Lipase variant

Abstract

To provide a lipase variant exhibiting excellent stability. Provided is a lipase variant that includes substitution of amino acid residues at one or more positions selected from positions corresponding to L16, G22, Y29, Y43, A45, T46, L50, S120, K123, R12 6, 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 the 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.

Description

Lipase variants

[0001] The present invention relates to lipase variants.

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

[0003] Detergent lipases have been employed in detergent compositions for removing oily stains. Patent Document 1 describes a method for cleaning oily stains by contacting a detergent containing a sulfosuccinic acid alkyl ester with the stains together with the lipase, and then leaving the mixture without applying external force. Furthermore, as described in Patent Document 2, dishwashing detergent compositions are sometimes used to clean the areas around stainless steel or resin sinks. To impart the ability to remove limescale and other deposits from around kitchen sinks, it is conceivable to incorporate a high concentration of a chelating agent such as citric acid. The composition of such detergents is characterized by a higher water content than typical detergents and a higher content of solvent than surfactants.

[0004] (Patent Document 1) JP 2021-17508 A (Patent Document 2) Japanese Patent No. 4776997 A

[0005] The present invention relates to the following 1) to 6): 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, F A variant of a parent lipase comprising substitutions of amino acid residues at one or more positions selected from positions corresponding to positions 234, S256, V260, L266, V269, N272, T274, K278 and R286, wherein the parent lipase or lipase variant has at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:4 or 6. 2) A polynucleotide encoding the lipase variant according to 1). 3) A vector or DNA fragment comprising 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 variant according to 1). 6) A lipase variant comprising L16, G22, Y29, Y43, A45, T46, L50, S120, K123, R126, T134, L142, A145 and G156 in the numbering of SEQ ID NO:4. , 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, wherein the lipase or the lipase after the amino acid residue substitution has at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 4 or 6. Detailed Description of the Invention

[0006] All patents, non-patent documents, and other publications cited herein are hereby incorporated by reference in their entirety.

[0007] As used herein, "lipase" refers to triacylglycerol lipase (EC 3.1.1.3), a group of enzymes that have the activity of hydrolyzing ester bonds in lipids to produce fatty acids. Lipase activity can be determined by measuring the rate of increase in absorbance associated with the release of 4-nitrophenol by hydrolysis of 4-nitrophenyl butyrate. Specific procedures for measuring lipase activity are described in detail in the Examples below.

[0008] Herein, the identity of an amino acid sequence or a nucleotide sequence is calculated by the Lipman-Pearson method (Science, 1985, 227:1435-1441). Specifically, the identity is calculated by performing an analysis using the Search homology program in the genetic information processing software GENETYX Ver. 12, with the unit size to compare (ktup) set to 2.

[0009] As used herein, "at least 80% identity" with respect to an amino acid sequence or a nucleotide sequence refers to identity of 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 93% or more, even more preferably 94% or more, even more preferably 95% or more, even more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more, and even more preferably 99.5% or more.

[0010] As used herein, a "corresponding position" on an amino acid sequence or a nucleotide sequence can be determined by aligning a target sequence with a reference sequence (e.g., the amino acid sequence set forth in SEQ ID NO: 4) to maximize homology. Alignment of amino acid sequences or nucleotide sequences can be performed using known algorithms, and the procedures are well known to those skilled in the art. For example, alignment can be performed using the Clustal W multiple alignment program (Thompson, J.D. et al., 1994, Nucleic Acids Res. 22:4673-4680) with default settings. Alternatively, revised versions of Clustal W, such as Clustal W2 and Clustal omega, can also be used. Clustal W, Clustal W2, and Clustal omega are available, for example, on the Clustal website operated by University College Dublin [www.clustal.org], the European Bioinformatics Institute (EBI [www.ebi.ac.uk / index.html]), and the website of the DNA Data Bank of Japan (DDBJ [www.ddbj.nig.ac.jp / searches-j.html]) operated by the National Institute of Genetics. The position of the target sequence aligned to any position in the reference sequence by the above-mentioned alignment is considered to be a "position corresponding to" that position.

[0011] Those skilled in the art can further fine-tune the alignment of amino acid sequences obtained above to optimize it. Such optimal alignment is preferably determined taking into account the similarity of the amino acid sequences, the frequency of inserted gaps, and the like. Here, amino acid sequence similarity refers to the percentage (%) of the number of positions at which identical or similar amino acid residues exist in both aligned amino acid sequences relative to the total number of amino acid residues in the two sequences. Similar amino acid residues refer to amino acid residues among the 20 amino acids that constitute proteins that have similar properties in terms of polarity and charge, resulting in so-called conservative substitutions. Groups of such similar amino acid residues are well known to those skilled in the art, and 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.

[0012] As used herein, the term "amino acid residue" refers to the 20 amino acid residues that make up 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).

[0013] As used herein, amino acid positions and variants are represented using the recognized IUPAC single-letter amino acid abbreviations, as follows: An amino acid at a given position is represented as [amino acid, position]. For example, valine at position 220 is represented as "V220." Amino acid "substitutions" are represented as [original amino acid, position, substituted amino acid]. For example, a substitution of valine at position 220 with histidine is represented as "V220H." Variants containing multiple modifications are represented by a plus sign ("+"). For example, "V220H+N272G" represents a substitution of valine at position 220 with histidine and asparagine at position 272 with glycine, respectively. When different modifications can be introduced at a single position, the different modifications are separated by a slash (" / "); for example, "G22C / M" represents a substitution of glycine at position 22 with cysteine ​​or methionine.

[0014] As used herein, the term "operably linked" between a gene and a regulatory region such as a promoter means that the gene and regulatory region are linked in such a way that the gene can be expressed under the control of the regulatory region. Procedures for "operably linking" a gene to a regulatory region are well known to those skilled in the art.

[0015] As used herein, "upstream" and "downstream" in relation 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 located on the 3' side of the promoter on the DNA sense strand, and "upstream" of a gene means the 5' region of the gene on the DNA sense strand.

[0016] The stability of lipases formulated in detergent compositions containing a higher water content and a higher concentration of solvents than surfactants, which are harsh environments for enzymes, was unknown. However, the inventors' investigations revealed that lipase stability is significantly reduced compared to when formulated in a typical detergent. Therefore, the present invention relates to providing a lipase that exhibits excellent stability.

[0017] The present inventors have obtained lipase variants that have improved stability in the presence of large amounts of water and / or solvents in amounts greater than surfactants, compared to the parent lipase.

[0018] The lipase variants of the present invention have improved stability compared to the parent lipase, and can be suitably incorporated into detergent compositions containing a large amount of water and / or a solvent in an amount greater than that of a surfactant.

[0019] <1. Lipase variant> The lipase variant of the present invention includes, 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, A variant of a parent lipase comprising a substitution of an amino acid residue at one or more positions selected from positions corresponding to R233, F234, S256, V260, L266, V269, N272, T274, K278 and R286, wherein the parent lipase or the lipase variant has at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4 or 6.

[0020] That is, the term "variant" refers to a polypeptide having lipase activity in which amino acid residues at one or more positions selected from the group consisting 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 in the parent lipase have been substituted. The substitution of amino acid residues at these predetermined positions is a modification for improving stability, and therefore the variant has improved stability compared to the parent lipase, more specifically, stability in the presence of a large amount of water and / or a solvent in an amount greater than that of a surfactant.

[0021] "Parent lipase" refers to the reference lipase to which modification is made to yield the variant of the invention, which in the present invention is a lipase having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 4 or 6. The parent lipase may be a naturally occurring (wild-type) polypeptide or a variant thereof.

[0022] An example of a parent lipase is a polypeptide having lipase activity and consisting of an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 4. Here, the lipase consisting of the amino acid sequence set forth in SEQ ID NO: 4 is a lipase in which the amino acid sequence constituting the lipase CnLip (NCBI Accession No. WP_061278013.1) derived from Cedecea neteri has been substituted with the amino acid residues I44T, L120S, S130A, A134T, A135V, L136M, and S137N (CnLipA having excellent detergency as described in Japanese Patent Application No. 2023-025141), and further with the amino acid residues F16L, I47L, A91V, E97D, and A149E, and is designated CnLipAm as shown in the Examples below. The above-mentioned CnLip and CnLipA can also be parent lipases for the mutants of the present invention.

[0023] Another example of a parent lipase is a polypeptide having lipase activity and consisting of an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 6. Here, the lipase consisting of the amino acid sequence set forth in SEQ ID NO: 6 is a lipase (CspLipA ​​with excellent detergency described in Japanese Patent Application No. 2023-025141) derived from Cedecea sp., in which the amino acid residues I45T, L121S, S131A, A135T, A136V, and S138N have been substituted in the amino acid sequence constituting the lipase CspLip (NCBI Accession No. WP_016537805.1), and this is the CspLipA ​​shown in the Examples below.

[0024] The "substitution" of an amino acid residue at a predetermined position refers to replacing an amino acid at a certain position with a different amino acid. In the present invention, the number of amino acid substitutions may be two or more, but from the viewpoint of stability, the number of substitutions is preferably two or more, more preferably 2 to 20, and even more preferably 3 to 10. From the viewpoint of stability and detergency, the mutant is preferably a lipase having at least 80% identity to the amino acid sequence shown in SEQ ID NO: 4 or 6. Furthermore, the mutant may contain any number of conservative amino acid substitutions as long as the properties of the mutant are maintained.

[0025] Preferred embodiments of substitutions of amino acid residues at 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 in the lipase variant of the present invention are shown below. That is, L16 is preferably substituted with F (L16F); G22 is preferably substituted with C or M (G22C / M); Y29 is preferably substituted with F (Y29F); Y43 is preferably substituted with W (Y43W); A45 is preferably substituted with V (A45V); T46 is preferably substituted with Q (T46Q); L50 is preferably substituted with I, M or V (L50I / M / V); S120 is preferably substituted with A or Q (S120A / Q); K123 is preferably substituted with I or T (K123I / T); R126 is preferably substituted with E (R126E); T134 is preferably substituted with A (T134A); L142 is preferably substituted with F (L142F); A145 is preferably substituted with C or Q (A145C / Q); G156 is preferably substituted with A (G156A); 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); and R286 is preferably substituted with M (R286M).

[0026] Next, preferred combinations of mutations that contribute to improved stability are shown in (A) to (D) below. Therefore, a mutant having at least any of the following combinations of mutations (A) to (D) contributes to improved stability. (A) A combination of N272C / G / T substitutions 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, D191A / E / V, L192F, D193A / E / V, L194F, D195A / E / V, L196F, D197A / E / V, L198F, D199A / E / V, L199A / E / V, L200F, D201A / E / V, L202F, D203A / E / V, L204F, D205A / E / V, L206F, D207A / E / V, L208F, D210F, D211A / E / V, L212F, D213A / E / V, L214F, D215A / E / V, L216F, D217A / E / V, L218F, D219A / E / V, L220F, D220F, D221A / E / V, L222F, D222F, D223A / E / V, L223A / E / V, L224F, D225A / E / V, L225 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 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 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.

[0027] More preferred combinations of mutations that contribute to improved stability are shown in (A') to (D') below. Therefore, a mutant having at least one of the following combinations of mutations (A') to (D') is a mutant that contributes more to improved stability. (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') A substitution of 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. From the viewpoint of stability, the following combinations of mutations are preferred among the combinations of mutations (A') to (D') above.(A') the substitution N272C in the numbering of 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') the substitution V220H in the numbering of 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') a substitution of V260C in the numbering of SEQ ID NO: 4 and one or more substitutions selected from L50I / V and L142F, or (C') a substitution of V260L in the numbering of 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') A substitution of L208C in the numbering of 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. Furthermore, from the viewpoint of stability, of the combinations of mutations (A') to (D') above, the following combinations of mutations are more preferred.(A') a substitution of N272C in the numbering of 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') a substitution of V220H in 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') a substitution of V260C in the numbering of SEQ ID NO: 4 and one or more substitutions selected from L50I / V and L142F, or a substitution of V260L in the numbering of SEQ ID NO: 4 and one or more substitutions selected from G22M, G156A, L208C, V220H, L228W, V269I / M and N272G; (D') a substitution of L208C in 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] Further preferred combinations of mutations that contribute to improved stability include, in the numbering of SEQ ID NO: 4, a V220H substitution and a N272C / G substitution, a V260L substitution and a V269I substitution, a L208C substitution and a V220H substitution and a N272C substitution, a L142F substitution and a V220H substitution and a N272G substitution, a V260L substitution and a V269I substitution and a N272G substitution, and a L208C substitution and a V220H substitution and a V269I substitution. and N272C substitution, L50V substitution, L142F substitution, V220H substitution, and N272G substitution, L50V substitution, V260L substitution, V269I substitution, and N272G substitution, L208C substitution, V220H substitution, S256N substitution, V269I substitution, and N272C substitution, or L50V substitution, L142F substitution, V220H substitution, V260L substitution, and N272G substitution. Therefore, a mutant having at least any of these combinations of mutations is a mutant that further contributes to improved stability.

[0029] More preferred mutations or combinations of mutations in the lipase variants 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, variants having at least any of the following mutations or combinations of mutations are variants that particularly contribute to improved stability.

[0030]

[0031]

[0032] In addition, the 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 are the same as those in the sequence In the numbering of No. 6, these correspond to positions 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, respectively. Thus, if a parent lipase has at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:6, the amino acids at 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 may be present. Substitution of amino acid residues can be interpreted as substitution of amino acid residues at positions corresponding to positions 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 shown in SEQ ID NO: 6, it preferably does not contain the substitutions T at position 124, E at position 187, and V at position 192. When the parent lipase has at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 4, the parent lipase preferably has the following amino acid residues at the positions substituted in the variant of the present invention, as numbered in 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, V260, L26 and more preferably has the amino acid residues 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.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 the following amino acid residues at the positions substituted in the variant of the present invention, as numbered in SEQ ID NO: 6: 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, L26 and more preferably has the amino acid residues 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.

[0033] The lipase variants of the present invention can be produced by substituting amino acid residues at one or more positions selected from the group consisting 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 in the parent lipase. wherein the parent lipase or lipase variant has at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:6.

[0034] 2. Polynucleotides Encoding Lipase Variants of the Present Invention The lipase variants of the present invention can be produced using various mutagenesis techniques known in the art. For example, they can be produced by mutating a polynucleotide encoding an amino acid residue to be substituted in a parent lipase gene (reference lipase gene) encoding the reference amino acid sequence to a polynucleotide encoding the substituted amino acid residue, and then expressing the variant from the mutant gene.

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

[0036] Introduction of a desired mutation into a parent gene can basically be carried out using various site-directed mutagenesis methods well known to those skilled in the art. Site-directed mutagenesis can be carried out by any method, such as inverse PCR or annealing. Commercially available site-directed mutagenesis kits (e.g., Stratagene's QuickChange II Site-Directed Mutagenesis Kit or QuickChange Multi Site-Directed Mutagenesis Kit) can also be used.

[0037] Site-specific mutagenesis of a parent gene can most commonly be performed using a mutagenesis primer containing the nucleotide mutation to be introduced. The mutagenesis primer anneals to a region of the parent gene containing a nucleotide sequence encoding the amino acid residue to be mutated, and is designed to contain a nucleotide sequence having a nucleotide sequence (codon) encoding the mutated amino acid residue in place of the nucleotide sequence (codon) encoding the amino acid residue to be mutated. Those skilled in the art can recognize and select the nucleotide sequences (codons) encoding the amino acid residues before and after mutation as appropriate based on standard textbooks, etc. Alternatively, site-specific mutagenesis can be performed using a method in which DNA fragments upstream and downstream of the mutation site are amplified separately using two complementary primers containing the nucleotide mutation to be introduced, and the resulting fragments are then ligated together using SOE (splicing by overlap extension)-PCR (Gene, 1989, 77(1): pp. 61-68).

[0038] Template DNA containing the parent gene can be prepared from a microorganism that produces the parent lipase by standard methods, either by extracting genomic DNA or by synthesizing cDNA by reverse transcription of extracted RNA. Alternatively, a corresponding nucleotide sequence can be chemically synthesized based on the amino acid sequence of the parent lipase and used as template DNA. DNA sequences containing base sequences encoding 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 mutagenesis primer can be prepared by a well-known oligonucleotide synthesis method such as the phosphoramidite method (Nucleic Acids Research, 1989, 17:7059-7071). Such primer synthesis can also be performed using, for example, a commercially available oligonucleotide synthesizer (manufactured by ABI, etc.). A primer set containing the mutagenesis primer can be used to perform site-specific mutagenesis as described above using a parent gene as template DNA to obtain a polynucleotide encoding the lipase variant of the present invention having the desired mutation.

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

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

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

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

[0044] Alternatively, the vector of the present invention may further incorporate a marker gene (e.g., a resistance gene to a drug such as ampicillin, neomycin, kanamycin, or chloramphenicol) for selecting a host into which the vector has been appropriately introduced. Alternatively, when an auxotrophic strain is used as the host, a gene encoding an enzyme that synthesizes the required nutrient may be incorporated into the vector as a marker gene. Furthermore, when a selective medium requiring a specific metabolism for growth is used, a gene related to that metabolism may be incorporated into the vector as a marker gene. An example of such a metabolism-related gene is the acetamidase gene for utilizing acetamide as a nitrogen source.

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

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

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

[0048] The vector can be introduced into the host by a method commonly used in the field, such as the protoplast method, electroporation, etc. Strains into which the vector has been appropriately introduced can be selected based on the expression of a marker gene, auxotrophy, etc., to obtain the desired transformant into which the vector has been introduced.

[0049] Alternatively, a fragment comprising a polynucleotide encoding the lipase variant of the present invention, a regulatory sequence, and a marker gene linked together can be directly introduced into the genome of a host. For example, a DNA fragment having sequences complementary to the host genome added to both ends of the linked fragment can be constructed by SOE-PCR or the like, and this can be introduced into the host to induce homologous recombination between the host genome and the DNA fragment, thereby introducing the polynucleotide encoding the lipase variant of the present invention into the genome of the host.

[0050] When the thus obtained transformant into which a polynucleotide encoding the lipase variant of the present invention or a vector containing the same has been introduced is cultured in an appropriate medium, the gene encoding the protein on the vector is expressed to produce the lipase variant of the present invention. The medium used to culture the transformant can be appropriately selected by those skilled in the art depending on the type of microorganism used as the transformant.

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

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

[0053] 5. Method for Improving Lipase Stability 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 large amount of water and / or a solvent in a larger amount than a surfactant. "Stability" refers to the ability to maintain lipase activity, and "improved stability" refers to an ability to maintain lipase activity that is improved compared to the parent lipase, and "improved stability in the presence of a large amount of water and / or a solvent in a larger amount than a surfactant" refers to an ability to maintain lipase activity in the presence of a large amount of water and / or a solvent in a larger amount than a surfactant, which is improved compared to the parent lipase. Here, "stability in the presence of a large amount of water" refers to the ability to maintain lipase activity when stored in a detergent composition containing 60 to 99% by mass of water, and "stability in the presence of a solvent in a larger amount than a surfactant" refers to the ability to maintain lipase activity in a detergent composition containing a solvent in a larger amount than the surfactant, or in a cleaning solution prepared by dissolving or diluting the detergent composition in water, which contains a solvent in a larger amount than the surfactant. Examples of the surfactant and solvent include those described below.

[0054] The stability of such lipases can be evaluated using methods well known in the art. For example, lipase stability 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 lipase activity before and after treatment for a predetermined period of time, and calculating the half-life of the activity based on the deactivation rate per unit time due to treatment, using the activity value of the sample before treatment as the initial activity. The value obtained by dividing the half-life of the lipase variant by the half-life of the parent lipase indicates the relative stability of the lipase variant. A ratio greater than 1 indicates that the lipase variant has improved stability compared to the parent lipase. Examples of cleaning solutions include, but are not limited to, model cleaning solutions having the compositions shown in Table 3 in the Examples below. The stability of the lipase variants of the present invention, as measured by relative stability when treated at a constant temperature for a certain period of time (e.g., 1 hour at 45°C), is 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 a lipase, which comprises improving the stability of a lipase by the following steps: 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, F22 in the numbering of SEQ ID NO: 4. and substituting amino acid residues at one or more positions selected from positions corresponding to positions 5, L228, R233, F234, S256, V260, L266, V269, N272, T274, K278, and R286, wherein the lipase or the lipase after the amino acid residue substitutions has at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 4 or 6. Details of the amino acid residue substitutions are the same as those in the production of the lipase mutant described above.

[0056] 6. Detergent Compositions The lipase variant of the present invention is useful as an enzyme to be incorporated into various detergent compositions, and is particularly useful as an enzyme to be incorporated into detergent compositions suitable for low-temperature cleaning. Here, "low temperature" refers to temperatures of 40°C or lower, 35°C or lower, 30°C or lower, or 25°C or lower, as well as temperatures of 5°C or higher, 10°C or higher, or 15°C or higher. Examples of temperatures also include temperatures between 5 and 40°C, 10 and 35°C, 15 and 30°C, and 15 and 25°C.

[0057] The amount of the lipase variant of the present invention to be incorporated into a detergent composition is not particularly limited as long as the lipase variant exhibits activity, but is, for example, preferably at least 0.1 mg, more preferably at least 1 mg, even more preferably at least 5 mg, and preferably at most 5,000 mg, even more preferably at most 1,000 mg, even more preferably at most 500 mg, per 1 kg of detergent composition. The amount is preferably 0.1 to 5,000 mg, more preferably 1 to 1,000 mg, and even more preferably 5 to 500 mg.

[0058] The detergent composition preferably contains, in addition to the lipase variant of the present invention, a surfactant, a sulfosuccinate ester or a salt thereof. Sulfosuccinate esters or salts thereof are known as components incorporated into detergent compositions (e.g., JP 2019-182911 A). The sulfosuccinate ester or salt thereof is preferably a branched alkyl sulfosuccinate ester having a branched alkyl group of 8 to 12 carbon atoms or a salt thereof, more preferably a branched alkyl sulfosuccinate ester having a branched alkyl group of 9 or 10 carbon atoms or a salt thereof, and even more preferably a branched alkyl sulfosuccinate ester having a branched alkyl group of 10 carbon atoms or a salt thereof. Furthermore, the sulfosuccinate ester or salt thereof is a dibranched alkyl sulfosuccinate ester or salt thereof, and is preferably a dibranched alkyl sulfosuccinate ester or salt thereof in which two branched alkyl groups each have from 8 to 12 carbon atoms, more preferably a dibranched alkyl sulfosuccinate ester or salt thereof in which two branched alkyl groups each have 9 or 10 carbon atoms, even more preferably a dibranched alkyl sulfosuccinate ester or salt thereof in which two branched alkyl groups each have 10 carbon atoms, and still more preferably bis-(2-propylheptyl)sulfosuccinate or salt thereof.

[0059] Examples of the salt include alkali metal salts and alkanolamine salts. Alkali metal salts or alkanolamine salts are preferred, and salts selected from sodium salts, potassium salts, triethanolamine salts, diethanolamine salts, and monoethanolamine salts are more preferred, with sodium salts being even more preferred.

[0060] Examples of sulfosuccinate esters or salts thereof include compounds represented by the following formula 1:

[0061]

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

[0063] In formula 1, R １ , R ２ are each preferably a branched alkyl group selected from a branched nonyl group, a branched decyl group, and a branched dodecyl group, and more preferably a branched decyl group. The branched decyl group is preferably a 2-propylheptyl group.

[0064] In formula 1, A １ O.A. ２ O is an alkyleneoxy group having 2 to 4 carbon atoms, preferably 2 or 3 carbon atoms in view of lubricity to water. １ O.A. ２ Each of these represents the average number of moles of O added, and is 0 or more and 10 or less, and from the viewpoint of lubricity in water, is preferably 6 or less, more preferably 4 or less, even more preferably 2 or less, with 0 being even more preferred.

[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. From the viewpoint of dispersibility in water, M is preferably an alkali metal ion or an alkanolammonium ion, more preferably a sodium ion, potassium ion, triethanolammonium ion, diethanolammonium ion, or monoethanolammonium ion, and even more preferably a sodium ion.

[0066] The sulfosuccinate ester or salt thereof is preferably a compound represented by the following formula 1-1: The compound of formula 1-1 is a compound in which x1 and x2 are each 0 in formula 1.

[0067]

[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.] R in Formula 1-1 １ , R ２ Specific and preferred examples of M are the same as those in Formula 1. In a preferred embodiment, the sulfosuccinate or a salt thereof is bis-(2-propylheptyl)sulfosuccinic acid or a salt thereof.

[0069] The amount of the sulfosuccinate ester or salt thereof in the detergent composition is preferably 0.01% by mass or more, more preferably 0.1% by mass or more, and preferably 30.0% by mass or less, more preferably 10.0% by mass or less, and even more preferably 2.0% by mass or less, preferably 0.01 to 30.0% by mass, more preferably 0.1 to 10.0% by mass, and even more preferably 0.1 to 2.0% by mass.

[0070] The detergent composition can also contain various enzymes in addition to the lipase variant of the present invention. Examples include hydrolases, oxidases, reductases, transferases, lyases, isomerases, ligases, synthetases, etc. Among these, lipases other than the lipase variant of the present invention, amylases, proteases, cellulases, keratinases, esterases, cutinases, pullulanases, pectinases, mannanases, glucosidases, glucanases, cholesterol oxidases, peroxidases, laccases, etc. are preferred, and proteases, cellulases, amylases, and lipases other than the lipase variant of the present invention are particularly 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 mutants of the KP43 protease described in JP-A-2020-145938 and JP-A-2022-118096. Examples of cellulases include Celluclean, Carezyme (registered trademark; Novozymes), KAC, alkaline cellulase produced by Bacillus sp. KSM-S237 strain described in JP-A-10-313859, and mutant alkaline cellulase described in JP-A-2003-313592 (all Kao products). Examples of amylases include Termamyl, Duramyl, Stainzyme, Stainzyme Plus, Amplify Prime (registered trademark; Novozymes), PREFERENZ, EFFECTENZ (registered trademark; DuPont), and KAM (Kao). Examples of the lipase include the YR288 amylase mutants described in JP 2022-60158 A and WO 2023 / 176970 A. Examples of the lipase include the amylase mutants described in JP 2019-500058 A and JP 2018-516553 A. Examples of the lipase include Lipolase and Lipex (registered trademark; Novozymes).In a detergent composition, one or more of the above-mentioned enzymes can be used in combination with the lipase variant of the present invention. Examples of combinations include, but are not limited to, a combination of the lipase variant of the present invention and a protease, a combination of the lipase variant of the present invention and an amylase, a combination of the lipase variant of the present invention, a protease, and an amylase, a combination of the lipase variant of the present invention and a lipase different from the lipase variant of the present invention and a protease, a combination of the lipase variant of the present invention, a lipase different from the lipase variant of the present invention, and an amylase, a combination of the lipase variant of the present invention, a lipase different from the lipase variant of the present invention, a protease, and an amylase, etc. Among these, a combination of the lipase variant of the present invention, a protease, and an amylase is preferred.

[0071] The detergent composition may contain known detergent components, and examples of such known detergent components include the following:

[0072] (1) Surfactant The surfactant is blended in the detergent composition in an amount of 0.5 to 90% by mass, preferably 10 to 45% by mass for powder detergent compositions and 20 to 90% by mass for liquid detergent compositions. When the detergent composition is a laundry detergent or an automatic dishwasher detergent, the surfactant is blended in generally 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 even more preferably 2 to 8% by mass.

[0073] The surfactant used in the detergent composition may be one or a combination of anionic surfactants, nonionic surfactants, amphoteric surfactants, and cationic surfactants other than the above-mentioned sulfosuccinate esters or salts thereof, with amphoteric surfactants being preferred.

[0074] The amphoteric surfactant is preferably an amine oxide surfactant or a betaine surfactant, and more preferably a tertiary amine oxide surfactant, a sulfobetaine surfactant, or a carbobetaine surfactant. Examples of tertiary amine oxide surfactants include tertiary amine oxide surfactants in which one of the groups bonded to the nitrogen atom is an alkyl group having from 8 to 18 carbon atoms, preferably an alkyl group having from 8 to 16 carbon atoms, more preferably an alkyl group having from 8 to 14 carbon atoms, optionally interrupted by an amide group or an ester group, and the remaining groups are alkyl groups having from 1 to 3 carbon atoms, preferably methyl groups. Suitable sulfobetaine surfactants are compounds having one alkyl group having from 10 to 18 carbon atoms, preferably 16 to 14 carbon atoms, two alkyl groups having from 1 to 3 carbon atoms, preferably methyl groups, and a 3-sulfopropyl group or a 2-hydroxy-3-sulfopropyl group. Furthermore, the carbobetaine surfactant is preferably a carbobetaine surfactant having one alkyl group having 10 or more and 18 or less, preferably 16 or less, and more preferably 14 or less, carbon atoms, which may be interrupted by an amide group or an ester group, two alkyl groups having 1 or more and 3 or less, preferably methyl groups, and one carboxyalkyl group, preferably carboxymethyl group.

[0075] (2) Divalent Metal Ion Scavenger The divalent metal ion scavenger is blended in an amount of 0.01 to 50% by mass, preferably 5 to 40% by mass. Examples of divalent metal ion scavengers used in the detergent composition include condensed phosphates such as tripolyphosphates, pyrophosphates, and orthophosphates, aluminosilicates such as zeolites, synthetic layered crystalline silicates, nitrilotriacetates, ethylenediaminetetraacetates, citrates, isocitrates, and polyacetalcarboxylates. Among these, crystalline aluminosilicates (synthetic zeolites) are particularly preferred, and of A-, X-, and P-type zeolites, A-type is particularly preferred. Synthetic zeolites with an average primary particle size of 0.1 to 10 μm, and particularly 0.1 to 5 μm, are preferably used.

[0076] (3) Alkaline Agent The alkaline agent is blended in an amount of 0.01 to 80% by mass, preferably 1 to 40% by mass. In the case of powder detergents, examples include alkali metal carbonates such as sodium carbonate, collectively known as dense ash or light ash, and amorphous alkali metal silicates such as JIS No. 1, No. 2, and No. 3. These inorganic alkaline agents are effective in forming the particle skeleton during detergent drying, resulting in a detergent that is relatively hard and has excellent fluidity. Other examples of alkalis include sodium sesquicarbonate and sodium bicarbonate, and phosphates such as tripolyphosphates also function as alkaline agents. In addition to the above alkaline agents, sodium hydroxide and mono-, di-, or triethanolamine can also be used as alkaline agents in liquid detergents, and they can also be used as counterions for the active agent.

[0077] (4) Anti-Redeposition Agent The anti-redeposition agent is blended in an amount of 0.001 to 10% by mass, preferably 1 to 5% by mass. Examples of anti-redeposition agents used in detergent compositions include polyethylene glycol, carboxylic acid polymers, polyvinyl alcohol, and polyvinylpyrrolidone. Among these, carboxylic acid polymers have the ability to prevent redeposition, as well as the ability to capture metal ions and disperse solid particle soils from clothing into the wash bath. Carboxylic acid polymers are homopolymers or copolymers of acrylic acid, methacrylic acid, itaconic acid, etc. Copolymers of the above monomers and maleic acid are preferred, with molecular weights of several thousand to 100,000 being preferred. In addition to the above carboxylic acid polymers, polymers such as polyglycidyl acid salts, cellulose derivatives such as carboxymethylcellulose, and aminocarboxylic acid polymers such as polyaspartic acid are also preferred because they have the ability to capture metal ions, disperse, and prevent redeposition.

[0078] (5) Bleaching Agents Bleaching agents such as hydrogen peroxide and percarbonates are preferably blended in an amount of 1 to 10% by mass. When using a bleaching agent, tetraacetylethylenediamine (TAED) or a bleaching activator such as that described in JP-A-6-316700 may be blended in an amount of 0.01 to 10% by mass.

[0079] (6) Fluorescent Agents Fluorescent agents used in the detergent composition include biphenyl-type fluorescent agents (e.g., Tinopal CBS-X) and stilbene-type fluorescent agents (e.g., DM-type fluorescent dyes). The fluorescent agent is preferably blended in an amount of 0.001 to 2% by mass.

[0080] (7) Chelating Agents Chelating agents are blended to, for example, facilitate the cleaning of stains or reduce water hardness during cleaning. Examples of such chelating agents include aminocarboxylic acid chelating agents, phosphonic acid chelating agents, hydroxycarboxylic acid chelating agents, and polycarboxylic acid chelating agents. Examples of aminocarboxylic acid 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), ethylenediaminesuccinic acid (EDDS), and salts thereof. Examples of phosphonic acid chelating agents include hydroxyethylidene diphosphonic acid (HEDP), nitrilotrismethylenephosphonic acid (NTMP), phosphonobutanetricarboxylic acid (PBTC), ethylenediaminetetramethylenephosphonic acid (EDTMP), and salts thereof. Examples of hydroxycarboxylic acid chelating agents include citric acid, malic acid, tartaric acid, gluconic acid, lactic acid, and salts thereof. Examples of polycarboxylic acid chelating agents include succinic acid, oxalic acid, glutaric acid, adipic acid, fumaric acid, malonic acid, and salts thereof.

[0081] (8) Other Components The detergent composition may contain solvents, builders, softeners, reducing agents (such as sulfites), foam inhibitors (such as silicones), fragrances, antibacterial and antifungal agents (such as Proxel (trade name) and benzoic acid), and other additives known in the field of 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; and monoalkoxy (methoxy, ethoxy, propoxy, butoxy), phenoxy, or benzooxy ethers of di- or tetraalkylene glycols having 2 to 4 carbon atoms in the alkylene glycol unit. Preferred solvents are water-soluble organic solvents having 2 or more carbon atoms, preferably 3 or more carbon atoms, and 10 or fewer carbon atoms, preferably 8 or fewer carbon atoms. Here, the term "water-soluble organic solvent" refers to a solvent having an octanol / water partition coefficient (LogPow) of 3.5 or less. Specific examples include 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 known as butyl diglycol, etc.), phenoxyethanol, phenoxytriethylene glycol, and phenoxyisopropanol. The solvent is preferably selected from ethanol, propylene glycol, dipropylene glycol, diethylene glycol monobutyl ether, phenoxyethanol, phenyl glycol, and phenoxyisopropanol. The solvent preferably has an alkoxy group, and further preferably contains one or more selected from the monoalkoxy, phenoxy, and benzooxy ethers of di- or tetraalkylene glycols having 2 to 4 carbon atoms in the alkylene glycol unit, and more preferably contains diethylene glycol monobutyl ether.

[0083] The detergent composition preferably has a water content of 60 to 99 mass% and / or a solvent content greater than the surfactant content, and more preferably has a water content of 60 to 99 mass% and a solvent content greater than the surfactant content.

[0084] The detergent composition can be produced by combining the lipase variant of the present invention obtained by the above-mentioned method with the above-mentioned known detergent components according to a conventional method. The detergent form can be selected depending on the intended use, and can be, for example, a liquid, powder, granule, paste, or solid.

[0085] The detergent composition thus obtained can be used as a clothing detergent, dish detergent, bleach, detergent for cleaning hard surfaces, drain cleaner, denture cleaner, disinfectant cleaner for medical instruments, etc., but is preferably a clothing detergent or dish detergent, and more preferably a laundry detergent (laundry laundry detergent), a hand-washing dish detergent, or a detergent for automatic dishwashers. The detergent composition is suitable for use at temperatures of 40°C or less, 35°C or less, 30°C or less, or 25°C or less, and 5°C or more, 10°C or more, or 15°C or more. The detergent composition is also suitable for use at temperatures of 5 to 40°C, 10 to 35°C, 15 to 30°C, or 15 to 25°C. Preferred modes of use include use in laundry washing at low temperatures (15 to 30°C), hand-washing dishes at low temperatures (15 to 30°C), and low-temperature washing in an automatic dishwasher (15 to 30°C).

[0086] The detergent composition of the present invention can be used to clean objects requiring stain removal (e.g., clothing, tableware, hard surfaces, drain pipes, dentures, medical instruments, etc.), i.e., to remove stains. Such a cleaning method comprises contacting the object requiring stain removal with the detergent composition of the present invention. Preferably, the stain is oil-containing stain, such as oil stain or complex stain containing proteins and carbohydrates in addition to oil.

[0087] In the cleaning method of the present invention, the object to be cleaned may be brought into contact with the detergent composition by immersing the object in water containing the detergent composition, or by directly applying the detergent composition to the object. In the method of the present invention, the object to be cleaned after the immersion or application of the detergent composition may be further washed by hand, scrubbing with a sponge, or in a washing machine, but this is not necessarily required.

[0088] Regarding the above-described embodiment, the present invention further discloses the following aspects: <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, F2 <2> A lipase variant of a parent lipase, comprising substitutions of 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, A278, and R286 in the numbering of SEQ ID NO: 4. The substitutions of amino acid residues at positions corresponding to positions 221, L223, A224, F225, L228, R233, F234, S256, V260, L266, V269, N272, T274, K278, and R286 are, respectively, L16F, G22C / M, Y29F, Y43W, A45V, T46Q, L 50I / 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, L22 The lipase variant according to <1>, which has the following amino acid sequences: 3A / 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 <2>, which contains 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 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 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 R286M. <4> The lipase variant according to <2>, which contains 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') a substitution of 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> The lipase variant according to <4>, wherein (A') to (D') are as follows: (A') the substitution N272C in the numbering of 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') the substitution V220H in the numbering of 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') a substitution of V260C in the numbering of SEQ ID NO: 4 and one or more substitutions selected from L50I / V and L142F, or (C') a substitution of V260L in the numbering of 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') a substitution of L208C in the numbering of 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 (A') to (D') are as follows: (A') a substitution of N272C in the numbering of 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') a substitution of V220H in 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') a substitution of V260C in the numbering of SEQ ID NO: 4 and one or more substitutions selected from L50I / V and L142F, or a substitution of V260L in the numbering of SEQ ID NO: 4 and one or more substitutions selected from G22M, G156A, L208C, V220H, L228W, V269I / M and N272G. (D') a substitution of L208C in 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>, which at least comprises a substitution of V220H and a substitution of N272C / G or a substitution of V260L and a substitution of V269I in the numbering of SEQ ID NO: 4. <8> The lipase variant according to <2>, which comprises at least a substitution of L208C, V220H, and N272C, a substitution of L142F, V220H, and N272G, or a substitution of V260L, V269I, and N272G in the numbering of SEQ ID NO: 4. <9> The lipase variant according to <2>, which comprises at least a substitution of L208C, V220H, V269I, and N272C, a substitution of L50V, L142F, V220H, and N272G, or a substitution of L50V, V260L, V269I, and N272G in the numbering of SEQ ID NO: 4.<10> The lipase variant according to <2>, which comprises at least the substitutions L208C, V220H, S256N, V269I, and N272C, or the substitutions L50V, L142F, V220H, V260L, and N272G in the numbering of SEQ ID NO: 4. <11> The lipase variant according to <1> or <2>, which comprises at least any of the substitutions or combinations of substitutions shown in Tables 1-1 to 1-5 and Tables 2-1 to 2-3 above. <12> When the parent lipase or the lipase variant has at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 6, the parent lipase or the lipase variant may have the following amino acid sequences in the numbering of SEQ ID NO: 4: L16, G22, Y29, Y43, A45, T46, L50, S120, K123, R126, T134, L142, A145, G156, K174, A181, K1 Substitution of amino acid residues at positions corresponding to positions 88, 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: 6, respectively. L17F, G23C / M, Y30F, Y44W, A46V, T476Q, L51I / M / V, S121A / Q, T124I, R127E, T135A, L143F, A1 46C / Q, G157A, K175D, A182K, K189Q, L191F, V192A / E, L209C, H210K, A213L, L216C, V221H, A22 2H, 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. <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 comprising the polynucleotide according to <14>. <16> A transformed cell comprising 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 Bacillus bacterium, preferably a Bacillus bacterium, more preferably Bacillus subtilis. <19> A method for producing a lipase variant, which comprises a step of culturing the transformed cell according to any one of <16> to <18>.

[0090] <20> A cleanser composition containing the lipase variant according to any one of <1> to <13>. <21> The cleanser composition according to <20>, further containing a sulfosuccinate ester or a salt thereof, preferably a branched alkyl sulfosuccinate ester having a branched alkyl group having from 8 to 12 carbon atoms or a salt thereof, more preferably a branched alkyl sulfosuccinate ester having a branched alkyl group having 9 or 10 carbon atoms or a salt thereof, and even more preferably a branched alkyl sulfosuccinate ester having a branched alkyl group having 10 carbon atoms or a salt thereof. <22> The detergent composition according to <20>, further comprising a sulfosuccinic acid diester or a salt thereof, preferably a di-branched alkyl sulfosuccinate or a salt thereof, wherein each of the two branched alkyl groups has from 8 to 12 carbon atoms, more preferably a di-branched alkyl sulfosuccinate or a salt thereof, wherein each of the two branched alkyl groups has from 9 to 10 carbon atoms, even more preferably a di-branched alkyl sulfosuccinate or a salt thereof, wherein each of the two branched alkyl groups has 10 carbon atoms, even 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 mass% and / or the solvent content is greater than the surfactant content, preferably the water content is 60 to 90 mass% and the solvent content is greater 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 cleaning composition according to any one of <20> to <24>, which is a powder or a liquid.

[0091] <26> A method for cleaning stains using the detergent composition according to any one of <20> to <25>. <27> The method according to <26>, comprising contacting an article to be cleaned with the detergent composition according to any one of <20> to <25>. <28> Use of the lipase variant according to any one of <1> to <13> for producing a detergent composition. <29> Use of the lipase variant according to any one of <1> to <13> for cleaning stains.

[0092] <30> 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, S2 56, V260, L266, V269, N272, T274, K278, and R286, wherein the lipase or the lipase after the amino acid residue substitution has at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 4 or 6. <31> 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, F2 34, S256, V260, L266, V269, N272, T274, K278 and R286, wherein the lipase or lipase variant has at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4 or 6.<32> 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 in the numbering of SEQ ID NO: 4, The substitutions of amino acid residues at positions corresponding to A221, L223, A224, F225, L228, R233, F234, S256, V260, L266, V269, N272, T274, K278, and R286 are, respectively, L16F, G22C / M, Y29F, Y43W, A45V, T46Q, L 50I / 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, L223 <33> The method according to <32>, wherein the substitutions 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: (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 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 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 R286M. <34> The method according to <32>, wherein the substitutions include 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') a substitution of 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. <35> The method according to <34>, wherein (A') to (D') are as follows:(A') the substitution N272C in the numbering of 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') the substitution V220H in the numbering of 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') a substitution of V260C in the numbering of SEQ ID NO: 4 and one or more substitutions selected from L50I / V and L142F, or (C') a substitution of V260L in the numbering of 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') a substitution of L208C in the numbering of 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. <36> The method according to <34>, wherein (A') to (D') are as follows: (A') a substitution of N272C in the numbering of 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') a substitution of V220H in 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') a substitution of V260C in the numbering of SEQ ID NO: 4 and one or more substitutions selected from L50I / V and L142F, or a substitution of V260L in the numbering of SEQ ID NO: 4 and one or more substitutions selected from G22M, G156A, L208C, V220H, L228W, V269I / M and N272G. (D') a substitution of L208C in 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 substitutions at least include a substitution of V220H and a substitution of N272C / G, or a substitution of V260L and a substitution of V269I in the numbering of SEQ ID NO: 4. <38> The method according to <32>, wherein the substitutions include at least the substitution of L208C, V220H, and N272C, the substitution of L142F, V220H, and N272G, or the substitution of V260L, V269I, and N272G in the numbering of SEQ ID NO: 4. <39> The method according to <32>, wherein the substitutions include at least the substitution of L208C, V220H, V269I, and N272C, the substitution of L50V, L142F, V220H, and N272G, or the substitution of L50V, V260L, V269I, and N272G in the numbering of SEQ ID NO: 4. <40> The method according to <32>, wherein the substitutions include at least the substitutions of L208C, V220H, S256N, V269I, and N272C, or the substitutions of L50V, L142F, V220H, V260L, and N272G in the numbering of SEQ ID NO: 4. <41> The method according to any one of <30> to <32>, wherein the substitutions include at least any of the substitutions or combinations of substitutions shown in Tables 1-1 to 1-5 and Tables 2-1 to 2-3 above.<42> When the lipase or the lipase after amino acid residue substitution of the lipase has at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 6, the numbering of the lipases in SEQ ID NO: 4 is L16, G22, Y29, Y43, A45, T46, L50, S120, K123, R126, T134, L142, A145, G156, and K17 Substitution of amino acid residues at positions corresponding to positions 4, 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 are respectively set forth in SEQ ID NO: 6. In the numbering of 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, V22 1H, 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.

[0093] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples.

[0094] Example 1 (1) Construction of Lipase Expression Plasmid Using the plasmid pHY-S237 described in Example 7 of WO2006 / 068148A1 as a template, an artificial gene CnLip I44T L120S S130A A134T A135V L136M S137N (hereinafter referred to as CnLipA, encoding the polynucleotide sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2) or CspLip I45T L121S S131A A135T A136V S138N described in Japanese Patent Application No. 2023-025141 was synthesized in addition to the full-length ORF of the alkaline cellulase gene. Plasmids pHY-CnLipA and pHY-CspLipA ​​were constructed by in-fusion reaction of A213M (hereinafter referred to as CspLipA, encoding the polynucleotide sequence of SEQ ID NO:5 and the amino acid sequence of SEQ ID NO:6). Using pHY-CnLipA as a template, five mutations were introduced by PCR site-directed mutagenesis using complementary primer pairs (Zheng, Lei, Ulrich Baumann, and Jean-Louis Reymond. Nucleic Acids Research 32.14 (2004): e115-e115.), creating plasmid pHY-CnLipAm, which expresses CnLipAm (encoding the polynucleotide sequence of SEQ ID NO:3 and the amino acid sequence of SEQ ID NO:4). Additional mutations were introduced into the lipase by PCR site-directed mutagenesis using complementary primer pairs.

[0095] (2) Preparation of Lipase Solution A lipase expression plasmid was introduced into the Bacillus subtilis strain 168ΔsigF described in JP 2003-47490 A by the protoplast method, and the resulting culture was 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, and the culture supernatant containing lipase was recovered by centrifugation.

[0096] (3) Evaluation of Stability in Model Cleaning Solutions 4-nitrophenyl butyrate (SIGMA) was used as a substrate. Lipase activity can be determined by measuring the rate of increase in absorbance associated with the liberation of 4-nitrophenol by the action of lipase. 4-nitrophenyl butyrate was added to 20 mM Tris-HCl (pH 7.0) at a final concentration of 2 mM and mixed to prepare a substrate solution. The lipase solution prepared by the method described in (2) above was added to the model cleaning solution shown in Table 3, and the mixture was incubated at 40, 45, 50, 55, or 60°C for the times listed in Tables 4 to 20. Activity was then measured using a 51x solution of 20 mM Tris-HCl (pH 7.0). The activity value of the sample before treatment at 40, 45, 50, 55, or 60°C was used as the initial activity, and the inactivation rate per unit time (h) due to treatment at 40, 45, 50, 55, or 60°C was calculated, from which the half-life (h) was calculated. The half-life (h) of each mutant was divided by the half-life (h) of the parent polypeptide to determine relative stability. The results are shown in Tables 4 to 20. Each table shows the relative stability of mutants in which the parent polypeptide (parent enzyme) at the top was modified with the mutations shown in the table. All mutants were shown to have improved stability compared to the parent polypeptide.

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132] (4) Evaluation of Detergency in Model Cleaning Solution The ability to remove triglycerides from hard surfaces was evaluated as detergency. Beef tallow (SIGMA, 03-0660) and rapeseed oil (SIGMA, 23-0450) were mixed at a weight ratio of 9:1, dissolved in three volumes of chloroform, and then colored with 0.2 wt% Sudan III to prepare a model soil. 10 μL of the model soil was dropped onto the bottom of a 96-well deep-well polypropylene plate, and the chloroform was evaporated and dried to prepare a soil plate. The lipase concentration of the culture supernatant from (2) was diluted with 20 mM Tris-HCl (pH 7.0) to the model cleaning solution listed in Table 3 to 200 mg / L. This solution was used as a lipase solution, and 1 / 50 of the model cleaning solution was added. The cleaning solution was slowly added in 300 μL portions to the soiled plate and allowed to stand at room temperature (approximately 22°C) for 15 minutes for immersion washing. 100 μL of the cleaning solution was dispensed into a new 96-well plate, avoiding contact with the soil at the bottom. The absorbance at 500 nm (A500) was measured to quantify the amount of Sudan III in the model soil solubilized in the cleaning solution by immersion washing. A500 corresponds to the amount of oil released into the cleaning solution and can be used as an indicator of cleaning power. The enzyme effect on cleaning power (ΔA500) was calculated by subtracting the A500 of the cleaning solution containing each lipase from the A500 of the cleaning solution containing 20 mM Tris-HCl (pH 7.0) instead of lipase. The results of the 15-minute wash are shown in Table 21. It was confirmed that the stabilized mutants of the present invention did not exhibit reduced cleaning power.

[0133]

[0134] Reference Example 1: Comparison of stability between model cleaning solutions and other formulations A commercially available liquid detergent (Kao Corporation, Attack ZERO (hereinafter referred to as detergent 1)) was left to stand 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 solutions shown in Table 3 and detergent 1 containing 0.5% Savinase, and the mixture was incubated at 45°C for 1 hour. Activity was measured using a solution diluted 51 times with Tris-HCl (pH 7.0). The activity of the sample before treatment at 45°C was defined as the initial activity, and the ratio of the activity after 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 Patent Document 1, whose suitability for cleaning is disclosed, lipase was found to be significantly destabilized compared to other formulations containing proteases that degrade proteins.

[0135]

Claims

1. 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 in the numbering of SEQ ID NO:4 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) the substitution of N272C / G / 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, D191A / E / V, L192A / E / V, L191B / E / V, L191C / E / V, L192C / E / V, L192D ... 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, comprising at least any one of the following substitutions (A') to (D'): (A') a substitution of 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 cleaning composition comprising the lipase variant according to any one of claims 1 to 4.

10. 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 in the numbering of SEQ ID NO:4 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.

Images