, V269I, N272C / G, K278A and R286M From the perspective of stability, among the combinations of the above mutations (A’) to (D’), the following combinations of mutations are preferred. (A’) Substitutions of N272C numbered according to SEQ ID NO: 4 and one or more substitutions selected from L16F, G22M, Y29F, Y43W, L50I / V, S120A / Q, K123I, R126E, L142F, G156A, A181K, K188Q, D191V, L208C, V220H, A221H, L223V, A224Q, F225T, R233K / T, F234W, S256N, V260A / C / L, V269I and T274Q (B’) Substitutions of V220H numbered according to SEQ ID NO: 4 and one or more substitutions selected from G22C / M, L50I / V, K123I / T, R126E, L142F, G156A, A181K, Q186E, D191V, L208C, M212L, A221H, L223V, L228W, R233K / Q, F234W, S256N, V260C / L, V269I / L / M, N272C / G, T274Q and K278A (C’) Substitutions of V260C numbered according to SEQ ID NO: 4 and one or more substitutions selected from L50I / V and L142F, or (C’) substitutions of V260L numbered according to SEQ ID NO: 4 and one or more substitutions selected from G22M, L50I / V, G156A, D191A / E, L208C, M212L, V220H, A224F / Q / Y, L228W, V269I / M and N272G (D’) Substitutions of L208C numbered according to SEQ ID NO: 4 and one or more substitutions selected from L16F, G22C, R126E, K188Q, D191A, V220H, L223V, A224Q / Y, L228W, S256N, V260A / C / L, V269I, N272C / G, K278A and R286M Also, from the viewpoint of stability, among the combinations of the mutations (A’) to (D’), the following combinations of mutations are more preferable. (A’) Substitutions of N272C numbered according to SEQ ID NO: 4 and one or more substitutions selected from G22M, K123I, G156A, K188Q, L208C, V220H, A221H, L223V, A224Q, F225T, R233T, S256N, V260A / C / L and V269I (B’) Substitution of V220H with the numbering of SEQ ID NO: 4 and one or more substitutions selected from G22C / M, G156A, L208C, M212L, L228W, S256N, V260C / L, V269I / L / M, and N272C / G (C’) Substitution of V260C with the numbering of SEQ ID NO: 4 and one or more substitutions selected from L50I / V and L142F, or substitution of V260L with the numbering of SEQ ID NO: 4 and one or more substitutions selected from G22M, G156A, L208C, V220H, L228W, V269I / M, and N272G (D’) Substitution of L208C with the numbering of SEQ ID NO: 4 and one or more substitutions selected from L16F, G22C, K188Q, V220H, L228W, S256N, V260A / C / L, V269I, and N272C / G

[0028] Even more preferred combinations of mutations that contribute to improved stability are substitution of V220H with the numbering of SEQ ID NO: 4 and substitution of N272C / G, substitution of V260L with the numbering of SEQ ID NO: 4 and substitution of V269I, substitution of L208C with the numbering of SEQ ID NO: 4 and substitution of V220H and substitution of N272C, substitution of L142F with the numbering of SEQ ID NO: 4 and substitution of V220H and substitution of N272G, substitution of V260L with the numbering of SEQ ID NO: 4 and substitution of V269I and N272G, substitution of L208C with the numbering of SEQ ID NO: 4 and substitution of V220H and substitution of V269I and substitution of N272C, substitution of L50V with the numbering of SEQ ID NO: 4 and substitution of L142F and substitution of V220H and substitution of N272G, substitution of L50V with the numbering of SEQ ID NO: 4 and substitution of V260L and substitution of V269I and substitution of N272G, substitution of L208C with the numbering of SEQ ID NO: 4 and substitution of V220H and substitution of S256N and substitution of V269I and substitution of N272C, or substitution of L50V with the numbering of SEQ ID NO: 4 and substitution of L142F and substitution of V220H and substitution of V260L and substitution of N272G. Therefore, mutants having at least any one of these combinations of mutations are mutants that further contribute to improved stability.

[0029] More preferred mutations or combinations of mutations in the lipase mutants of the present invention are shown in Table 1 (Tables 1-1 to 1-5) and Table 2 (Tables 2-1 to 2-3) below. Therefore, mutants having at least any one of the following mutations or combinations of mutations are mutants that particularly contribute to improved stability.

[0030]

Table 1

[0031]

Table 2

[0032] Furthermore, in the numbering of SEQ ID NO: 4, the positions at L16, G22, Y29, Y43, A45, T46, L50, S120, K123, R126, T134, L142, A145, G156, K174, A181, Q186, K188, L190, D191, L208, H209, M212, L215, V220, A221, L223, A224, F225, L228, R233, F234, S256, V260, L266, V269, N272, T274, K278, and R286 correspond to the positions at L17, G23, Y30, Y44, A46, T47, L51, S121, T124, R127, T135, L143, A146, G157, K175, A182, E187, K189, L191, V192, L209, H210, A213, L216, V221, A222, L224, A225, F226, L229, R234, F235, S257, V261, L267, V270, N273, T275, Q279, and R287 in the numbering of SEQ ID NO: 6. Therefore, when the parent lipase has at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 6, the substitution of amino acid residues at the positions corresponding to the positions at L16, G22, Y29, Y43, A45, T46, L50, S120, K123, R126, T134, L142, A145, G156, K174, A181, Q186, K188, L190, D191, L208, H209, M212, L215, V220, A221, L223, A224, F225, L228, R233, F234, S256, V260, L266, V269, N272, T274, K278, and R286 in the numbering of SEQ ID NO: 4 can be read as the substitution of amino acid residues at the positions corresponding to the positions at L17, G23, Y30, Y44, A46, T47, L51, S121, T124, R127, T135, L143, A146, G157, K175, A182, E187, K189, L191, V192, L209, H210, A213, L216, V221, A222, L224, A225, F226, L229, R234, F235, S257, V261, L267, V270, N273, T275, Q279, and R287 in the numbering of SEQ ID NO: 6, and the amino acid residues after substitution are preferably those described above.However, when the parent lipase has the amino acid sequence represented by SEQ ID NO: 6, it is preferably free from the substitution of T at position 124, the substitution of E at position 187, and the substitution of V at position 192. When the parent lipase has at least 80% sequence identity to the amino acid sequence represented by SEQ ID NO: 4, the parent lipase preferably has, as the amino acid residues at the positions substituted in the variant of the present invention, the corresponding amino acid residues among L16, G22, Y29, Y43, A45, T46, L50, S120, K123, R126, T134, L142, A145, G156, K174, A181, Q186, K188, L190, D191, L208, H209, M212, L215, V220, A221, L223, A224, F225, L228, R233, F234, S256, V260, L266, V269, N272, T274, K278 and R286 numbered in SEQ ID NO: 4, and more preferably has the amino acid residues of L16, G22, Y29, Y43, A45, T46, L50, S120, K123, R126, T134, L142, A145, G156, K174, A181, Q186, K188, L190, D191, L208, H209, M212, L215, V220, A221, L223, A224, F225, L228, R233, F234, S256, V260, L266, V269, N272, T274, K278 and R286 numbered in SEQ ID NO: 4.When the parent lipase has at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 6, the parent lipase preferably has, as the amino acid residues at the positions substituted in the variant of the present invention, L17, G23, Y30, Y44, A46, T47, L51, S121, T124, R127, T135, L143, A146, G157, K175, A182, E187, K189, L191, V192, L209, H210, A213, L216, V221, A222, L224, A225, F226, L229, R234, F235, S257, V261, L267, V270, N273, T275, Q279 and R287 numbered as in SEQ ID NO: 6, and more preferably has the amino acid residues of L17, G23, Y30, Y44, A46, T47, L51, S121, T124, R127, T135, L143, A146, G157, K175, A182, E187, K189, L191, V192, L209, H210, A213, L216, V221, A222, L224, A225, F226, L229, R234, F235, S257, V261, L267, V270, N273, T275, Q279 and R287 numbered as in SEQ ID NO: 6.

[0033] The lipase variant of the present invention can be produced by substituting the amino acid residue(s) at one or more positions selected from the positions corresponding to positions L16, G22, Y29, Y43, A45, T46, L50, S120, K123, R126, T134, L142, A145, G156, K174, A181, Q186, K188, L190, D191, L208, H209, M212, L215, V220, A221, L223, A224, F225, L228, R233, F234, S256, V260, L266, V269, N272, T274, K278 and R286 numbered as in SEQ ID NO: 4 in the parent lipase. Here, the parent lipase or lipase variant has at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 4 or 6.

[0034] <2. Polynucleotide encoding the lipase variant of the present invention> The lipase variant of the present invention can be produced using various mutagenesis techniques known in the art. For example, a polynucleotide encoding an amino acid residue to be substituted in a parental lipase gene (reference lipase gene) encoding its reference amino acid sequence is mutated into a polynucleotide encoding the amino acid residue after substitution, and further, the variant can be produced by expressing the variant from the mutated gene.

[0035] In the present invention, as a means for mutating the amino acid residue of the parental lipase, various mutagenesis techniques known in the art can be used. For example, in a polynucleotide encoding the amino acid sequence of the parental lipase (hereinafter also referred to as the parental gene), by mutating the nucleotide sequence encoding the amino acid residue to be mutated into the nucleotide sequence encoding the amino acid residue after mutation, a polynucleotide encoding the lipase variant of the present invention can be obtained.

[0036] The introduction of the desired mutation into the parental gene can basically be carried out using various site-directed mutagenesis methods well-known to those skilled in the art. The site-directed mutagenesis method can be carried out by any method such as, for example, the inverse PCR method or the annealing method. It is also possible to use a commercially available site-directed mutagenesis kit (for example, QuickChange II Site-Directed Mutagenesis Kit or QuickChange Multi Site-Directed Mutagenesis Kit manufactured by Stratagene).

[0037] Site-directed mutagenesis of the parental gene can most commonly be carried out using a mutagenic primer containing the nucleotide mutation to be introduced. The mutagenic primer should be designed to anneal to a region containing the nucleotide sequence encoding the amino acid residue to be mutated in the parental gene and to have a nucleotide sequence containing a nucleotide sequence (codon) encoding the amino acid residue after mutation 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 mutation based on ordinary textbooks and the like. Alternatively, site-directed mutagenesis can also be carried out using a method in which DNA fragments amplified upstream and downstream of the mutation site are separately amplified using two complementary primers containing the nucleotide mutation to be introduced, and then ligated together by SOE (splicing by overlap extension)-PCR (Gene, 1989, 77(1): p61-68).

[0038] The template DNA containing the parental gene can be prepared by extracting genomic DNA from the microorganism producing the above-described parental lipase by a conventional method, or by extracting RNA and synthesizing cDNA by reverse transcription. Alternatively, based on the amino acid sequence of the parental lipase, the corresponding nucleotide sequence can be chemically synthesized and used as the template DNA. The DNA sequences containing the base sequences encoding the lipases consisting of the amino acid sequences shown in SEQ ID NOs: 4 and 6 are shown in SEQ ID NOs: 3 and 5, respectively.

[0039] The mutagenic 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 carried out using, for example, a commercially available oligonucleotide synthesizer (such as those manufactured by ABI). By using a primer set containing the mutagenic primer and performing site-directed mutagenesis as described above with the parental gene as the template DNA, a polynucleotide encoding the lipase mutant of the present invention having the desired mutation can be obtained.

[0040] The polynucleotide encoding the lipase variant of the present invention may contain single-stranded or double-stranded DNA, cDNA, RNA or other artificial nucleic acids. The DNA, cDNA and RNA may be chemically synthesized. In addition to the open reading frame (ORF), the polynucleotide may also contain the nucleotide sequence of the untranslated region (UTR). Further, the polynucleotide may be codon-optimized according to the species of the transformant for producing the mutant polypeptide of the present invention. Information on the codons used by various organisms is available from the Codon Usage Database ([www.kazusa.or.jp / codon / ]).

[0041] <3. Vector or DNA fragment> The polynucleotide encoding the obtained lipase variant of the present invention can be incorporated into a vector. The type of vector containing the polynucleotide is not particularly limited, and it may be any vector such as a plasmid, phage, phagemid, cosmid, virus, YAC vector, shuttle vector, etc. Further, the vector is preferably, but not limited to, a vector that can be amplified in bacteria, preferably in bacteria of the genus Bacillus (e.g., Bacillus subtilis or its mutant strain), and more preferably an expression vector that can induce the expression of the transgene in bacteria of the genus Bacillus. Among them, a shuttle vector, which is a vector that can replicate in both bacteria of the genus Bacillus and other organisms, can be preferably used for the recombinant production of the lipase variant of the present invention. Examples of preferred vectors include, but are not limited to, pHA3040SP64, pHSP64R or pASP64 (Japanese Patent No. 3492935), pHY300PLK (an expression vector capable of transforming both Escherichia coli and Bacillus subtilis; Jpn J Genet, 1985, 60:235-243), pAC3 (Nucleic Acids Res, 1988, 16:8732), etc. shuttle vectors; pUB110 (J Bacteriol, 1978, 134:318-329), pTA10607 (Plasmid, 1987, 18:8-15), etc. plasmid vectors available for the transformation of bacteria of the genus Bacillus, and the like. Also, plasmid vectors derived from Escherichia coli (e.g., pET22b(+), pBR322, pBR325, pUC57, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.) can be used.

[0042] The above vector may contain a DNA region including a DNA replication initiation region or an origin of replication. Alternatively, in the above vector, a control sequence such as a promoter region, a terminator region, or a secretion signal region for secreting the expressed protein extracellularly may be operably linked upstream of the polynucleotide encoding the lipase variant of the present invention (i.e., the lipase variant gene).

[0043] The types of control sequences such as the above-mentioned promoter region, terminator region, and secretion signal region are not particularly limited, and depending on the host to be introduced, commonly used promoters and secretion signal sequences can be appropriately selected and used. For example, preferred examples of control sequences that can be incorporated into a vector include the promoter of the cellulase gene of Bacillus sp. KSM-S237 strain, the secretion signal sequence, and the like.

[0044] Alternatively, the vector of the present invention may further incorporate a marker gene (for example, a drug resistance gene such as ampicillin, neomycin, kanamycin, chloramphenicol, etc.) for selecting a host into which the vector has been appropriately introduced. Alternatively, when a auxotrophic strain is used as the host, a gene encoding a synthetic enzyme for the required nutrient may be incorporated into the vector as a marker gene. Or alternatively, when a selective medium that requires a specific metabolism for growth is used, a gene related to the metabolism may be incorporated into the vector as a marker gene. Examples of such metabolism-related genes include the acetamidase gene for utilizing acetamide as a nitrogen source.

[0045] The ligation of the polynucleotide encoding the lipase variant of the present invention with the control sequence and the marker gene can be carried out by a method known in the art such as the SOE (splicing by overlap extension)-PCR method (Gene, 1989, 77: 61-68). The procedure for introducing the ligated fragment into the vector is well known in the art.

[0046] <4. Transformed Cells> The transformed cells of the present invention can be obtained by introducing a vector containing the polynucleotide encoding the lipase variant of the present invention into a host, or by introducing a DNA fragment containing the polynucleotide encoding the lipase variant of the present invention into the genome of the host.

[0047] Examples of host cells include microorganisms such as bacteria and filamentous fungi. Examples of bacteria include bacteria belonging to Escherichia coli, Staphylococcus, Enterococcus, Listeria, and Bacillus. Among these, Escherichia coli and Bacillus bacteria are preferred, Bacillus bacteria are more preferred, and Bacillus subtilis (for example, Bacillus subtilis Marburg No. 168 (Bacillus subtilis 168 strain) or its mutant strain) is even more preferred. Examples of Bacillus mutant strains include the protease nona-deficient strain KA8AX described in J. Biosci. Bioeng., 2007, 104(2): 135-143, and the D8PA strain described in Biotechnol. Lett., 2011, 33(9): 1847-1852, in which the protein folding efficiency was improved in the protease octa-deficient strain. Examples of filamentous fungi include Trichoderma, Aspergillus, Rhizopus, and the like.

[0048] As a method for introducing a vector into a host, methods commonly used in the art such as the protoplast method and the electroporation method can be used. By selecting a strain into which the introduction has been appropriately performed using the expression of a marker gene, auxotrophy, etc. as indicators, a target transformant into which the vector has been introduced can be obtained.

[0049] Alternatively, a fragment obtained by ligating a polynucleotide encoding the lipase mutant of the present invention, a control sequence, and a marker gene can also be directly introduced into the host genome. For example, by the SOE-PCR method or the like, a DNA fragment having sequences complementary to the host genome added to both ends of the above ligation fragment is constructed, and this is introduced into the host to cause homologous recombination between the host genome and the DNA fragment, whereby the polynucleotide encoding the lipase mutant of the present invention is introduced into the host genome.

[0050] When the polynucleotide encoding the lipase variant of the present invention obtained as described above or the transformant introduced with the vector containing the same 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 according to the type of microorganism of the transformant.

[0051] Alternatively, the lipase variant of the present invention may be expressed from the polynucleotide encoding the lipase variant of the present invention or its transcription product using a cell-free translation system. The "cell-free translation system" is a suspension obtained by mechanically disrupting the host cells and adding reagents such as amino acids necessary for protein translation to constitute an in vitro transcription-translation system or an in vitro translation system.

[0052] The lipase variant of the present invention produced in the above culture or cell-free translation system can be isolated or purified by using general methods used for protein purification, such as centrifugation, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc., alone or in appropriate combination. The protein recovered from the culture may be further purified by known means.

[0053] <5. Method for improving the stability of lipase> The lipase variant of the present invention thus obtained has improved stability compared to the parent lipase, more specifically, improved stability in the presence of a larger amount of solvent than a large amount of water and / or surfactant. "Stability" means the ability to maintain lipase activity, "improved stability" means the ability to maintain lipase activity improved compared to the parent lipase, and "improved stability in the presence of a larger amount of solvent than a large amount of water and / or surfactant" means the ability to maintain lipase activity improved compared to the parent lipase in the presence of a larger amount of solvent than a large amount of water and / or surfactant. Here, the stability in the presence of a large amount of water means the ability to maintain lipase activity when stored in a detergent composition with a water content of 60 to 99% by mass, and the stability in the presence of a solvent in an amount larger than that of the surfactant means the ability to maintain lipase activity in a detergent composition in which the solvent content is higher than the surfactant content or in a cleaning solution in which the solvent content is higher than the surfactant content prepared by dissolving or diluting the detergent composition in water. Examples of the surfactant and the solvent include the surfactants and solvents described below.

[0054] Such stability of lipase can be evaluated using methods well-known in the art. For example, the stability of lipase can be evaluated by adding lipase to an appropriate detergent composition or a cleaning solution prepared by dissolving or diluting the detergent composition in water, measuring the lipase activity before and after treatment for a predetermined time, taking the activity value of the sample before treatment as the initial activity, and calculating the half-life of the activity based on the inactivation rate per unit time by the treatment. The value obtained by dividing the half-life of the lipase mutant by the half-life of the parent lipase indicates the relative stability of the lipase mutant, and when it exceeds 1, the lipase mutant is evaluated to have improved stability compared to the parent lipase. Examples of the cleaning solution include, but are not limited to, the model cleaning solution having the composition shown in Table 3 of the following examples. The stability of the lipase mutant of the present invention can be, as the relative stability during treatment at a constant temperature for a constant time (for example, 45°C for 1 hour), preferably 1.05 or more, more preferably 1.1 or more, and even more preferably 1.2 or more.

[0055] The present invention also provides a method for improving the stability of lipase. The method for improving the stability of lipase according to the present invention includes a step of substituting amino acid residues at one or more positions selected from the positions corresponding to positions L16, G22, Y29, Y43, A45, T46, L50, S120, K123, R126, T134, L142, A145, G156, K174, A181, Q186, K188, L190, D191, L208, H209, M212, L215, V220, A221, L223, A224, F225, L228, R233, F234, S256, V260, L266, V269, N272, T274, K278, and R286 in lipase numbered according to SEQ ID NO: 4, wherein the lipase or the lipase after amino acid residue substitution has at least 80% sequence identity with the amino acid sequence represented by SEQ ID NO: 4 or 6. Details of the substitution of amino acid residues are the same as in the case of the production of the lipase mutants described above.

[0056] <6. Detergent Composition> The lipase mutants of the present invention are useful as enzymes for formulating various detergent compositions, and are particularly useful as enzymes for formulating detergent compositions suitable for low-temperature washing. Here, examples of "low temperature" include 40°C or lower, 35°C or lower, 30°C or lower, 25°C or lower, and also 5°C or higher, 10°C or higher, 15°C or higher. Also, 5 to 40°C, 10 to 35°C, 15 to 30°C, 15 to 25°C can be mentioned.

[0057] The amount of the lipase mutant of the present invention incorporated into the detergent composition is not particularly limited as long as the lipase mutant exhibits activity. For example, it is preferably 0.1 mg or more, more preferably 1 mg or more, still more preferably 5 mg or more per 1 kg of the detergent composition, and preferably 5000 mg or less, more preferably 1000 mg or less, still more preferably 500 mg or less. Also, it is preferably 0.1 to 5000 mg, more preferably 1 to 1000 mg, and still more preferably 5 to 500 mg.

[0058] The detergent composition preferably contains, in addition to the lipase variant of the present invention, a sulfosuccinic acid ester or a salt thereof, which is a surfactant. The sulfosuccinic acid ester or a salt thereof is known as a component to be formulated in a detergent composition (for example, JP-A-2019-182911). As the sulfosuccinic acid ester or a salt thereof, a sulfosuccinic acid branched alkyl ester or a salt thereof having a branched alkyl group having 8 to 12 carbon atoms is preferable, a sulfosuccinic acid branched alkyl ester or a salt thereof having a branched alkyl group having 9 or 10 carbon atoms is more preferable, and a sulfosuccinic acid branched alkyl ester or a salt thereof having a branched alkyl group having 10 carbon atoms is even more preferable. Further, the sulfosuccinic acid ester or a salt thereof is a sulfosuccinic acid di-branched alkyl ester or a salt thereof, and a sulfosuccinic acid di-branched alkyl ester or a salt thereof in which two branched alkyl groups are each a branched alkyl group having 8 to 12 carbon atoms is preferable, a sulfosuccinic acid di-branched alkyl ester or a salt thereof in which two branched alkyl groups are each a branched alkyl group having 9 or 10 carbon atoms is more preferable, a sulfosuccinic acid di-branched alkyl ester or a salt thereof in which two branched alkyl groups are each a branched alkyl group having 10 carbon atoms is even more preferable, and bis-(2-propylheptyl)sulfosuccinic acid or a salt thereof is even more preferable.

[0059] Examples of the salt include an alkali metal salt, an alkanolamine salt, etc., an alkali metal salt or an alkanolamine salt is preferable, and a salt selected from a sodium salt, a potassium salt, a triethanolamine salt, a diethanolamine salt, and a monoethanolamine salt is more preferable, and a sodium salt is even more preferable.

[0060] Examples of the sulfosuccinic acid ester or a salt thereof include a compound represented by the following formula 1.

[0061]

Chemical formula

[0062] 〔In formula 1, R 1 , R 2is a branched alkyl group having 8 to 12 carbon atoms, respectively, and A 1 O, A 2 O is an alkyleneoxy group having 2 to 4 carbon atoms, respectively, x1 and x2 are the average number of moles added, and are numbers of 0 or more and 10 or less, respectively, and M is a cation. ]]

[0063] In Formula 1, R 1 , R 2 is 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.

[0064] In Formula 1, A 1 O, A 2 O is an alkyleneoxy group having 2 to 4 carbon atoms, and preferably an alkyleneoxy group having 2 or 3 carbon atoms from the viewpoint of lubricity with respect to water. In Formula 1, x1 and x2 represent the average number of moles added of A 1 O, A 2 O, and are numbers of 0 or more and 10 or less, preferably 6 or less, more preferably 4 or less, still more preferably 2 or less, and even more preferably 0 from the viewpoint of lubricity with respect to water.

[0065] 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. M is preferably an alkali metal ion or an alkanolammonium ion from the viewpoint of dispersibility in water, more preferably sodium ion, potassium ion, triethanolammonium ion, diethanolammonium ion, or monoethanolammonium ion, and still more preferably sodium ion.

[0066] The sulfosuccinic acid ester or its salt 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.

[0067]

Chemical formula

[0068] 〔In formula 1-1, R 1 , R 2 are each a branched alkyl group having 8 to 12 carbon atoms, and M is a cation.〕 Specific examples and preferred examples of R 1 , R 2 , and M in formula 1-1 are the same as those in formula 1. In a preferred embodiment, the sulfosuccinic acid ester or its salt is bis-(2-propylheptyl)sulfosuccinic acid or its salt.

[0069] The blending amount of the sulfosuccinic acid ester 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, still more preferably 2.0% by mass or less. Also, it is preferably 0.01 to 30.0% by mass, more preferably 0.1 to 10.0% by mass, and still more preferably 0.1 to 2.0% by mass.

[0070] In addition to the lipase variant of the present invention, the detergent composition can also use various enzymes in combination. For example, hydrolases, oxidases, reductases, transferases, lyases, isomerases, ligases, synthetases, etc. Among these, lipases, amylases, proteases, cellulases, keratinases, esterases, cutinases, pullulanases, pectinases, mannanases, glucosidases, glucanases, cholesterol oxidases, peroxidases, laccases, etc., which are different from the lipase variant of the present invention, are preferred, and particularly proteases, cellulases, amylases, and lipases different from the lipase variant of the present invention are preferred. Examples of proteases include commercially available Alcalase, Esperase, Everlase, Savinase, Kannase, Progress Uno (registered trademark; Novozymes), PREFERENZ, EFFECTENZ, EXCELLENZ (registered trademark; DuPont), Lavergy (registered trademark; BASF), and KAP (Kao). Alternatively, examples include variants of the KP43 protease described in JP 2020-145938 A and Japanese Patent Application No. 2022-118096. Examples of cellulases include Celluclean, Carezyme (registered trademark; Novozymes), KAC, an alkaline cellulase produced by Bacillus sp. KSM-S237 described in JP 10-313859 A, and a mutant alkaline cellulase described in JP 2003-313592 A (both from Kao). Examples of amylases include Termamyl, Duramyl, Stainzyme, Stainzyme Plus, Amplify Prime (registered trademark; Novozymes), PREFERENZ, EFFECTENZ (registered trademark; DuPont), and KAM (Kao). Alternatively, examples include variants of the YR288 amylase described in JP 2022-60158 A and WO2023 / 176970. Alternatively, examples include variants of the amylases described in JP 2019-500058 T and JP 2018-516553 T. Examples of lipases include Lipolase, Lipex (registered trademark; Novozymes). In the detergent composition, in addition to the lipase variant of the present invention, one or more of the above enzymes can be used in combination. Examples of the combination modes include, but are not limited to, the combination of the lipase variant of the present invention and protease, the combination of the lipase variant of the present invention and amylase, the combination of the lipase variant of the present invention, protease and amylase, the combination of the lipase variant of the present invention, a lipase different from the lipase variant of the present invention and protease, the combination of the lipase variant of the present invention, a lipase different from the lipase variant of the present invention and amylase, the combination of the lipase variant of the present invention, a lipase different from the lipase variant of the present invention, protease and amylase, etc. Among them, it is preferable to use the lipase variant of the present invention, protease and amylase in combination.

[0071] Known detergent components can be incorporated into the detergent composition, and examples of such known detergent components include the following.

[0072] (1) Surfactant The surfactant is incorporated in the detergent composition in an amount of 0.5 to 90% by mass, and particularly preferably 10 to 45% by mass for the powdered detergent composition and 20 to 90% by mass for the liquid detergent composition. When the detergent composition is a laundry detergent for clothes or a detergent for an automatic dishwashing machine, the surfactant is generally incorporated in an amount of 0.5 to 20% by mass, preferably 1 to 15% by mass, more preferably 1.5 to 10% by mass, and still more preferably 2 to 8% by mass.

[0073] Examples of the surfactant used in the detergent composition include one or a combination of anionic surfactants, nonionic surfactants, amphoteric surfactants, and cationic surfactants other than the above-mentioned sulfosuccinic acid ester or its salt, and preferably an amphoteric surfactant.

[0074] 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 carboxybetaine type surfactant is more preferred. As the tertiary amine oxide type surfactant, one of the groups bonded to the nitrogen atom may be an alkyl group having 8 to 18 carbon atoms, preferably 8 to 16 carbon atoms, more preferably 8 to 14 carbon atoms, which may be interrupted by an amide group or an ester group, and the rest is an alkyl group having 1 to 3 carbon atoms, preferably a methyl group. Examples of the tertiary amine oxide type surfactant include those having such a structure. As the sulfobetaine type surfactant, a compound having one alkyl group having 10 to 18 carbon atoms, preferably 16 or less, more preferably 14 or less, two alkyl groups having 1 to 3 carbon atoms, preferably methyl groups, and a 3-sulfopropyl group or a 2-hydroxy-3-sulfopropyl group is suitable. As the carboxybetaine type surfactant, a carboxybetaine type surfactant having one alkyl group having 10 to 18 carbon atoms, preferably 16 or less, more preferably 14 or less, which may be interrupted by an amide group or an ester group, two alkyl groups having 1 to 3 carbon atoms, preferably methyl groups, and one carboxyalkyl group, preferably a carboxymethyl group is preferred.

[0075] (2) Divalent metal ion capturer The divalent metal ion capturer is blended in an amount of 0.01 to 50% by mass, preferably 5 to 40% by mass. Examples of the divalent metal ion capturer used in the detergent composition include condensed phosphates such as tripolyphosphate, pyrophosphate, and orthophosphate, aluminosilicates such as zeolite, synthetic layered crystalline silicate, nitrilotriacetate, ethylenediaminetetraacetate, citrate, isocitrate, polyacetal carboxylate, and the like. Among these, crystalline aluminosilicate (synthetic zeolite) is particularly preferred, and among A-type, X-type, and P-type zeolites, A-type is particularly preferred. The synthetic zeolite having an average primary particle size of 0.1 to 10 μm, particularly 0.1 to 5 μm is preferably used.

[0076] (3) Alkali agent The alkali agent is formulated in an amount of 0.01 to 80% by mass, preferably 1 to 40% by mass. In the case of a powder detergent, examples of the alkali agent include alkali metal carbonates such as sodium carbonate, which is collectively referred to as dense ash or light ash, and amorphous alkali metal silicates such as JIS No. 1, No. 2, and No. 3. These inorganic alkali agents are effective in forming the particle skeleton during detergent drying, and a detergent that is relatively hard and has excellent fluidity can be obtained. Examples of other alkalis include sesquicarbonate and sodium hydrogen carbonate, and phosphates such as tripolyphosphate also have the function of an alkali agent. In addition, as the alkali agent used in a liquid detergent, in addition to the above alkali agents, sodium hydroxide, and mono-, di- or triethanolamine can be used, and they can also be used as the counter ion of the surfactant.

[0077] (4) Redeposition inhibitor The redeposition inhibitor is formulated in an amount of 0.001 to 10% by mass, preferably 1 to 5% by mass. Examples of the redeposition inhibitor used in the detergent composition include polyethylene glycol, carboxylic acid polymers, polyvinyl alcohol, and polyvinyl pyrrolidone. Among these, the carboxylic acid polymer has, in addition to the redeposition prevention ability, a function of capturing metal ions and an action of dispersing solid particle stains from clothing into the washing bath. The carboxylic acid polymer is a homopolymer or copolymer of acrylic acid, methacrylic acid, itaconic acid, etc. As the copolymer, a copolymer of the above monomers and maleic acid is preferable, and those having a molecular weight of several thousand to 100,000 are preferable. In addition to the above carboxylic acid polymers, polymers such as polyglycidylates, cellulose derivatives such as carboxymethyl cellulose, and aminocarboxylic acid polymers such as polyaspartic acid are also preferable because they have the functions of a metal ion capturer, a dispersant, and redeposition prevention ability.

[0078] (5) Bleaching agent For example, it is preferable to formulate 1 to 10% by mass of a bleaching agent such as hydrogen peroxide or percarbonate. When using a bleaching agent, 0.01 to 10% by mass of a bleach activator such as tetraacetylethylenediamine (TAED) or as described in JP-A-6-316700 can be formulated.

[0079] (6) Fluorescent agent Examples of the fluorescent agent used in the detergent composition include biphenyl type fluorescent agents (such as Tinopal CBS-X) and stilbene type fluorescent agents (such as DM type fluorescent dyes). It is preferable to blend the fluorescent agent in an amount of 0.001 to 2% by mass.

[0080] (7) Chelating agent The chelating agent is blended, for example, for easy cleaning of dirt and reduction of water hardness during washing. Examples of such chelating agents include aminocarboxylic acid-based chelating agents, phosphonic acid-based chelating agents, hydroxycarboxylic acid-based chelating agents, polyvalent carboxylic acid-based chelating agents, etc. Examples of aminocarboxylic acid-based chelating agents include ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), hydroxyethylethylenediaminetriacetic acid (HEDTA), diethylenetriaminepentaacetic acid (DTPA), methylglycine diacetic acid (MGDA), triethylenetetraminehexaacetic acid (TTHA), glutamic acid diacetic acid (GLDA), hydroxyethyliminodiacetic acid (HIDA), dihydroxyethylglycine (DHEG), aspartic acid diacetic acid (ASDA), ethylenediamine succinic acid (EDDS) and their salts, etc. Examples of phosphonic acid-based chelating agents include hydroxyethylidene diphosphonic acid (HEDP), nitrilotris(methylene phosphonic acid) (NTMP), phosphonobutane tricarboxylic acid (PBTC), ethylenediamine tetramethylene phosphonic acid (EDTMP) and their salts, etc. Examples of hydroxycarboxylic acid-based chelating agents include citric acid, malic acid, tartaric acid, gluconic acid, lactic acid and their salts, etc. Examples of polyvalent carboxylic acid-based chelating agents include succinic acid, oxalic acid, glutaric acid, adipic acid, fumaric acid, malonic acid and their salts, etc.

[0081] (8) Other components The detergent composition can contain solvents, builders, softeners, reducing agents (such as sulfites), defoaming agents (such as silicone), fragrances, antibacterial and antifungal agents (such as Proxel [trade name], benzoic acid, etc.), and other additives known in the field of detergents.

[0082] 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; monoalkoxy (methoxy, ethoxy, propoxy, butoxy), phenoxy or benzooxy ethers of di- to tetraalkylene glycols having 2 to 4 carbon atoms in the alkylene glycol unit. As the solvent, water-soluble organic solvents having 2 or more carbon atoms, preferably 3 or more carbon atoms, and 10 or less carbon atoms, preferably 8 or less carbon atoms are preferred. 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 referred to as butyl diglycol, etc.), phenoxyethanol, phenoxy triethylene glycol, phenoxyisopropanol can be mentioned. As the solvent, a solvent selected from ethanol, propylene glycol, dipropylene glycol, diethylene glycol monobutyl ether, phenoxyethanol, phenyl glycol, and phenoxyisopropanol is preferred. The solvent preferably has an alkoxy group, and more preferably contains one or more selected from the above-mentioned monoalkoxy, phenoxy or benzooxy ethers of di- to tetraalkylene glycols having 2 to 4 carbon atoms in the alkylene glycol unit. It is more preferred to contain diethylene glycol monobutyl ether as the solvent.

[0083] As the detergent composition, those having a water content of 60 to 99% by mass and / or having a higher solvent content than the surfactant content are preferred, and those having a water content of 60 to 99% by mass and a higher solvent content than the surfactant content are more preferred.

[0084] The detergent composition can be produced according to a conventional method by combining the lipase mutant of the present invention obtained by the above method and the above-known cleaning components. The form of the detergent can be selected according to the use, and for example, it can be made into a liquid, powder, granule, paste, solid, etc.

[0085] The thus-obtained detergent composition can be used as a laundry detergent, dishwashing detergent, bleaching agent, hard surface cleaning detergent, drain pipe cleaning detergent, denture cleaning detergent, sterilizing detergent for medical instruments, etc. Preferably, laundry detergents and dishwashing detergents are mentioned, and more preferably, laundry detergents for laundry (laundry detergents for washing), dishwashing detergents for hand washing, and detergents for automatic dishwashers are mentioned. In addition, the detergent composition is suitable for use at 40°C or lower, 35°C or lower, 30°C or lower, 25°C or lower, and 5°C or higher, 10°C or higher, 15°C or higher. It is also suitable for use at 5 to 40°C, 10 to 35°C, 15 to 30°C, 15 to 25°C. Preferred usage modes include use in low-temperature (15 to 30°C) washing in laundry, use in low-temperature (15 to 30°C) in dishwashing by hand, and use in low-temperature (15 to 30°C) washing by an automatic dishwasher.

[0086] By using the detergent composition of the present invention, it is possible to wash an object to be washed that requires removal of dirt (for example, clothes, tableware, hard surfaces, drain pipes, dentures, medical instruments, etc.), that is, to remove dirt. Such a cleaning method includes bringing the object to be washed that requires removal of dirt into contact with the detergent composition of the present invention. Preferably, the dirt is dirt containing oil, and examples include oil stains and composite stains containing proteins and carbohydrates in addition to oil.

[0087] In the cleaning method of the present invention, in order to bring the object to be cleaned into contact with the cleaning composition, the object to be cleaned may be immersed in water in which the cleaning composition is dissolved, or the cleaning composition may be directly applied 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 cleaning composition may be further hand-washed, scrubbed with a sponge, washed with a washing machine, etc., but this is not necessarily required.

[0088] Regarding the above-described embodiments, the present invention further discloses the following aspects. <1>A variant of a parent lipase comprising substitution of one or more amino acid residues at positions selected from positions corresponding to positions L16, G22, Y29, Y43, A45, T46, L50, S120, K123, R126, T134, L142, A145, G156, K174, A181, Q186, K188, L190, D191, L208, H209, M212, L215, V220, A221, L223, A224, F225, L228, R233, F234, S256, V260, L266, V269, N272, T274, K278 and R286 in the numbering of SEQ ID NO: 4, wherein the parent lipase or the lipase variant has at least 80% sequence identity to the amino acid sequence represented by SEQ ID NO: 4 or 6. <2>The lipase variant according to <1>, wherein the substitution of the amino acid residues at the positions corresponding to the positions of L16, G22, Y29, Y43, A45, T46, L50, S120, K123, R126, T134, L142, A145, G156, K174, A181, Q186, K188, L190, D191, L208, H209, M212, L215, V220, A221, L223, A224, F225, L228, R233, F234, S256, V260, L266, V269, N272, T274, K278 and R286 in the numbering of SEQ ID NO: 4 are L16F, G22C / M, Y29F, Y43W, A45V, T46Q, L50I / M / V, S120A / Q, K123I / T, R126E, T134A, L142F, A145C / Q, G156A, K174D, A181K, Q186E, K188Q, L190F, D191A / E / V, L208C, H209K, M212L, L215C, V220H, A221H, L223A / E / S / V, A224F / Q / Y, F225T, L228W, R233K / Q / T, F234W, S256N, V260A / C / F / G / L / M / T, L266P, V269I / L / M, N272C / G / T, T274I / Q, K278A and R286M, respectively. <3>The lipase variant according to <2>, comprising at least one of the following substitutions (A) to (D). (A) The substitution of N272C / G / T in the numbering of SEQ ID NO: 4 and one or more substitutions selected from L16F, G22C / M, Y29F, Y43W, A45V, T46Q, L50I / M / V, S120A / Q, K123I / T, R126E, T134A, L142F, A145C / Q, G156A, K174D, A181K, Q186E, K188Q, L190F, D191A / E / V, L208C, H209K, M212L, L215C, V220H, A221H, L223A / E / S / V, A224F / Q / Y, F225T, L228W, R233K / Q / T, F234W, S256N, V260A / C / F / G / L / M / T, L266P, V269I / L / M, T274I / Q, K278A and R286M (B) One or more substitutions selected from the substitution of V220H with the numbering of SEQ ID NO: 4 and L16F, G22C / M, Y29F, Y43W, A45V, T46Q, L50I / M / V, S120A / Q, K123I / T, R126E, T134A, L142F, A145C / Q, G156A, K174D, A181K, Q186E, K188Q, L190F, D191A / E / V, L208C, H209K, M212L, L215C, A221H, L223A / E / S / V, A224F / Q / Y, F225T, L228W, R233K / Q / T, F234W, S256N, V260A / C / F / G / L / M / T, L266P, V269I / L / M, N272C / G / T, T274I / Q, K278A, and R286M (C) One or more substitutions selected from the substitution of V260A / C / F / G / L / M / T with the numbering of SEQ ID NO: 4 and L16F, G22C / M, Y29F, Y43W, A45V, T46Q, L50I / M / V, S120A / Q, K123I / T, R126E, T134A, L142F, A145C / Q, G156A, K174D, A181K, Q186E, K188Q, L190F, D191A / E / V, L208C, H209K, M212L, L215C, V220H, A221H, L223A / E / S / V, A224F / Q / Y, F225T, L228W, R233K / Q / T, F234W, S256N, L266P, V269I / L / M, N272C / G / T, T274I / Q, K278A, and R286M (D) One or more substitutions selected from the substitution of L208C with the numbering of SEQ ID NO: 4 and L16F, G22C / M, Y29F, Y43W, A45V, T46Q, L50I / M / V, S120A / Q, K123I / T, R126E, T134A, L142F, A145C / Q, G156A, K174D, A181K, Q186E, K188Q, L190F, D191A / E / V, H209K, M212L, L215C, V220H, A221H, L223A / E / S / V, A224F / Q / Y, F225T, L228W, R233K / Q / T, F234W, S256N, V260A / C / F / G / L / M / T, L266P, V269I / L / M, N272C / G / T, T274I / Q, K278A, and R286M The lipase variant according to <2>, which comprises at least any one of the following substitutions (A’) to (D’). (A’) One or more substitutions selected from N272C / G substitution at position numbered in SEQ ID NO: 4 and L16F, G22M, Y29F, Y43W, L50I / V, S120A / Q, K123I / T, R126E, L142F, G156A, A181K, Q186E, K188Q, L190F, D191V, L208C, M212L, V220H, A221H, L223V, A224Q, F225T, L228W, R233K / T, F234W, S256N, V260A / C / L, V269I / L, T274Q and K278A (B’) One or more substitutions selected from V220H substitution at position numbered in SEQ ID NO: 4 and L16F, G22C / M, Y43W, L50I / V, S120Q, K123I / T, R126E, L142F, G156A, A181K, Q186E, K188Q, L190F, D191A / E / V, L208C, M212L, A221H, L223V, A224Q, F225T, L228W, R233K / Q, F234W, S256N, V260A / C / L, V269I / L / M, N272C / G, T274Q and K278A (C’) One or more substitutions selected from V260A / C / L substitution at position numbered in SEQ ID NO: 4 and L16F, G22M, T46Q, L50I / V, K123I, R126E, L142F, G156A, L190F, D191A / E, L208C, M212L, V220H, A224F / Q / Y, F225T, L228W, R233K / Q, R234W, V269I / M, N272C / G and K278A (D’) One or more substitutions selected from L208C substitution at position numbered in SEQ ID NO: 4 and L16F, G22C, L50I / V, K123I, R126E, K188Q, L190F, D191A, M212L, V220H, L223V, A224Q / Y, F225T, L228W, R233K, F234W, S256N, V260A / C / L, V269I, N272C / G, K278A and R286M <5>The lipase variant according to <4>, wherein the said (A’) to (D’) are as follows. (A’) Substitutions of N272C numbered according to SEQ ID NO: 4 and one or more substitutions selected from L16F, G22M, Y29F, Y43W, L50I / V, S120A / Q, K123I, R126E, L142F, G156A, A181K, K188Q, D191V, L208C, V220H, A221H, L223V, A224Q, F225T, R233K / T, F234W, S256N, V260A / C / L, V269I and T274Q (B’) Substitutions of V220H numbered according to SEQ ID NO: 4 and one or more substitutions selected from G22C / M, L50I / V, K123I / T, R126E, L142F, G156A, A181K, Q186E, D191V, L208C, M212L, A221H, L223V, L228W, R233K / Q, F234W, S256N, V260C / L, V269I / L / M, N272C / G, T274Q and K278A (C’) Substitutions of V260C numbered according to SEQ ID NO: 4 and one or more substitutions selected from L50I / V and L142F, or substitutions of V260L numbered according to SEQ ID NO: 4 and one or more substitutions selected from G22M, L50I / V, G156A, D191A / E, L208C, M212L, V220H, A224F / Q / Y, L228W, V269I / M and N272G (D’) Substitutions of L208C numbered according to SEQ ID NO: 4 and one or more substitutions selected from L16F, G22C, R126E, K188Q, D191A, V220H, L223V, A224Q / Y, L228W, S256N, V260A / C / L, V269I, N272C / G, K278A and R286M <6>The lipase variant according to <4>, wherein the said (A’) to (D’) are as follows. (A’) Substitutions of N272C numbered according to SEQ ID NO: 4 and one or more substitutions selected from G22M, K123I, G156A, K188Q, L208C, V220H, A221H, L223V, A224Q, F225T, R233T, S256N, V260A / C / L and V269I (B’) Substitution of V220H with the numbering of SEQ ID NO: 4 and one or more substitutions selected from G22C / M, G156A, L208C, M212L, L228W, S256N, V260C / L, V269I / L / M, and N272C / G (C’) Substitution of V260C with the numbering of SEQ ID NO: 4 and one or more substitutions selected from L50I / V and L142F, or substitution of V260L with the numbering of SEQ ID NO: 4 and one or more substitutions selected from G22M, G156A, L208C, V220H, L228W, V269I / M, and N272G (D’) Substitution of L208C with the numbering of SEQ ID NO: 4 and one or more substitutions selected from L16F, G22C, K188Q, V220H, L228W, S256N, V260A / C / L, V269I, and N272C / G <7> The lipase variant according to <2>, comprising at least substitution of V220H with the numbering of SEQ ID NO: 4 and substitution of N272C / G or substitution of V260L and substitution of V269I. <8> The lipase variant according to <2>, comprising at least substitution of L208C with the numbering of SEQ ID NO: 4, substitution of V220H, and substitution of N272C, substitution of L142F, substitution of V220H, and substitution of N272G, or substitution of V260L, substitution of V269I, and substitution of N272G. <9> The lipase variant according to <2>, comprising at least substitution of L208C with the numbering of SEQ ID NO: 4, substitution of V220H, substitution of V269I, and substitution of N272C, substitution of L50V, substitution of L142F, substitution of V220H, and substitution of N272G, or substitution of L50V, substitution of V260L, substitution of V269I, and substitution of N272G. <10> The lipase variant according to <2>, comprising at least substitution of L208C with the numbering of SEQ ID NO: 4, substitution of V220H, substitution of S256N, substitution of V269I, and substitution of N272C, or substitution of L50V, substitution of L142F, substitution of V220H, substitution of V260L, and substitution of N272G. <11> The lipase variant according to <1> or <2>, comprising at least any substitution or combination of substitutions shown in Table 1-1 to 1-5 and Table 2-1 to 2-3 above. <12>If the parent lipase or the lipase variant has at least 80% sequence identity to the amino acid sequence represented by SEQ ID NO: 6, the substitutions of the amino acid residues at the positions corresponding to the positions of L16, G22, Y29, Y43, A45, T46, L50, S120, K123, R126, T134, L142, A145, G156, K174, A181, K188, L190, D191, L208, H209, M212, L215, V220, A221, L223, A224, F225, L228, R233, F234, S256, V260, L266, V269, N272, T274, K278 and R286 in the numbering of SEQ ID NO: 4 are, respectively, L17F, G23C / M, Y30F, Y44W, A46V, T476Q, L51I / M / V, S121A / Q, T124I, R127E, T135A, L143F, A146C / Q, G157A, K175D, A182K, K189Q, L191F, V192A / E, L209C, H210K, A213L, L216C, V221H, A222H, L224A / E / S / V, A225F / Q / Y, F226T, L229W, R234K / Q / T, F235W, S257N, V261A / C / F / G / L / M / T, L267P, V270I / L / M, N273C / G / T, T275I / Q, Q279A and R287M in the numbering of SEQ ID NO: 6, the lipase variant according to any one of <1> to <11>. <13>The lipase variant according to any one of <1> to <12>, which has improved stability compared to the parent lipase.

[0089] <14>A polynucleotide encoding the lipase variant according to any one of <1> to <13>. <15>A vector or DNA fragment containing the polynucleotide according to <14>. <16>A transformed cell containing the vector or DNA fragment according to <15>. <17>The transformed cell according to <16>, which is a microorganism. <18>The transformed cell according to <17>, which is Escherichia coli or a bacterium belonging to the genus Bacillus, preferably a bacterium belonging to the genus Bacillus, more preferably Bacillus subtilis. A method for producing a lipase mutant, comprising the step of culturing the transformed cell according to any one of <19><16> to <18>.

[0090] A detergent composition containing the lipase mutant according to any one of <20><1> to <13>. <21>The detergent composition according to <20>, further containing a sulfosuccinate ester or a salt thereof, preferably a branched alkyl sulfosuccinate ester having a branched alkyl group with 8 to 12 carbon atoms or a salt thereof, more preferably a branched alkyl sulfosuccinate ester having a branched alkyl group with 9 or 10 carbon atoms or a salt thereof, still more preferably a branched alkyl sulfosuccinate ester having a branched alkyl group with 10 carbon atoms or a salt thereof. <22>The detergent composition according to <20>, further containing a sulfosuccinate diester or a salt thereof, preferably a branched dialkyl sulfosuccinate ester or a salt thereof in which each of the two branched alkyl groups is a branched alkyl group with 8 to 12 carbon atoms, more preferably a branched dialkyl sulfosuccinate ester or a salt thereof in which each of the two branched alkyl groups is a branched alkyl group with 9 or 10 carbon atoms, still more preferably a branched dialkyl sulfosuccinate ester or a salt thereof in which each of the two branched alkyl groups is a branched alkyl group with 10 carbon atoms, and still more preferably bis-(2-propylheptyl) sulfosuccinic acid or a salt thereof. <23>The detergent composition according to any one of <20> to <22>, wherein the water content is 60 to 90% by mass and / or the solvent content is higher than the surfactant content, preferably the water content is 60 to 90% by mass and the solvent content is higher than the surfactant content. <24>The detergent composition according to any one of <20> to <23>, which is a laundry detergent or a dishwashing detergent. <25>The detergent composition according to any one of <20> to <24>, which is in powder or liquid form.

[0091] <26>A method for cleaning stains, using the detergent composition according to any one of <20> to <25>. The method according to <26>, comprising contacting the object to be washed with the detergent composition according to any one of <20> to <25>. Use of the lipase variant according to any one of <1> to <13> for the production of a detergent composition. Use of the lipase variant according to any one of <1> to <13> for the cleaning of stains.

[0092] A method for improving the stability of lipase, comprising a step of substituting amino acid residues at one or more positions selected from the positions corresponding to L16, G22, Y29, Y43, A45, T46, L50, S120, K123, R126, T134, L142, A145, G156, K174, A181, Q186, K188, L190, D191, L208, H209, M212, L215, V220, A221, L223, A224, F225, L228, R233, F234, S256, V260, L266, V269, N272, T274, K278 and R286 in the numbering of SEQ ID NO: 4, wherein the lipase or the lipase after amino acid residue substitution has at least 80% sequence identity to the amino acid sequence shown by SEQ ID NO: 4 or 6. A method for producing a variant of a parent lipase, comprising a step of substituting amino acid residues at one or more positions selected from the positions corresponding to L16, G22, Y29, Y43, A45, T46, L50, S120, K123, R126, T134, L142, A145, G156, K174, A181, Q186, K188, L190, D191, L208, H209, M212, L215, V220, A221, L223, A224, F225, L228, R233, F234, S256, V260, L266, V269, N272, T274, K278 and R286 in the numbering of SEQ ID NO: 4, wherein the lipase or the lipase variant has at least 80% sequence identity to the amino acid sequence shown by SEQ ID NO: 4 or 6. <32>The method according to <30> or <31>, wherein the substitution of the amino acid residues at the positions corresponding to the positions of L16, G22, Y29, Y43, A45, T46, L50, S120, K123, R126, T134, L142, A145, G156, K174, A181, Q186, K188, L190, D191, L208, H209, M212, L215, V220, A221, L223, A224, F225, L228, R233, F234, S256, V260, L266, V269, N272, T274, K278 and R286 in the numbering of SEQ ID NO: 4 are L16F, G22C / M, Y29F, Y43W, A45V, T46Q, L50I / M / V, S120A / Q, K123I / T, R126E, T134A, L142F, A145C / Q, G156A, K174D, A181K, Q186E, K188Q, L190F, D191A / E / V, L208C, H209K, M212L, L215C, V220H, A221H, L223A / E / S / V, A224F / Q / Y, F225T, L228W, R233K / Q / T, F234W, S256N, V260A / C / F / G / L / M / T, L266P, V269I / L / M, N272C / G / T, T274I / Q, K278A and R286M, respectively. <33>The method according to <32>, wherein the substitution comprises at least one of the following substitutions (A) to (D). (A) The substitution of N272C / G / T in the numbering of SEQ ID NO: 4 and one or more substitutions selected from L16F, G22C / M, Y29F, Y43W, A45V, T46Q, L50I / M / V, S120A / Q, K123I / T, R126E, T134A, L142F, A145C / Q, G156A, K174D, A181K, Q186E, K188Q, L190F, D191A / E / V, L208C, H209K, M212L, L215C, V220H, A221H, L223A / E / S / V, A224F / Q / Y, F225T, L228W, R233K / Q / T, F234W, S256N, V260A / C / F / G / L / M / T, L266P, V269I / L / M, T274I / Q, K278A and R286M (B) One or more substitutions selected from the substitution of V220H with the numbering of SEQ ID NO: 4 and L16F, G22C / M, Y29F, Y43W, A45V, T46Q, L50I / M / V, S120A / Q, K123I / T, R126E, T134A, L142F, A145C / Q, G156A, K174D, A181K, Q186E, K188Q, L190F, D191A / E / V, L208C, H209K, M212L, L215C, A221H, L223A / E / S / V, A224F / Q / Y, F225T, L228W, R233K / Q / T, F234W, S256N, V260A / C / F / G / L / M / T, L266P, V269I / L / M, N272C / G / T, T274I / Q, K278A and R286M (C) One or more substitutions selected from the substitution of V260A / C / F / G / L / M / T with the numbering of SEQ ID NO: 4 and L16F, G22C / M, Y29F, Y43W, A45V, T46Q, L50I / M / V, S120A / Q, K123I / T, R126E, T134A, L142F, A145C / Q, G156A, K174D, A181K, Q186E, K188Q, L190F, D191A / E / V, L208C, H209K, M212L, L215C, V220H, A221H, L223A / E / S / V, A224F / Q / Y, F225T, L228W, R233K / Q / T, F234W, S256N, L266P, V269I / L / M, N272C / G / T, T274I / Q, K278A and R286M (D) One or more substitutions selected from the substitution of L208C with the numbering of SEQ ID NO: 4 and L16F, G22C / M, Y29F, Y43W, A45V, T46Q, L50I / M / V, S120A / Q, K123I / T, R126E, T134A, L142F, A145C / Q, G156A, K174D, A181K, Q186E, K188Q, L190F, D191A / E / V, H209K, M212L, L215C, V220H, A221H, L223A / E / S / V, A224F / Q / Y, F225T, L228W, R233K / Q / T, F234W, S256N, V260A / C / F / G / L / M / T, L266P, V269I / L / M, N272C / G / T, T274I / Q, K278A and R286M <34>The method according to <32>, wherein the substitution comprises at least one substitution selected from the following (A') to (D'). (A') Substitution of N272C / G with the numbering of SEQ ID NO: 4 and one or more substitutions selected from L16F, G22M, Y29F, Y43W, L50I / V, S120A / Q, K123I / T, R126E, L142F, G156A, A181K, Q186E, K188Q, L190F, D191V, L208C, M212L, V220H, A221H, L223V, A224Q, F225T, L228W, R233K / T, F234W, S256N, V260A / C / L, V269I / L, T274Q, and K278A (B') Substitution of V220H with the numbering of SEQ ID NO: 4 and one or more substitutions selected from L16F, G22C / M, Y43W, L50I / V, S120Q, K123I / T, R126E, L142F, G156A, A181K, Q186E, K188Q, L190F, D191A / E / V, L208C, M212L, A221H, L223V, A224Q, F225T, L228W, R233K / Q, F234W, S256N, V260A / C / L, V269I / L / M, N272C / G, T274Q, and K278A (C') Substitution of V260A / C / L with the numbering of SEQ ID NO: 4 and one or more substitutions selected from L16F, G22M, T46Q, L50I / V, K123I, R126E, L142F, G156A, L190F, D191A / E, L208C, M212L, V220H, A224F / Q / Y, F225T, L228W, R233K / Q, R234W, V269I / M, N272C / G, and K278A (D') Substitution of L208C with the numbering of SEQ ID NO: 4 and one or more substitutions selected from L16F, G22C, L50I / V, K123I, R126E, K188Q, L190F, D191A, M212L, V220H, L223V, A224Q / Y, F225T, L228W, R233K, F234W, S256N, V260A / C / L, V269I, N272C / G, K278A, and R286M <35>The method according to <34>, wherein (A') to (D') are as follows. (A’) One or more substitutions selected from the substitution of N272C with the numbering of SEQ ID NO: 4 and L16F, G22M, Y29F, Y43W, L50I / V, S120A / Q, K123I, R126E, L142F, G156A, A181K, K188Q, D191V, L208C, V220H, A221H, L223V, A224Q, F225T, R233K / T, F234W, S256N, V260A / C / L, V269I and T274Q (B’) One or more substitutions selected from the substitution of V220H with the numbering of SEQ ID NO: 4 and G22C / M, L50I / V, K123I / T, R126E, L142F, G156A, A181K, Q186E, D191V, L208C, M212L, A221H, L223V, L228W, R233K / Q, F234W, S256N, V260C / L, V269I / L / M, N272C / G, T274Q and K278A (C’) One or more substitutions selected from the substitution of V260C with the numbering of SEQ ID NO: 4 and L50I / V and L142F, or (C’) one or more substitutions selected from the substitution of V260L with the numbering of SEQ ID NO: 4 and G22M, L50I / V, G156A, D191A / E, L208C, M212L, V220H, A224F / Q / Y, L228W, V269I / M and N272G (D’) One or more substitutions selected from the substitution of L208C with the numbering of SEQ ID NO: 4 and L16F, G22C, R126E, K188Q, D191A, V220H, L223V, A224Q / Y, L228W, S256N, V260A / C / L, V269I, N272C / G, K278A and R286M <36>The method according to <34>, wherein the above (A’) to (D’) are as follows. (A’) One or more substitutions selected from the substitution of N272C with the numbering of SEQ ID NO: 4 and G22M, K123I, G156A, K188Q, L208C, V220H, A221H, L223V, A224Q, F225T, R233T, S256N, V260A / C / L and V269I (B’) Substitution of V220H with the numbering of SEQ ID NO: 4 and one or more substitutions selected from G22C / M, G156A, L208C, M212L, L228W, S256N, V260C / L, V269I / L / M, and N272C / G (C’) Substitution of V260C with the numbering of SEQ ID NO: 4 and one or more substitutions selected from L50I / V and L142F, or substitution of V260L with the numbering of SEQ ID NO: 4 and one or more substitutions selected from G22M, G156A, L208C, V220H, L228W, V269I / M, and N272G (D’) Substitution of L208C with the numbering of SEQ ID NO: 4 and one or more substitutions selected from L16F, G22C, K188Q, V220H, L228W, S256N, V260A / C / L, V269I, and N272C / G <37>The method according to <32>, wherein the substitution at least includes substitution of V220H with the numbering of SEQ ID NO: 4 and substitution of N272C / G, or substitution of V260L with the numbering of SEQ ID NO: 4 and substitution of V269I. <38>The method according to <32>, wherein the substitution at least includes substitution of L208C with the numbering of SEQ ID NO: 4, substitution of V220H, and substitution of N272C, substitution of L142F, substitution of V220H, and substitution of N272G, or substitution of V260L with the numbering of SEQ ID NO: 4, substitution of V269I, and substitution of N272G. <39>The method according to <32>, wherein the substitution at least includes substitution of L208C with the numbering of SEQ ID NO: 4, substitution of V220H, substitution of V269I, and substitution of N272C, substitution of L50V, substitution of L142F, substitution of V220H, and substitution of N272G, or substitution of L50V, substitution of V260L, substitution of V269I, and substitution of N272G. <40>The method according to <32>, wherein the substitution at least includes substitution of L208C with the numbering of SEQ ID NO: 4, substitution of V220H, substitution of S256N, substitution of V269I, and substitution of N272C, or substitution of L50V, substitution of L142F, substitution of V220H, substitution of V260L, and substitution of N272G. <41>The method according to any one of <30> to <32>, wherein the substitution at least includes any substitution or combination of substitutions shown in Table 1-1 to 1-5 and Table 2-1 to 2-3 above. <42>If the lipase or the lipase after amino acid residue substitution of the lipase has at least 80% sequence identity with the amino acid sequence represented by SEQ ID NO: 6, the substitution of the amino acid residues at the positions corresponding to the positions of L16, G22, Y29, Y43, A45, T46, L50, S120, K123, R126, T134, L142, A145, G156, K174, A181, K188, L190, D191, L208, H209, M212, L215, V220, A221, L223, A224, F225, L228, R233, F234, S256, V260, L266, V269, N272, T274, K278 and R286 in the numbering of SEQ ID NO: 4 are, respectively, L17F, G23C / M, Y30F, Y44W, A46V, T476Q, L51I / M / V, S121A / Q, T124I, R127E, T135A, L143F, A146C / Q, G157A, K175D, A182K, K189Q, L191F, V192A / E, L209C, H210K, A213L, L216C, V221H, A222H, L224A / E / S / V, A225F / Q / Y, F226T, L229W, R234K / Q / T, F235W, S257N, V261A / C / F / G / L / M / T, L267P, V270I / L / M, N273C / G / T, T275I / Q, Q279A and R287M in the numbering of SEQ ID NO: 6, the method according to any one of <30> to <41>.

Example

[0093] Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited thereto.

[0094] Example 1 (1) Construction of lipase expression plasmid Using the plasmid pHY-S237 described in Example 7 of WO2006 / 068148A1 as a template, the entire ORF of the alkaline cellulase gene was replaced by artificial gene synthesis with CnLip I44T L120S S130A A134T A135V L136M S137N (hereinafter referred to as CnLipA, encoding the polynucleotide of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2) or CspLip I45T L121S S131A A135T A136V S138N A213M (hereinafter referred to as CspLipA, encoding the polynucleotide of SEQ ID NO: 5 and the amino acid sequence of SEQ ID NO: 6) described in Japanese Patent Application No. 2023-025141 by In-Fusion reaction to construct plasmids pHY-CnLipA and pHY-CspLipA. Using pHY-CnLipA as a template, five mutations were introduced by site-directed mutagenesis by PCR using a complementary primer pair (Zheng, Lei, Ulrich Baumann, and Jean-Louis Reymond. Nucleic Acids Research 32.14 (2004): e115-e115.) to create a plasmid pHY-CnLipAm expressing CnLipAm (encoding the polynucleotide of SEQ ID NO: 3 and the amino acid sequence of SEQ ID NO: 4). For further mutagenesis of lipase, site-directed mutagenesis by PCR using a complementary primer pair was used.

[0095] (2) Preparation of lipase solution The lipase expression plasmid was introduced into the Bacillus subtilis strain 168ΔsigF strain described in JP 2003-47490 A by the protoplast method, and cultured in 2×L-maltose medium (2% tryptone, 1% yeast extract, 1% NaCl, 7.5% maltose, 7.5 ppm manganese sulfate pentahydrate, 0.04% calcium chloride dihydrate, 15 ppm tetracycline; % is (w / v)%) at 30 °C for 3 days. Then, the culture supernatant containing lipase was recovered by centrifugation.

[0096] (3) Stability evaluation in model cleaning solution 4-Nitrophenyl butyrate (SIGMA) was used as a substrate. The lipase activity can be determined by measuring the rate of increase in absorbance associated with the release of 4-nitrophenol due to the action of lipase. A substrate solution was prepared by adding 4-nitrophenyl butyrate to 20 mM Tris-HCl (pH 7.0) at a final concentration of 2 mM and mixing them. The lipase solution prepared by the method described in (2) was added to the model cleaning solution shown in Table 3, and after incubating at 40, 45, 50, 55, or 60 °C for the time described in Tables 4 to 20, the activity was measured using a solution diluted 51-fold with 20 mM Tris-HCl (pH 7.0). Using the activity value of the sample before treatment at 40, 45, 50, 55, or 60 °C as the initial activity, the inactivation rate per unit time (h) due to treatment at 40, 45, 50, 55, or 60 °C was calculated, and the half-life (h) was calculated therefrom. The relative stability was determined by dividing the half-life (h) of each mutant by the half-life (h) of the parent polypeptide. The results are shown in Tables 4 to 20. Each table shows the relative stability of the mutants obtained by introducing the mutations shown in the table into the parent polypeptide (parent enzyme) at the topmost row. It was shown that the stability of all mutants was improved compared to the parent polypeptide.

[0097]

Table 3

[0098]

Table 4

[0099]

Table 5-1

[0100]

Table 5-2

[0101]

Table 5-3

[0102]

Table 5-4

[0103]

Table 5-5

[0104]

Table 6-1

[0105]

Table 6-2

[0106]

Table 6-3

[0107]

Table 7-1

[0108]

Table 7-2

[0109]

Table 8-1

[0110]

Table 8-2

[0111]

Table 9

[0112]

Table 10-1

[0113]

Table 10-2

[0114]

Table 11

[0115]

Table 12

[0116]

Table 13

[0117]

Table 14

[0118]

Table 15-1

[0119]

Table 15-2

[0120]

Table 15-3

[0121]

Table 15-4

[0122]

Table 16-1

[0123]

Table 16-2

[0124]

Table 16-3

[0125]

Table 17-1

[0126]

Table 17-2

[0127]

Table 17-3

[0128]

Table 17-4

[0129]

Table 18

[0130]

Table 19

[0131]

Table 20

[0132] (4) Evaluation of detergency in the model cleaning solution The ability to remove triglycerides on a hard surface was evaluated as detergency. Tallow (SIGMA, 03-0660) and rapeseed oil (SIGMA, 23-0450) were mixed at a weight ratio of 9:1, dissolved in three times the amount of chloroform, and then colored with 0.2 wt% Sudan III to obtain a model stain. 10 μL of the model stain was dropped into the bottom of each well of a 96-well deep well plate made of polypropylene, and the chloroform was evaporated and dried to obtain a stained plate. The lipase solution was prepared by diluting the culture supernatant of (2) to a lipase concentration of 200 mg / L with 20 mM Tris-HCl (pH 7.0) in the model cleaning solution described in Table 3, and 1 / 50 volume was added to the model cleaning solution. 300 μL of the cleaning solution was gently added to the stained plate and allowed to stand at room temperature (about 22 °C) for 15 minutes for immersion cleaning. 100 μL of the cleaning solution was aliquoted without touching the stain at the bottom and transferred to a new 96-well plate. To quantify Sudan III in the model stain solubilized in the cleaning solution by immersion cleaning, the absorbance at 500 nm (A500) was measured. A500 corresponds to the amount of oil released into the cleaning solution and can be used as an index of detergency. The enzymatic effect of detergency (ΔA500) was determined by subtracting the A500 of the cleaning solution added with 20 mM Tris-HCl (pH 7.0) instead of lipase from the A500 of the cleaning solution containing each lipase. The cleaning results for 15 minutes are shown in Table 21. It was confirmed that the detergency of the stabilized mutant of the present invention did not decrease.

[0133]

Table 21

[0134] Reference Example 1: Comparison of stability in model cleaning solution and other formulations A commercially available liquid detergent (Kao Corporation, Attack ZERO (hereinafter referred to as Detergent 1)) was left standing in a boiling water bath for 30 minutes to inactivate the enzymes contained therein. Savinase (SIGMA, P3111) was added to this detergent to a final concentration of 0.5% and mixed well. CnLipAm prepared by the method described in (2) was added to the model cleaning solution shown in Table 3 and Detergent 1 containing 0.5% Savinase, and after incubating at 45°C for 1 hour, activity measurement was performed using a solution diluted 51-fold with 20 mM Tris-HCl (pH 7.0). The activity value of the sample before the 45°C treatment was defined as the initial activity, and the ratio of the activity value after the treatment to the initial activity was defined as the residual activity (%). The results are shown in Table 22. In the model cleaning solution described in Table 3, in which the suitability for cleaning is disclosed in Patent Document 1, a phenomenon was confirmed in which the lipase was significantly destabilized even when compared with other formulations containing proteases that decompose proteins.

[0135]

Table 22

Claims

1. In the numbering of SEQ ID NO:4, L16, G22, Y29, Y43, A45, T46, L50, S120, K123, R126, T134, L142, A145, G156, K174, A181, Q186, K188, L190, D191, L208, H209, M212, L215, V220, A221, L223, A224, F225, L228, R233, F23 4, S256, V260, L266, V269, N272, T274, K278 and R286, wherein said parent lipase or said lipase variant has at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4 or 6.

2. In the numbering of SEQ ID NO: 4, L16, G22, Y29, Y43, A45, T46, L50, S120, K123, R126, T134, L142, A145, G156, K174, A181, Q186, K188, L190, D191, L208, H209, M212, L215, V220, A22 The amino acid residues at the positions corresponding to the positions of L1, L223, A224, F225, L228, R233, F234, S256, V260, L266, V269, N272, T274, K278, and R286 are substituted with L16F, G22C / M, Y29F, Y43W, A45V, T46Q, L50, I / M / V, S120A / Q, K123I / T, R126E, T134A, L142F, A145C / Q, G156A, K174D, A181K, Q 186E, K188Q, L190F, D191A / E / V, L208C, H209K, M212L, L215C, V220H, A221H, L223 2. The lipase variants of claim 1, which are A / E / S / V, A224F / Q / Y, F225T, L228W, R233K / Q / T, F234W, S256N, V260A / C / F / G / L / M / T, L266P, V269I / L / M, N272C / G / T, T274I / Q, K278A and R286M.

3. The lipase variant according to claim 2, which comprises at least any one of the following substitutions (A) to (D): (A) Substitutions N272C / G / T and L16F, G22C / M, Y29F, Y43W, A45V, T46Q, L50I / M / V, S120A / Q, K123I / T, R126E, T134A, L142F, A145C / Q, G156A, K174D, A181K, Q186E, K188Q, L190F, D191A / E / V, L 208C, H209K, M212L, L215C, V220H, A221H, L223A / E / S / V, A224F / Q / Y, F225T, L228W, R233K / Q / T, F234W, S256N, V260A / C / F / G / L / M / T, L266P, V269I / L / M, T274I / Q, K278A, and R286M (B) Substitution of V220H in the numbering of SEQ ID NO:4 with L16F, G22C / M, Y29F, Y43W, A45V, T46Q, L50I / M / V, S120A / Q, K123I / T, R126E, T134A, L142F, A145C / Q, G156A, K174D, A181K, Q186E, K188Q, L190F, D191A / E / V, L208C , H209K, M212L, L215C, A221H, L223A / E / S / V, A224F / Q / Y, F225T, L228W, R233K / Q / T, F234W, S256N, V260A / C / F / G / L / M / T, L266P, V269I / L / M, N272C / G / T, T274I / Q, K278A, and one or more substitutions selected from R286M. (C) Substitutions V260A / C / F / G / L / M / T in the numbering of SEQ ID NO:4 and L16F, G22C / M, Y29F, Y43W, A45V, T46Q, L50I / M / V, S120A / Q, K123I / T, R126E, T134A, L142F, A145C / Q, G156A, K174D, A181K, Q186E, K188Q, L190F, D19 One or more substitutions selected from 1A / E / V, L208C, H209K, M212L, L215C, V220H, A221H, L223A / E / S / V, A224F / Q / Y, F225T, L228W, R233K / Q / T, F234W, S256N, L266P, V269I / L / M, N272C / G / T, T274I / Q, K278A, and R286M (D) Substitution of L208C in the numbering of SEQ ID NO:4 with L16F, G22C / M, Y29F, Y43W, A45V, T46Q, L50I / M / V, S120A / Q, K123I / T, R126E, T134A, L142F, A145C / Q, G156A, K174D, A181K, Q186E, K188Q, L190F, D191A / E / V, H209K , M212L, L215C, V220H, A221H, L223A / E / S / V, A224F / Q / Y, F225T, L228W, R233K / Q / T, F234W, S256N, V260A / C / F / G / L / M / T, L266P, V269I / L / M, N272C / G / T, T274I / Q, K278A, and one or more substitutions selected from R286M.

4. The lipase variant according to claim 2, which comprises at least any one of the following substitutions (A') to (D'): (A') the substitution N272C / G in the numbering of SEQ ID NO: 4 and one or more substitutions selected from L16F, G22M, Y29F, Y43W, L50I / V, S120A / Q, K123I / T, R126E, L142F, G156A, A181K, Q186E, K188Q, L190F, D191V, L208C, M212L, V220H, A221H, L223V, A224Q, F225T, L228W, R233K / T, F234W, S256N, V260A / C / L, V269I / L, T274Q and K278A (B') the substitution V220H in the numbering of SEQ ID NO: 4 and one or more substitutions selected from L16F, G22C / M, Y43W, L50I / V, S120Q, K123I / T, R126E, L142F, G156A, A181K, Q186E, K188Q, L190F, D191A / E / V, L208C, M212L, A221H, L223V, A224Q, F225T, L228W, R233K / Q, F234W, S256N, V260A / C / L, V269I / L / M, N272C / G, T274Q, and K278A (C') the substitution V260A / C / L in the numbering of SEQ ID NO: 4 and one or more substitutions selected from L16F, G22M, T46Q, L50I / V, K123I, R126E, L142F, G156A, L190F, D191A / E, L208C, M212L, V220H, A224F / Q / Y, F225T, L228W, R233K / Q, R234W, V269I / M, N272C / G and K278A; (D') the substitution L208C in the numbering of SEQ ID NO: 4 and one or more substitutions selected from L16F, G22C, L50I / V, K123I, R126E, K188Q, L190F, D191A, M212L, V220H, L223V, A224Q / Y, F225T, L228W, R233K, F234W, S256N, V260A / C / L, V269I, N272C / G, K278A and R286M.

5. A polynucleotide encoding the lipase variant according to any one of claims 1 to 4.

6. A vector or DNA fragment comprising the polynucleotide of claim 5.

7. A transformed cell containing the vector or DNA fragment according to claim 6.

8. The transformed cell according to claim 7, which is a microorganism.

9. A detergent composition comprising the lipase variant according to any one of claims 1 to 4.

10. In the numbering of SEQ ID NO:4, L16, G22, Y29, Y43, A45, T46, L50, S120, K123, R126, T134, L142, A145, G156, K174, A181, Q186, K188, L190, D191, L208, H209, M212, L215, V220, A221, L223, A224, F225, L228, R233, F234, S256 5. A method for improving the stability of a lipase, comprising a step of substituting amino acid residues at one or more positions selected from positions corresponding to positions 1, 2, 3, 4, 5, 6, V260, L266, V269, N272, T274, K278 and R286, wherein the lipase or the lipase after amino acid residue substitution has at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 4 or 6.

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