Lipase stability improvement method

Abstract

Provided is a lipase stability improvement method. Provided are: a lipase complex which includes two or more polypeptide molecules, in which the two or more polypeptide molecules are linked via a disulfide bond, and in which at least one of the two or more polypeptide molecules is a polypeptide exhibiting lipase activity and comprising an amino acid sequence which has at least 70% identity with an amino acid sequence represented by SEQ ID NO: 4, 6, 8, 10, 14, 16, 18, or 20; a lipase complex production method; and a lipase stability improvement method comprising a step for complexing a lipase.

Description

Method for improving the stability of lipase

[0001] The present invention relates to a method for improving the stability of a lipase.

[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] On the other hand, amino acid sequences are sometimes modified to improve the function of proteins, including enzymes. Non-Patent Document 1 describes that the side chains of cysteine ​​residues present in proteins exist in different oxidation states depending on the pH and redox potential of the environment, and that changes in the oxidation state can result in significant site-specific changes in the properties of the amino acid, thereby changing the structure and function. Non-Patent Document 2 also describes avoiding the introduction of cysteine ​​residues in protein design. Because the introduction of cysteine ​​residues may thus interfere with the maintenance of protein structure and function, it is often treated as an undesirable mutation.

[0005] (Patent Document 1) Japanese Patent Application Publication No. 2021-17508 (Patent Document 2) Patent Application Publication No. 4776997 (Non-Patent Document 1) Diego Garrido Ruiz et al. Biochemistry 2022, 61(20): 2165-2176 (Non-Patent Document 2) Taihei Murakami et al. Antibodies 2022, 11(1), 10

[0006] The present invention relates to the following 1) to 8): 1) A lipase complex comprising two or more polypeptide molecules, the two or more polypeptide molecules being linked via disulfide bonds, and at least one of the two or more polypeptide molecules being a polypeptide having lipase activity that consists of an amino acid sequence having at least 70% identity to the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 14, 16, 18, or 20. 2) A method for producing a lipase complex, the method comprising the step of linking, via disulfide bonds, two or more polypeptide molecules, the two or more polypeptide molecules including at least one polypeptide having lipase activity that consists of an amino acid sequence having at least 70% identity to the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 14, 16, 18, or 20 and in which the amino acid residues at one or more positions selected from positions 208 and 272 in SEQ ID NO: 4 are cysteine. 3) A method for improving lipase stability, comprising the step of linking, via a disulfide bond, two or more polypeptide molecules, each including at least one polypeptide molecule having lipase activity, the polypeptide comprising an amino acid sequence having at least 70% identity to the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 14, 16, 18, or 20 and in which cysteine ​​residues are present at one or more positions selected from positions 208 and 272 in SEQ ID NO: 4. 4) A polypeptide having lipase activity, the polypeptide comprising an amino acid sequence having at least 70% identity to the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 14, 16, 18, or 20 and in which cysteine ​​residues are present at one or more positions selected from positions 208 and 272 in SEQ ID NO: 4. 5) A polynucleotide encoding the polypeptide according to 4). 6) A vector or DNA fragment comprising the polynucleotide according to 5). 7) A transformed cell containing the vector or DNA fragment according to 6). 8) A detergent composition containing one or both of the complex described in 1) and the polypeptide described in 4).

[0007] SDS-PAGE results of the monomer fraction or complex fraction of each lipase in the presence or absence of a reducing agent. A matrix showing the identity of the amino acid sequence of each lipase. Detailed Description of the Invention

[0008] All patents, non-patent documents, and other publications cited herein are hereby incorporated by reference in their entirety.

[0009] As used herein, "lipase" refers to triacylglycerol lipase (EC 3.1.1.3), a group of enzymes that have lipase activity that hydrolyzes 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.

[0010] 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.

[0011] As used herein, "at least 70% identity" with respect to an amino acid sequence or a nucleotide sequence refers to identity of 70% or more, preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, even more preferably 93% or more, even more preferably 94% or more, even more preferably 95% or more, even more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more, and even more preferably 99.5% or more.

[0012] 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.

[0013] 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.

[0014] 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).

[0015] 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, leucine at position 208 is represented as "L208." Amino acid "substitutions" are represented as [original amino acid, position, substituted amino acid]. For example, a substitution of leucine at position 208 with cysteine ​​is represented as "L208C." Variants containing multiple modifications are represented by a plus sign ("+"). For example, "L208C+N272C" represents a substitution of leucine at position 208 with cysteine ​​and asparagine at position 272 with cysteine, 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.

[0016] 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.

[0017] 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.

[0018] As used herein, a "parent" polypeptide of a given mutant polypeptide refers to a polypeptide that has a predetermined mutation in an amino acid residue thereof that results in the mutant polypeptide. In other words, a "parent" polypeptide is a polypeptide before the mutation is introduced into the mutant polypeptide.

[0019] As used herein, the term "lipase complex" refers to two or more polypeptide molecules, including at least one molecule of a polypeptide having lipase activity (lipase), joined together via a disulfide bond. The individual polypeptides constituting the lipase complex may be of the same or different species, with no particular limitation on whether the other polypeptides have lipase activity, as long as at least one polypeptide molecule is a lipase. However, the complex may be composed of lipases of the same or different species, preferably lipases of the same species, and more preferably lipases of the same species. The number of polypeptides constituting the complex is not particularly limited, but is preferably two. Therefore, the lipase complex may preferably be a dimer consisting of two molecules of lipases of the same or different species, i.e., a lipase dimer, more preferably a lipase dimer consisting of two molecules of lipases of the same species. Hereinafter, the lipase complex may be simply referred to as a "complex."

[0020] The stability of lipases formulated in detergent compositions containing a higher water content and a higher concentration of solvents than surfactants, which is a harsh environment 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 a method for improving lipase stability and to providing a lipase exhibiting excellent stability.

[0021] The present inventors have found that when an amino acid residue at a specific position in the amino acid sequence of a parent lipase is substituted with cysteine, the resulting lipase variant can form a complex containing the lipase variant, and in particular, the complex has improved stability in the presence of a large amount of water and / or a solvent in an amount greater than a surfactant, compared to the parent lipase, i.e., the stability of the lipase can be improved by complexing the lipase.

[0022] According to the present invention, lipase stability can be improved by complexing the lipase. A polypeptide having lipase activity capable of forming a lipase complex exhibits excellent stability, particularly in the form of a complex, and can be suitably incorporated into a detergent composition containing a large amount of water and / or a solvent in an amount greater than that of a surfactant.

[0023] <1. Polypeptide Having Lipase Activity> A polypeptide having lipase activity capable of forming a lipase complex of the present invention (referred to as "polypeptide of the present invention") is a polypeptide having lipase activity, consisting of an amino acid sequence having at least 70% identity with the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 14, 16, 18, or 20. The polypeptide of the present invention has cysteines involved in disulfide bonds in the amino acid sequence. The polypeptide of the present invention is preferably a polypeptide having lipase activity, consisting of an amino acid sequence having at least 70% identity with the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 14, 16, 18, or 20, and in which the amino acid residues at one or more positions selected from positions 208 and 272 in the numbering based on SEQ ID NO: 4 are cysteines.

[0024] In one embodiment, the polypeptide of the present invention is a naturally occurring (wild-type) polypeptide having lipase activity, which consists of an amino acid sequence having at least 70% identity to the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 14, 16, 18, or 20. Such naturally occurring polypeptides can have good stability in the form of a complex formed via disulfide bonds. In another embodiment, the polypeptide of the present invention is a mutant polypeptide having lipase activity, which consists of an amino acid sequence having at least 70% identity to the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 14, 16, 18, or 20 (the mutant polypeptides of the polypeptides of the present invention may be particularly referred to as "mutant polypeptides of the present invention"). Such mutant polypeptides can have good stability in the form of a complex formed via disulfide bonds. Here, stability more specifically refers to stability in the presence of a large amount of water and / or a solvent in an amount greater than that of a surfactant.

[0025] In a preferred embodiment, the polypeptide of the present invention is a naturally occurring (wild-type) polypeptide having lipase activity, which consists of an amino acid sequence that has at least 70% identity to the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 14, 16, 18, or 20 and in which the amino acid residues at one or more positions selected from positions 208 and 272 in the numbering based on SEQ ID NO: 4 are cysteine. The cysteines at these positions contribute to the complex formation and stability of the lipase. Thus, such naturally occurring polypeptides can form complexes and have good stability in the form of a single molecule, particularly in the form of a complex. In another preferred embodiment, the polypeptide of the present invention is a mutant polypeptide having lipase activity, which consists of an amino acid sequence in which amino acid residues at one or more positions selected from positions 208 and 272 in the numbering of SEQ ID NO: 4 in the amino acid sequence of a parent lipase have been substituted with cysteine, the mutant polypeptide having lipase activity having at least 70% identity to the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 14, 16, 18, or 20, and in which amino acid residues at one or more positions selected from positions 208 and 272 in the numbering of SEQ ID NO: 4 are cysteine. The substitution of amino acid residues at the above-mentioned predetermined positions with cysteine ​​is a modification that contributes to complex formation of the lipase and improves the stability of the lipase. Therefore, a polypeptide obtained by substituting amino acid residues at the above-mentioned predetermined positions in the amino acid sequence of a parent lipase, i.e., a mutant polypeptide having lipase activity and consisting of the amino acid sequence after the amino acid residue substitution, can form a complex and has improved stability in its simple form compared to the parent lipase, and particularly has much improved stability in its complex form compared to the parent lipase.Generally, the introduction of cysteine ​​into a protein is often regarded as an undesirable mutation because it may interfere with the maintenance of the protein's structure and function, and the effect of lipase conjugation on stability was unknown. Therefore, it was quite surprising that lipase conjugation, particularly lipase conjugation based on substituting an amino acid residue at a specific position in the lipase amino acid sequence with cysteine, improved stability. More specifically, stability here refers to stability in the presence of a large amount of water and / or a solvent in an amount greater than that of a surfactant.

[0026] In the present invention, a parent lipase is a polypeptide having lipase activity and consisting of an amino acid sequence having at least 70% identity to the amino acid sequence set forth in SEQ ID NO: 4, 6, 8, 10, 14, 16, 18, or 20. The parent lipase may be a naturally occurring (wild-type) polypeptide or a variant thereof.

[0027] An example of a parent lipase is a polypeptide having lipase activity and consisting of an amino acid sequence having at least 70% 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 following 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 following amino acid residues: F16L, I47L, A91V, E97D, and A149E, and is designated CnLipAm as shown in the Examples below. The parent lipase of the present invention may also be the CnLip and CnLipA. The parent lipase, which is a polypeptide having lipase activity and consisting of an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO:4, preferably has a leucine at position 208 in the numbering of SEQ ID NO:4 and an asparagine at position 272 in the numbering of SEQ ID NO:4, and more preferably has a leucine at position 208 and an asparagine at position 272 in the numbering of SEQ ID NO:4.

[0028] Another example of the parent lipase is a polypeptide having lipase activity and consisting of an amino acid sequence at least 70% 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 (the highly productive mutant SspLip_m described in Japanese Patent Application No. 2022-149382) in which the amino acid residue E118D has been substituted in the amino acid sequence constituting the lipase SspLip (NCBI Accession No. WP_025122441.1) derived from Serratia sp. The parent lipase, which is a polypeptide having lipase activity and consisting of an amino acid sequence having at least 70% identity to the amino acid sequence set forth in SEQ ID NO:6, preferably has a leucine at position 208 in the numbering of SEQ ID NO:4 and a proline at position 272 in the numbering of SEQ ID NO:4, and more preferably has a leucine at position 208 and a proline at position 272 in the numbering of SEQ ID NO:4. In the amino acid sequence of SEQ ID NO:6, the positions corresponding to positions 208 and 272 in the numbering of SEQ ID NO:4 are positions 210 and 274, respectively.

[0029] Another example of the parent lipase is a polypeptide having lipase activity and consisting of an amino acid sequence having at least 70% identity to the amino acid sequence shown in SEQ ID NO: 8. Here, the lipase consisting of the amino acid sequence shown in SEQ ID NO: 8 is a lipase (the highly productive mutant EtLip_m described in Japanese Patent Application No. 2022-149382) in which the amino acid residue I222G has been substituted in the amino acid sequence constituting the lipase EtLip (NCBI Accession No. WP_115457195.1) derived from Enterobacillus tribolii. The parent lipase, which is a polypeptide having lipase activity and consisting of an amino acid sequence having at least 70% identity to the amino acid sequence set forth in SEQ ID NO:8, preferably has a phenylalanine at position 208 in the numbering of SEQ ID NO:4 and a threonine at position 272 in the numbering of SEQ ID NO:4, more preferably has a phenylalanine at position 208 and a threonine at position 272 in the numbering of SEQ ID NO:4. In the amino acid sequence of SEQ ID NO:8, the positions corresponding to positions 208 and 272 in the numbering of SEQ ID NO:4 are positions 210 and 274, respectively.

[0030] Another example of a parent lipase is a polypeptide having lipase activity and consisting of an amino acid sequence having at least 70% identity to the amino acid sequence set forth in SEQ ID NO: 10. Here, the lipase consisting of the amino acid sequence set forth in SEQ ID NO: 10 is a lipase (CspLipA ​​with excellent detergency described in Japanese Patent Application No. 2023-025141) 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) derived from Cedecea sp. The parent lipase, which is a polypeptide having lipase activity and consisting of an amino acid sequence having at least 70% identity to the amino acid sequence set forth in SEQ ID NO:10, preferably has a leucine at position 208 in the numbering of SEQ ID NO:4 and an asparagine at position 272 in the numbering of SEQ ID NO:4, and more preferably has a leucine at position 208 and an asparagine at position 272 in the numbering of SEQ ID NO:4. In the amino acid sequence of SEQ ID NO:10, the positions corresponding to positions 208 and 272 in the numbering of SEQ ID NO:4 are positions 209 and 273, respectively.

[0031] Another example of a parent lipase is a polypeptide having lipase activity and consisting of an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 14. Here, the lipase consisting of the amino acid sequence set forth in SEQ ID NO: 14 is KAL (JP 2023-156914 A). A parent lipase that is a polypeptide having lipase activity and consisting of an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 14 preferably has a leucine at position 208 in the numbering of SEQ ID NO: 4 and a serine at position 272 in the numbering of SEQ ID NO: 4, and more preferably has a leucine at position 208 and a serine at position 272 in the numbering of SEQ ID NO: 4. In the amino acid sequence of SEQ ID NO: 14, the positions corresponding to positions 208 and 272 in the numbering of SEQ ID NO: 4 are positions 210 and 274, respectively.

[0032] Another example of a parent lipase is a polypeptide having lipase activity and consisting of an amino acid sequence having at least 70% identity to the amino acid sequence set forth in SEQ ID NO: 16. Here, the lipase consisting of the amino acid sequence set forth in SEQ ID NO: 16 is the ancestral lipase AncLip10 (JP 2022-66571 A). Here, the ancestral lipase refers to a lipase consisting of an amino acid sequence estimated to have been possessed by a common ancestor, which is derived from the existing sequences of lipases of each organism derived from the common ancestor based on a rooted phylogenetic tree representing protein evolution. The amino acid sequence of an ancestral lipase can be determined by ancestral sequence reconstruction (ASR) based on the existing sequence of the lipase. The parent lipase, which is a polypeptide having lipase activity and consisting of an amino acid sequence having at least 70% identity to the amino acid sequence set forth in SEQ ID NO: 16, preferably has a leucine at position 208 in the numbering of SEQ ID NO: 4 and a threonine at position 272 in the numbering of SEQ ID NO: 4, more preferably has a leucine at position 208 and a threonine at position 272 in the numbering of SEQ ID NO: 4. In the amino acid sequence of SEQ ID NO: 16, the positions corresponding to positions 208 and 272 in the numbering of SEQ ID NO: 4 are positions 209 and 273, respectively.

[0033] Another example of a parent lipase is a polypeptide having lipase activity and consisting of an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 18. Here, the lipase consisting of the amino acid sequence set forth in SEQ ID NO: 18 is a lipase derived from Enterobacteriaceae (NCBI Accession No. WP_045783583.1). The parent lipase, which is a polypeptide having lipase activity and consisting of an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 18, preferably has a leucine at position 208 in the numbering of SEQ ID NO: 4 and a serine at position 272 in the numbering of SEQ ID NO: 4, and more preferably has a leucine at position 208 and a serine at position 272 in the numbering of SEQ ID NO: 4. In the amino acid sequence of SEQ ID NO: 18, the positions corresponding to positions 208 and 272 in the numbering of SEQ ID NO: 4 are positions 208 and 272, respectively.

[0034] Another example of a parent lipase is a polypeptide having lipase activity and consisting of an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 20. Here, the lipase consisting of the amino acid sequence set forth in SEQ ID NO: 20 is a lipase derived from Chania multitudinisentens (NCBI Accession No. WP_037407093.1). The parent lipase, which is a polypeptide having lipase activity and consisting of an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 20, preferably has isoleucine at position 208 in the numbering of SEQ ID NO: 4 and glycine at position 272 in the numbering of SEQ ID NO: 4, and more preferably has isoleucine at position 208 and glycine at position 272 in the numbering of SEQ ID NO: 4. In the amino acid sequence of SEQ ID NO:20, the positions corresponding to positions 208 and 272 in the numbering of SEQ ID NO:4 are positions 208 and 272, respectively.

[0035] The amino acid sequence represented by SEQ ID NO: 4, 6, 8, 10, 14, 16, 18, or 20 has at least 70% identity with at least one amino acid sequence among the amino acid sequences represented by SEQ ID NOs: 4, 6, 8, 10, 14, 16, 18, and 20 excluding itself ( FIG. 2 ). For example, the amino acid sequences represented by SEQ ID NOs: 6, 8, 10, 14, 16, 18, and 20 all have at least 70% identity with the amino acid sequence represented by SEQ ID NO: 4. On the other hand, none of the amino acid sequences represented by SEQ ID NOs: 4, 6, 8, 10, 14, 16, 18, or 20 has at least 70% identity with the amino acid sequence of lipase PvLip (SEQ ID NO: 12), which is a reference example in the Examples described below.

[0036] In terms of stability, the polypeptide of the present invention is preferably a polypeptide having lipase activity which has at least 70% identity with the amino acid sequence shown in SEQ ID NO:4 and in which one or more amino acid residues selected from positions 208 and 272 in the numbering of SEQ ID NO:4 are cysteine, or a polypeptide having lipase activity which has at least 70% identity with the amino acid sequence shown in SEQ ID NO:6, 8, 10, 14, 16, 18, or 20 and in which the amino acid residue at position 208 in the numbering of SEQ ID NO:4 is cysteine; more preferably a polypeptide having lipase activity which has at least 70% identity with the amino acid sequence shown in SEQ ID NO:4 and in which one or more amino acid residues selected from positions 208 and 272 in the numbering of SEQ ID NO:4 are cysteine, or a polypeptide having lipase activity which has at least 70% identity with the amino acid sequence shown in SEQ ID NO:6, 8, or 10 and in which the amino acid residue at position 208 in the numbering of SEQ ID NO:4 is cysteine.

[0037] The polypeptide of the present invention may contain, as long as the formation of a complex is not prevented, phenylalanine at a position corresponding to position 16 in the numbering of SEQ ID NO: 4, cysteine ​​or methionine at a position corresponding to position 22, phenylalanine at a position corresponding to position 29, isoleucine or valine at a position corresponding to position 50, alanine or glutamine at a position corresponding to position 120, isoleucine or threonine at a position corresponding to position 123, glutamic acid at a position corresponding to position 126, phenylalanine at a position corresponding to position 142, alanine at a position corresponding to position 156, lysine at a position corresponding to position 181, It may have one or more, preferably one, amino acid residue selected from glutamic acid at a position corresponding to position 186, glutamine at a position corresponding to position 188, valine at a position corresponding to position 191, histidine at a position corresponding to position 220, histidine at a position corresponding to position 221, glutamine at a position corresponding to position 224, threonine at a position corresponding to position 233, asparagine at a position corresponding to position 256, alanine, cysteine ​​or leucine at a position corresponding to position 260, isoleucine at a position corresponding to position 269, and glutamine at a position corresponding to position 274.

[0038] 2. Polynucleotides Encoding the Polypeptides of the Present Invention The polypeptides of the present invention can be produced by expressing the polypeptides from polynucleotides encoding them. The polynucleotides can be prepared by extracting genomic DNA from a microorganism that produces the target lipase using standard methods, or by extracting RNA and synthesizing cDNA by reverse transcription. Alternatively, a corresponding nucleotide sequence can be chemically synthesized based on the amino acid sequence of the target lipase and used as a polynucleotide encoding the polypeptide of the present invention.

[0039] Alternatively, the polypeptides of the present invention can be produced using various mutagenesis techniques known in the art, for example, by mutating a polynucleotide encoding an amino acid residue to be mutated in a parent lipase gene (reference lipase gene) encoding the reference amino acid sequence to a polynucleotide encoding the mutated amino acid residue, and then expressing the mutant polypeptide from the mutant gene.

[0040] 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 mutant polypeptide 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 a parent lipase (hereinafter also referred to as a parent gene) to a nucleotide sequence encoding the mutated amino acid residue.

[0041] 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.

[0042] 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).

[0043] 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, 6, 8, 10, 14, 16, 18, and 20 are shown in SEQ ID NOS: 3, 5, 7, 9, 13, 15, 17, and 19, respectively.

[0044] Mutation primers can be prepared by well-known oligonucleotide synthesis methods, 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.). By using a primer set containing the mutation primers and carrying out the above-described site-specific mutagenesis using a parent gene as template DNA, a polynucleotide encoding the mutant polypeptide of the present invention having the desired mutation can be obtained.

[0045] The polynucleotide encoding the polypeptide 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 polypeptide of the present invention. Information on codons used by various organisms is available from the Codon Usage Database ([www.kazusa.or.jp / codon / ]).

[0046] 3. Vector or DNA Fragment The obtained polynucleotide encoding the polypeptide 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 polypeptide 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.

[0047] 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 polypeptide of the present invention.

[0048] 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.

[0049] 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.

[0050] The polynucleotide encoding the polypeptide 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.

[0051] 4. Transformed Cells The transformed cells of the present invention can be obtained by introducing a vector containing a polynucleotide encoding the polypeptide of the present invention into a host, or by introducing a DNA fragment containing a polynucleotide encoding the polypeptide of the present invention into the genome of a host.

[0052] 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.

[0053] 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.

[0054] Alternatively, a fragment comprising a polynucleotide encoding the polypeptide of the present invention, a regulatory sequence, and a marker gene can be directly introduced into the genome of a host. For example, a DNA fragment in which sequences complementary to the host genome are added to both ends of the above-mentioned ligated fragment is constructed by SOE-PCR or the like, and this is introduced into a host to induce homologous recombination between the host genome and the DNA fragment, thereby introducing the polynucleotide encoding the polypeptide of the present invention into the genome of the host.

[0055] When the thus obtained transformant into which a polynucleotide encoding the polypeptide 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 polypeptide 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.

[0056] Alternatively, the polypeptide of the present invention may be expressed from a polynucleotide encoding the polypeptide 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.

[0057] 5. Lipase Complex The polypeptide of the present invention can form a lipase complex by bonding via a disulfide bond, preferably under non-reducing conditions. The lipase complex formed by the polypeptide of the present invention is a complex containing at least one molecule of the polypeptide of the present invention. Examples of such complexes include complexes in which homologous or heterologous polypeptides of the present invention are bonded via disulfide bonds, and complexes in which the polypeptide of the present invention is bonded via a disulfide bond with a component other than the polypeptide of the present invention. The component other than the polypeptide of the present invention that constitutes the complex is a polypeptide capable of forming a disulfide bond with a cysteine ​​in the polypeptide of the present invention, and examples include polypeptides containing cysteine ​​that can be produced by a host (e.g., Bacillus subtilis) upon expression of the polypeptide of the present invention. The lipase complex formed by the polypeptide of the present invention is preferably a complex containing two or more polypeptide molecules each containing at least one molecule of the polypeptide of the present invention, more preferably a lipase dimer consisting of two molecules of the same or heterologous polypeptide of the present invention, and even more preferably a lipase dimer consisting of two molecules of the same polypeptide of the present invention.That is, the lipase complex formed by the polypeptide of the present invention is preferably a complex comprising two or more polypeptide molecules, the two or more polypeptide molecules being linked by disulfide bonds, and at least one of the two or more polypeptide molecules being a polypeptide having lipase activity consisting of an amino acid sequence having at least 70% identity with the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 14, 16, 18, or 20; more preferably a complex comprising two or more polypeptide molecules, the two or more polypeptide molecules being linked by disulfide bonds, and at least one of the two or more polypeptide molecules being a polypeptide having lipase activity consisting of an amino acid sequence having at least 70% identity with the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 14, 16, 18, or 20, and in which the amino acid residues at one or more positions selected from positions 208 and 272 in the numbering of SEQ ID NO: 4 are cysteine; More preferred is a lipase dimer comprising two identical polypeptide molecules linked by a disulfide bond, wherein the two polypeptide molecules each independently have at least 70% identity to the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 14, 16, 18, or 20 and have cysteine ​​amino acid residues at one or more positions selected from positions 208 and 272 in the numbering of SEQ ID NO: 4; and even more preferred is a lipase dimer comprising two identical polypeptide molecules linked by a disulfide bond, wherein the polypeptide is a polypeptide having lipase activity and has an amino acid sequence which has at least 70% identity to the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 14, 16, 18, or 20 and have cysteine ​​amino acid residues at one or more positions selected from positions 208 and 272 in the numbering of SEQ ID NO: 4.

[0058] 6. Method for Producing a Lipase Conjugate The present invention also provides a method for producing a lipase conjugate. The method for producing a lipase conjugate of the present invention comprises the step of linking two or more polypeptide molecules, each including at least one polypeptide molecule of the present invention, via a disulfide bond. Formation of a conjugate via a disulfide bond between cysteines in the two or more polypeptide molecules can be achieved, for example, by expressing the polypeptide of the present invention under non-reducing conditions using a transformed cell of the present invention or by maintaining the polypeptide of the present invention under non-reducing conditions. Here, "non-reducing conditions" refers to an environment in which an oxidizing force capable of forming a disulfide bond is present, such as an environment in which a reducing agent such as dithiothreitol (DTT) is not present in an amount that would prevent disulfide bond formation. Since the cytoplasm is generally maintained in a reducing state, it is preferred to express the polypeptide of the present invention using a transformed cell of the present invention by culturing it under conditions in which the polypeptide is transferred to an oxidizing environment such as the endoplasmic reticulum or periplasm, or under conditions in which the polypeptide is released extracellularly by secretion or lysis after culture. Alternatively, it is preferred to express or maintain the polypeptide of the present invention using a transformed cell of the present invention under aeration and agitation conditions to promote air oxidation. Furthermore, complex formation can be achieved by expressing the polypeptide of the present invention under non-reducing conditions using a cell-free translation system or by maintaining the polypeptide of the present invention under non-reducing conditions. To enhance the oxidizing power in a cell-free translation system, it is preferable to express the polypeptide of the present invention using a cell-free translation system with the addition of chaperones such as glutathione, disulfide bond isomerase (DsbC), and protein disulfide isomerase (PDI). Alternatively, to promote air oxidation, it is preferable to express or maintain the polypeptide of the present invention using a cell-free translation system under aeration and agitation.

[0059] The method for producing a lipase conjugate of the present invention may further comprise the step of providing a polypeptide of the present invention prior to the above-mentioned binding step. In a preferred embodiment, this step comprises substituting, with cysteine, amino acid residues at specific positions in the amino acid sequence of at least one parent lipase, which amino acid residues contribute to integration of the polypeptides. In a more preferred embodiment, this step comprises substituting, with cysteine, amino acid residues at one or more positions selected from positions 208 and 272 in numbering based on SEQ ID NO: 4, in a polypeptide having lipase activity and consisting of an amino acid sequence having at least 70% identity to at least one amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 14, 16, 18, or 20.

[0060] Substitution of such an amino acid residue at a predetermined position with cysteine ​​is a modification that contributes to complex formation of the lipase. From the viewpoint of improving stability, the substitution site of the amino acid residue is preferably one or more positions selected from positions 208 and 272 in the numbering of SEQ ID NO: 4 in a polypeptide consisting of an amino acid sequence having at least 70% identity with the amino acid sequence shown in SEQ ID NO: 4 and having lipase activity, or position 208 in the numbering of SEQ ID NO: 4 in a polypeptide consisting of an amino acid sequence having at least 70% identity with the amino acid sequence shown in SEQ ID NO: 6, 8, 10, 14, 16, 18, or 20 and having lipase activity.

[0061] In the step of providing the polypeptide of the present invention, in addition to the substitution of the above amino acid residues, substitution of one or more, preferably one, amino acid residues selected from positions corresponding to positions 16, 22, 29, 50, 120, 123, 126, 142, 156, 181, 186, 188, 191, 220, 221, 224, 233, 256, 260, 269, and 274 in the numbering of SEQ ID NO: 4 may be performed, as long as the substitution does not interfere with the formation of a complex. Preferred embodiments of each substitution include substitution of the amino acid residue at position 16 in the numbering of SEQ ID NO: 4 with phenylalanine, substitution of the amino acid residue at position 22 with cysteine ​​or methionine, substitution of the amino acid residue at position 29 with phenylalanine, substitution of the amino acid residue at position 50 with isoleucine or valine, substitution of the amino acid residue at position 120 with alanine or glutamine, substitution of the amino acid residue at position 123 with isoleucine or threonine, substitution of the amino acid residue at position 126 with glutamic acid, substitution of the amino acid residue at position 142 with phenylalanine, substitution of the amino acid residue at position 156 with alanine, and substitution of the amino acid residue at position 181 with lysine. Conservative amino acid substitutions include substitution of the amino acid residue at position 186 with glutamic acid, substitution of the amino acid residue at position 188 with glutamine, substitution of the amino acid residue at position 191 with valine, substitution of the amino acid residue at position 220 with histidine, substitution of the amino acid residue at position 221 with histidine, substitution of the amino acid residue at position 224 with glutamine, substitution of the amino acid residue at position 233 with threonine, substitution of the amino acid residue at position 256 with asparagine, substitution of the amino acid residue at position 260 with alanine, cysteine, or leucine, substitution of the amino acid residue at position 269 with isoleucine, and substitution of the amino acid residue at position 274 with glutamine. Conservative amino acid substitutions include any number of conservative amino acid substitutions, as long as they do not interfere with the formation of the complex.

[0062] The polypeptide of the present invention can be isolated or purified, either alone or in the form of a complex, by a general method used for protein purification, such as centrifugation, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc., either alone or in appropriate combinations. The protein recovered from the culture may be further purified by known means.

[0063] 7. Method for Improving Lipase Stability The polypeptide of the present invention has good detergency and has improved stability in the form of a complex compared to the form of a monolayer, more specifically, improved stability in the form of a complex compared to the form of a monolayer in the presence of a large amount of water and / or a solvent compared to a surfactant. "Stability" refers to the ability to maintain lipase activity, and "improved stability in the form of a complex compared to the form of a monolayer" refers to the improved ability of the complex to maintain lipase activity compared to the monolayer, and "improved stability in the form of a complex compared to the form of a monolayer in the presence of a large amount of water and / or a solvent compared to a surfactant" refers to the improved ability of the complex to maintain lipase activity compared to the monolayer in the presence of a large amount of water and / or a solvent compared to a surfactant.

[0064] The variant polypeptides of the present invention have improved stability compared to the parent lipase in the form of a single molecule, without impairing the detergency of the parent lipase, and further have much improved stability compared to the parent lipase in the form of a complex, more specifically, improved stability in the presence of a large amount of solvent compared to a large amount of water and / or surfactant. "Stability" refers to the ability to maintain lipase activity, "improved stability compared to the parent lipase" refers to the ability to maintain lipase activity improved compared to the parent lipase, and "improved stability compared to the parent lipase in the presence of a large amount of solvent compared to a large amount of water and / or surfactant" refers to the ability to maintain lipase activity improved compared to the parent lipase in the presence of a large amount of water and / or solvent compared to a large amount of surfactant.

[0065] Here, "stability in the presence of a large amount of water" means the ability to maintain lipase activity when stored in a detergent composition having a water content of 60 to 99% by mass, and "stability in the presence of a solvent in an amount greater than that of the surfactant" means the ability to maintain lipase activity in a detergent composition having a solvent content greater than that of the surfactant, or in a cleaning liquid prepared by dissolving or diluting the detergent composition in water and having a solvent content greater than that of the surfactant. Examples of surfactants and solvents include those described below.

[0066] 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 inactivation 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 mutant polypeptide by the half-life of the parent lipase indicates the relative stability of the mutant polypeptide, and when this value exceeds 1, the mutant polypeptide is evaluated to have 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 2 in the Examples below. The stability of a lipase complex (lipase dimer) containing the polypeptide of the present invention, relative to the polypeptide of the present invention alone, when treated at 45°C for 1 hour, can be preferably 1.3 or more, more preferably 1.5 or more. The stability of the mutant polypeptide of the present invention, expressed relative to the parent lipase when treated at 45° C. for 1 hour, can be preferably 1.5 or higher, more preferably 1.7 or higher. The stability of a lipase complex (lipase dimer) containing the mutant polypeptide of the present invention, expressed relative to the parent lipase when treated at 45° C. for 1 hour, can be preferably 2 or higher, more preferably 3 or higher.

[0067] The present invention also provides a method for improving lipase stability. The method for improving lipase stability of the present invention comprises the step of complexing lipase. Lipase complexing is a process for preparing a lipase complex by combining two or more polypeptide molecules, each including at least one molecule of a polypeptide having lipase activity, via a disulfide bond between the cysteines in the two or more polypeptide molecules. Specifically, the method for improving lipase stability of the present invention comprises the step of linking two or more polypeptide molecules, each including at least one polypeptide of the present invention, via a disulfide bond. Complex formation via a disulfide bond between the cysteines in the two or more polypeptide molecules can be achieved, for example, by expressing the polypeptide of the present invention under non-reducing conditions using a transformed cell of the present invention or by maintaining the polypeptide of the present invention under non-reducing conditions. The non-reducing conditions and preferred embodiments thereof are as described above.

[0068] The method for improving the stability of a lipase of the present invention may further comprise the step of providing a polypeptide of the present invention prior to the above-mentioned binding step. In a preferred embodiment, this step comprises substituting, with cysteine, amino acid residues at specific positions in the amino acid sequence of at least one parent lipase, which amino acid residues contribute to integration of the polypeptides. In a more preferred embodiment, this step comprises substituting, with cysteine, amino acid residues at one or more positions selected from positions 208 and 272 in numbering based on SEQ ID NO: 4, in a polypeptide having lipase activity and consisting of an amino acid sequence having at least 70% identity to at least one amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 14, 16, 18, or 20.

[0069] The substitution of an amino acid residue at a predetermined position with a cysteine ​​contributes to complex formation of the lipase and is a modification for improving the stability of the lipase. Therefore, a polypeptide obtained by substituting an amino acid residue at the predetermined position in the amino acid sequence of a parent lipase, i.e., a mutant polypeptide having lipase activity and comprising the amino acid sequence after the amino acid residue substitution, can form a complex and has improved stability in the form of a single molecule compared to the parent lipase, and particularly has significantly improved stability in the form of a complex compared to the parent lipase. From the viewpoint of improving stability, the substitution site is preferably at one or more positions selected from positions 208 and 272 in the numbering of SEQ ID NO: 4 for a polypeptide having lipase activity and comprising an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 4, or at position 208 in the numbering of SEQ ID NO: 4 for a polypeptide having lipase activity and comprising an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 6, 8, 10, 14, 16, 18, or 20.

[0070] In the step of providing the polypeptide of the present invention, in addition to the substitution of the above amino acid residues, substitution of one or more, preferably one, amino acid residues selected from positions corresponding to positions 16, 22, 29, 50, 120, 123, 126, 142, 156, 181, 186, 188, 191, 220, 221, 224, 233, 256, 260, 269, and 274 in the numbering of SEQ ID NO: 4 may be performed, as long as the substitution does not interfere with the formation of a complex. Preferred embodiments of each substitution include substitution of the amino acid residue at position 16 in the numbering of SEQ ID NO: 4 with phenylalanine, substitution of the amino acid residue at position 22 with cysteine ​​or methionine, substitution of the amino acid residue at position 29 with phenylalanine, substitution of the amino acid residue at position 50 with isoleucine or valine, substitution of the amino acid residue at position 120 with alanine or glutamine, substitution of the amino acid residue at position 123 with isoleucine or threonine, substitution of the amino acid residue at position 126 with glutamic acid, substitution of the amino acid residue at position 142 with phenylalanine, substitution of the amino acid residue at position 156 with alanine, and substitution of the amino acid residue at position 181 with lysine. Conservative amino acid substitutions include substitution of the amino acid residue at position 186 with glutamic acid, substitution of the amino acid residue at position 188 with glutamine, substitution of the amino acid residue at position 191 with valine, substitution of the amino acid residue at position 220 with histidine, substitution of the amino acid residue at position 221 with histidine, substitution of the amino acid residue at position 224 with glutamine, substitution of the amino acid residue at position 233 with threonine, substitution of the amino acid residue at position 256 with asparagine, substitution of the amino acid residue at position 260 with alanine, cysteine, or leucine, substitution of the amino acid residue at position 269 with isoleucine, and substitution of the amino acid residue at position 274 with glutamine. Conservative amino acid substitutions include any number of conservative amino acid substitutions, as long as they do not interfere with the formation of the complex.

[0071] The polypeptide of the present invention can be isolated or purified, either alone or in the form of a complex, by a general method used for protein purification, such as centrifugation, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc., either alone or in appropriate combinations. The protein recovered from the culture may be further purified by known means.

[0072] 8. Detergent Compositions The complex of the polypeptide and lipase 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. From the viewpoint of stability, it is preferable to use either one or both of the polypeptide and lipase complex of the present invention as the enzyme to be incorporated into the detergent composition, and it is more preferable to use at least the lipase complex of the present invention. Hereinafter, the polypeptide and lipase complex of the present invention will be collectively referred to as the polypeptide of the present invention. Herein, "low temperature" refers to 40°C or lower, 35°C or lower, 30°C or lower, or 25°C or lower, as well as 5°C or higher, 10°C or higher, or 15°C or higher. Examples of temperatures include 5 to 40°C, 10 to 35°C, 15 to 30°C, and 15 to 25°C.

[0073] The amount of the polypeptide of the present invention to be incorporated into a detergent composition is not particularly limited as long as the polypeptide exhibits activity, but is, for example, preferably 0.1 mg or more, more preferably 1 mg or more, even more preferably 5 mg or more, and preferably 5,000 mg or less, even more preferably 1,000 mg or less, even more preferably 500 mg or less, per 1 kg of detergent composition. The amount is also preferably 0.1 to 5,000 mg, more preferably 1 to 1,000 mg, and even more preferably 5 to 500 mg.

[0074] The detergent composition preferably contains, in addition to the polypeptide of the present invention, a sulfosuccinate ester or a salt thereof, which is a surfactant. Sulfosuccinate esters or salts thereof are known as components incorporated into detergent compositions (for example, 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.

[0075] 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.

[0076] Examples of sulfosuccinate esters or salts thereof include compounds represented by the following formula 1:

[0077]

[0078] [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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083]

[0084] [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.

[0085] 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.

[0086] The detergent composition can also contain various enzymes in addition to the polypeptide of the present invention. Examples of such enzymes include hydrolases, oxidases, reductases, transferases, lyases, isomerases, ligases, synthetases, etc. Among these, lipases other than the lipase variants of the present invention, amylases, proteases, cellulases, keratinases, esterases, cutinases, pullulanases, pectinases, mannanases, glucosidases, glucanases, cholesterol oxidases, peroxidases, laccases, etc. are preferred, with proteases, cellulases, amylases, and lipases other than the polypeptide of the present invention being 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 the detergent composition, one or more of the above enzymes can be used in combination with the polypeptide etc. of the present invention. Examples of combinations include, but are not limited to, a combination of the polypeptide etc. of the present invention and a protease, a combination of the polypeptide etc. of the present invention and an amylase, a combination of the polypeptide etc. of the present invention, a protease and an amylase, a combination of a lipase different from the polypeptide etc. of the present invention and the polypeptide etc. of the present invention, a combination of the polypeptide etc. of the present invention and a lipase different from the polypeptide etc. of the present invention and an amylase, a combination of the polypeptide etc. of the present invention, a lipase different from the polypeptide etc. of the present invention, a protease and an amylase, etc., and among these, a combination of the polypeptide etc. of the present invention, a protease and an amylase is preferred.

[0087] The detergent composition may contain known detergent components, and examples of such known detergent components include the following:

[0088] (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.

[0089] 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.

[0090] 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.

[0091] (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.

[0092] (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.

[0093] (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.

[0094] (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.

[0095] (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.

[0096] (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.

[0097] (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.

[0098] 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.

[0099] 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.

[0100] The detergent composition can be produced by combining the polypeptide of the present invention obtained by the above method with the above-mentioned known detergent components according to a conventional method. The form of the detergent can be selected depending on the application, and can be, for example, a liquid, powder, granule, paste, solid, etc.

[0101] 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).

[0102] By using the detergent composition of the present invention, it is possible to clean objects requiring stain removal (e.g., clothes, tableware, hard surfaces, drain pipes, dentures, medical instruments, etc.), i.e., 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 an oil-containing stain, and examples of the stain include oil stains and complex stains containing proteins and carbohydrates in addition to oil.

[0103] 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.

[0104] In addition to the above-described embodiments, the present invention also discloses the following aspects: <1> A lipase complex comprising two or more polypeptide molecules, the two or more polypeptide molecules being linked via disulfide bonds, and at least one of the two or more polypeptide molecules being a polypeptide having lipase activity consisting of an amino acid sequence having at least 70% identity to the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 14, 16, 18, or 20. <2> The lipase complex according to <1>, wherein the polypeptide having lipase activity consists of an amino acid sequence having at least 70% identity to the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 14, 16, 18, or 20 and in which the amino acid residues at one or more positions selected from positions 208 and 272 in SEQ ID NO: 4 are cysteine. <3> A lipase complex according to <1> or <2>, which is composed of two polypeptide molecules, the two polypeptide molecules being linked via a disulfide bond, and the two polypeptide molecules are each independently a polypeptide having lipase activity, which has an amino acid sequence that has at least 70% identity with the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 14, 16, 18 or 20 and in which the amino acid residues at one or more positions selected from positions corresponding to 208 and 272 in the numbering of SEQ ID NO: 4 are cysteine. <4> A lipase complex according to <1> or <2>, which is composed of two identical polypeptide molecules, the two polypeptide molecules being linked via a disulfide bond, and which has lipase activity and is composed of an amino acid sequence that has at least 70% identity with the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 14, 16, 18 or 20 and in which the amino acid residues at one or more positions selected from positions 208 and 272 in the numbering of SEQ ID NO: 4 are cysteine.

[0105] <5> A method for producing a lipase complex, the method comprising a step of linking, via a disulfide bond, two or more polypeptide molecules, each including at least one polypeptide molecule having lipase activity, the polypeptide molecule having an amino acid sequence having at least 70% identity to the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, or 18. <6> The method according to <5>, wherein the polypeptide having lipase activity has an amino acid sequence having at least 70% identity to the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, or 18, and in which the amino acid residues at one or more positions selected from positions 208 and 272 in the numbering of SEQ ID NO: 4 are cysteine. <7> The method according to <5> or <6>, wherein the binding step involves binding two polypeptide molecules via a disulfide bond, and the two polypeptide molecules are each independently a polypeptide having lipase activity consisting of an amino acid sequence that has at least 70% identity to the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 14, 16, 18, or 20 and in which cysteine ​​residues are present at one or more positions selected from positions 208 and 272 in the numbering of SEQ ID NO: 4. <8> The method according to <5> or <6>, wherein the binding step involves binding two identical polypeptide molecules via a disulfide bond, and the polypeptides are polypeptides having lipase activity consisting of an amino acid sequence that has at least 70% identity to the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 14, 16, 18, or 20 and in which cysteine ​​residues are present at one or more positions selected from positions 208 and 272 in the numbering of SEQ ID NO: 4. <9> The method according to any one of <5> to <8>, further comprising, prior to the binding step, a step of providing a polypeptide having lipase activity, which consists of an amino acid sequence that has at least 70% identity with the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16 or 18 and in which the amino acid residues at one or more positions selected from positions corresponding to 208 and 272 in the numbering of SEQ ID NO: 4 are cysteine.<10> A method for producing a lipase complex, comprising: substituting, with cysteine, amino acid residues at one or more positions selected from positions corresponding to 208 and 272 in the numbering of SEQ ID NO: 4, in a polypeptide having lipase activity and consisting of an amino acid sequence having at least 70% identity to the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, or 18; and bonding, via a disulfide bond, two or more polypeptide molecules, each including at least one molecule of the polypeptide after the amino acid residue substitution. <11> The method according to <10>, wherein the binding step involves binding two molecules of the polypeptide after amino acid residue substitution via a disulfide bond, and the two molecules of the polypeptide after amino acid residue substitution are each independently a polypeptide having lipase activity consisting of an amino acid sequence that has at least 70% identity to the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 14, 16, 18, or 20 and in which the amino acid residues at one or more positions selected from positions 208 and 272 in SEQ ID NO: 4 are cysteine. <12> The method according to <10>, wherein the binding step involves binding two molecules of the same polypeptide after amino acid residue substitution via a disulfide bond. <13> The method according to any one of <5> to <12>, wherein the binding step is performed under non-reducing conditions, preferably by expressing or maintaining the polypeptide under non-reducing conditions.

[0106] <14> A method for improving the stability of a lipase, comprising a step of complexing the lipase. <15> The method according to <14>, comprising a step of conjugating, via a disulfide bond, two or more polypeptide molecules, each including at least one polypeptide molecule having lipase activity, the polypeptide consisting of an amino acid sequence having at least 70% identity to the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, or 18. <16> The method according to <15>, wherein the polypeptide having lipase activity consists of an amino acid sequence having at least 70% identity to the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, or 18, and in which the amino acid residues at one or more positions selected from positions 208 and 272 in SEQ ID NO: 4 are cysteine. <17> The method according to <15> or <16>, wherein the binding step involves binding two polypeptide molecules via a disulfide bond, and the two polypeptide molecules are each independently a polypeptide having lipase activity consisting of an amino acid sequence that has at least 70% identity with the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 14, 16, 18, or 20 and in which cysteine ​​residues are present at one or more positions selected from positions 208 and 272 in SEQ ID NO: 4. <18> The method according to <15> or <16>, wherein the binding step involves binding two identical polypeptide molecules via a disulfide bond, and the polypeptides are each a polypeptide having lipase activity consisting of an amino acid sequence that has at least 70% identity with the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 14, 16, 18, or 20 and in which cysteine ​​residues are present at one or more positions selected from positions 208 and 272 in SEQ ID NO: 4. <19> The method according to any one of <15> to <18>, further comprising, prior to the binding step, a step of providing a polypeptide having lipase activity, which consists of an amino acid sequence that has at least 70% identity with the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16 or 18 and in which the amino acid residues at one or more positions selected from positions corresponding to 208 and 272 in the numbering of SEQ ID NO: 4 are cysteine.<20> A method for improving the stability of a lipase, comprising: substituting, with cysteine, amino acid residues at one or more positions selected from positions corresponding to 208 and 272 in the numbering of SEQ ID NO: 4, in a polypeptide having lipase activity and consisting of an amino acid sequence having at least 70% identity to the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, or 18; and bonding, via a disulfide bond, two or more polypeptide molecules, each including at least one molecule of the polypeptide after the amino acid residue substitution. <21> The method according to <20>, wherein the binding step involves binding two molecules of the polypeptide after amino acid residue substitution via a disulfide bond, and the two molecules of the polypeptide after amino acid residue substitution are each independently a polypeptide having lipase activity consisting of an amino acid sequence that has at least 70% identity to the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 14, 16, 18, or 20 and in which the amino acid residues at one or more positions selected from positions 208 and 272 in SEQ ID NO: 4 are cysteine. <22> The method according to <20>, wherein the binding step involves binding two molecules of the same polypeptide after amino acid residue substitution via a disulfide bond. <23> The method according to any one of <15> to <22>, wherein the binding step is performed under non-reducing conditions, preferably by expressing or maintaining the polypeptide under non-reducing conditions.

[0107] <24> A polypeptide having lipase activity, which has at least 70% identity to the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 14, 16, 18, or 20, and which contains cysteine ​​at one or more positions selected from positions corresponding to positions 208 and 272 in the numbering of SEQ ID NO: 4. <25> A polypeptide having a phenylalanine at position corresponding to position 16 in the numbering of SEQ ID NO: 4, a cysteine ​​or methionine at position 22, a phenylalanine at position corresponding to position 29, an isoleucine or valine at position 50, an alanine or glutamine at position 120, an isoleucine or threonine at position 123, a glutamic acid at position 126, a phenylalanine at position 142, an alanine at position 156, a lysine at position 181, a glutamic acid at position 186, a cysteine ​​or methionine at position 22, a phenylalanine at position 29, an isoleucine or valine at position 50, an alanine or glutamine at position 120, an isoleucine or threonine at position 123, a glutamic acid at position 126, a phenylalanine at position 142, an alanine at position 156, a lysine at position 181, a glutamic acid at position 186, a cysteine ​​or methionine at position 2 ... The complex according to any one of <1> to <4> or the polypeptide according to <24>, which has one or more amino acid residues selected from glutamine at a position corresponding to position 88, valine at a position corresponding to position 191, histidine at a position corresponding to position 220, histidine at a position corresponding to position 221, glutamine at a position corresponding to position 224, threonine at a position corresponding to position 233, asparagine at a position corresponding to position 256, alanine, cysteine ​​or leucine at a position corresponding to position 260, isoleucine at a position corresponding to position 269, and glutamine at a position corresponding to position 274. <26> The lipase complex according to any one of <1> to <4> and <25> or the polypeptide according to <24> or <25>, wherein the polypeptide having lipase activity is a mutant polypeptide.

[0108] <27> A polynucleotide encoding the polypeptide according to any one of <24> to <26>. <28> A vector or DNA fragment comprising the polynucleotide according to <27>. <29> A transformed cell comprising the vector or DNA fragment according to <28>. <30> The transformed cell according to <29>, which is a microorganism. <31> The transformed cell according to <29>, which is Escherichia coli or a Bacillus bacterium, preferably a Bacillus bacterium, more preferably Bacillus subtilis. <32> A method for producing a polypeptide having lipase activity, which comprises a step of culturing the transformed cell according to any one of <29> to <31>.

[0109] <33> A detergent composition comprising one or both of the lipase complex according to any one of <1> to <4>, <25> and <26> and the polypeptide according to any one of <24> to <26>, preferably the lipase complex according to any one of <1> to <4>, <25> and <26>, or the lipase complex according to any one of <1> to <4>, <25> and <26> and the polypeptide according to any one of <24> to <26>. <34> The detergent composition according to <33>, further comprising 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. <35> The detergent composition according to <33>, further comprising a sulfosuccinic acid diester or a salt thereof, preferably a sulfosuccinic acid di-branched alkyl ester or a salt thereof, wherein each of the two branched alkyl groups has from 8 to 12 carbon atoms, more preferably a sulfosuccinic acid di-branched alkyl ester or a salt thereof, wherein each of the two branched alkyl groups has from 9 to 10 carbon atoms, even more preferably a sulfosuccinic acid di-branched alkyl ester 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. <36> The detergent composition according to any one of <33> to <35>, wherein the water content is 60 to 99 mass% and / or the solvent content is greater than the surfactant content, preferably the water content is 60 to 99 mass% and the solvent content is greater than the surfactant content. <37> The detergent composition according to any one of <33> to <36>, which is a laundry detergent or a dishwashing detergent. <38> The detergent composition according to any one of <33> to <37>, which is a powder or a liquid.

[0110] <39> A method for cleaning stains using the detergent composition according to any one of <33> to <38>. <40> The method according to <39>, comprising contacting an object to be cleaned with the detergent composition according to any one of <33> to <38>. <41> Use of one or both of the lipase complex according to any one of <1> to <4>, <25> and <26> and the polypeptide according to any one of <24> to <26>, preferably the lipase complex according to any one of <1> to <4>, <25> and <26>, or the lipase complex according to any one of <1> to <4>, <25> and <26> and the polypeptide according to any one of <24> to <26>, for the production of a detergent composition. <42> Use of one or both of the lipase complex according to any one of <1> to <4>, <25> and <26> and the polypeptide according to any one of <24> to <26>, preferably the lipase complex according to any one of <1> to <4>, <25> and <26>, or the lipase complex according to any one of <1> to <4>, <25> and <26> and the polypeptide according to any one of <24> to <26>, for cleaning dirt.

[0111] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples.

[0112] Example 1 (1) Construction of Lipase Expression Plasmid Using the plasmid pHY-S237 described in Example 7 of WO2006 / 068148A1 as a template, the full-length ORF of an alkaline cellulase gene was replaced by an artificial gene synthesized, 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), described in Japanese Patent Application No. 2023-025141, by in-fusion reaction, to construct the plasmid pHY-CnLipA. Using pHY-CnLipA as a template, five mutations were introduced by site-specific PCR using complementary primer pairs (Zheng, Lei, Ulrich Baumann, and Jean-Louis Reymond. Nucleic Acids Research 32.14 (2004): e115.), to construct a plasmid pHY-CnLipAm expressing CnLipAm (encoding the polynucleotide sequence of SEQ ID NO:3 and the amino acid sequence of SEQ ID NO:4). Using the plasmid pHY-S237 described in Example 7 of WO2006 / 068148A1 as a template, the entire ORF of the alkaline cellulase gene was artificially synthesized SspLip_m (polynucleotide of SEQ ID NO: 5, encoding the amino acid sequence of SEQ ID NO: 6), EtLip_m (polynucleotide of SEQ ID NO: 7, encoding the amino acid sequence of SEQ ID NO: 8), or CspLip I45T L121S S131A A135T A136V S138N A213M (hereinafter referred to as CspLipA, polynucleotide of SEQ ID NO: 9, encoding the amino acid sequence of SEQ ID NO: 10) by in-fusion reaction to construct the plasmids pHY-SspLip_m, pHY-EtLip_m, and pHY-CspLipA. For further mutation introduction into the lipase, site-directed mutagenesis by PCR using complementary primer pairs was used.

[0113] (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 mixture 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 4 days, and the culture supernatant containing lipase was recovered by centrifugation.

[0114] (3) SDS-PAGE of lipase mutants. Culture supernatant containing CnLipAm (parent enzyme), CnLipAm L208C mutant, or CnLipAm N272C mutant prepared by the method described in (2) was mixed with equal volumes of Laemmli Sample Buffer (BIO-RAD) containing or not containing 100 mM DTT as a reducing agent, and the mixture was then heat-treated at 99°C for 5 minutes to prepare a sample. Mini-PROTEIN TGX Stain-Free gel (BIO-RAD) was used. 5 μL of sample was applied to each well and electrophoresed at 210 V for 25 minutes. Precision Plus protein unstained standard (BIO-RAD) was used as a molecular weight marker. Protein band intensities were calculated using the ChemiDoc MP Imaging System. The monomer ratio (%) of each mutant was calculated by dividing the band intensity under non-reducing conditions by the band intensity under reducing conditions (Table 1). The decreased monomer ratio in the mutants revealed that they formed a complex via disulfide formation in the culture supernatant.

[0115]

[0116] (4) Purification of lipase complex by gel filtration chromatography The culture supernatant containing CnLipAm (parent enzyme), CnLipAm L208C mutant, or CnLipAm N272C mutant prepared by the method described in (2) was subjected to Amicon Ultra 10K (Merck Millipore) and buffer exchanged to 20 mM Tris-HCl (pH 7.0), 2 mM CaCl, 0.01% Triton-X100, and 150 mM NaCl to prepare a lipase sample for gel filtration. The lipase sample for gel filtration was subjected to gel filtration chromatography under the conditions shown below to obtain a fraction sample. (Conditions) Apparatus: AKTA pure (GE healthcare) Column: HiLoad 26 / 600 Superdex 200 pg (cytiva) Buffer: 20 mM Tris-HCl (pH 7.0), 2 mM CaCl2, 0.01% Triton-X100, 150 mM NaCl Flow rate: 1 mL / min Elute fraction size: 1.5 mL Length of elution: 1.3 CV

[0117] 4-Nitrophenyl butyrate (SIGMA) was used as a substrate to measure lipase activity in the fraction samples. Lipase activity can be determined by measuring the rate of increase in absorbance associated with the release of 4-nitrophenol by the action of lipase. 4-Nitrophenyl butyrate was added to 20 mM Tris-HCl (pH 7.0) at a final concentration of 2 mM and mixed to prepare the substrate solution. The fraction samples were diluted 51-fold with 20 mM Tris-HCl (pH 7.0) and subjected to activity measurements. Lipase activity was used as an indicator to identify fractions containing CnLipAm L208C monomer, CnLipAm L208C complex 1, CnLipAm L208C complex 2, CnLipAm N272C monomer, CnLipAm N272C complex 1, CnLipAm N272C complex 2, and CnLipAm (parent enzyme) monomer. The fraction solutions containing lipase monomers or complexes were subjected to SDS-PAGE as described in (3) (Figure 1). A clear band was observed at twice the predicted molecular weight for CnLipAm L208C complex 1, revealing that this fraction contained a homodimer. CnLipAm N272C complex 2 contained a band with a molecular weight twice that of the predicted molecular weight, indicating that it contained a homodimer, and furthermore, the presence of complexes formed via disulfide bonds with components contained in the culture supernatant. CnLipAm L208C complex 2 and CnLipAm N272C complex 1 were confirmed to form complexes with components contained in the culture supernatant via disulfide bonds.

[0118] (5) Evaluation of Stability in Model Washing Solution 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 release of 4-nitrophenol by the action of lipase. A mixture of 20 mM Tris-HCl (pH 7.0) and 4-nitrophenyl butyrate at a final concentration of 2 mM was used as the substrate solution. A fraction solution containing lipase monomer or complex was added to the model washing solution shown in Table 2, incubated at 45°C for 1 hour, and then diluted 51 times with 20 mM Tris-HCl (pH 7.0) for activity measurement. The activity value of the sample before 45°C treatment was used as the initial activity, and the inactivation rate per unit time (h) due to 45°C treatment was calculated, from which the half-life (h) was calculated. Relative stability was determined by dividing the half-life (h) of each mutant by the half-life (h) of the parent polypeptide. The results are shown in Table 3. It was revealed that the CnLipAm L208C and CnLipAm N272C mutants were significantly stabilized by forming complexes compared to the parent enzymes.

[0119]

[0120]

[0121] (6) 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 each well of a 96-well polypropylene deep-well plate, and the chloroform was evaporated and dried to prepare a soil plate. The lipase concentration obtained by gel filtration chromatography in (4) was diluted to 200 mg / L with 20 mM Tris-HCl (pH 7.0), 2 mM CaCl2, 0.01% Triton-X100, and 150 mM NaCl to prepare a lipase solution. This solution was added in an amount of 1 / 25 of the model cleaning solution. 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 20 minutes for immersion cleaning. 100 μL of the cleaning solution was transferred to a new 96-well plate without touching 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 cleaning. The 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), 2 mM CaCl2, 0.01% Triton-X100, and 150 mM NaCl instead of lipase. The results of the 20-minute cleaning are shown in Table 4. It was revealed that the CnLipAm L208C and CnLipAm N272C mutants maintained their detergency even after complex formation.

[0122]

[0123] (7) Evaluation of Lipase Variant Stability 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 release of 4-nitrophenol by the action of lipase. 20 mM Tris-HCl (pH 7.0) was mixed with 4-nitrophenyl butyrate at a final concentration of 2 mM to prepare a substrate solution. Culture supernatants containing SspLip_m, EtLip_m, CspLipA, and the SspLip_m L210C, EtLip_m F210C, and CspLipA ​​L209C mutants prepared by the method described in (2) were added to the model washing solutions shown in Table 2 and incubated at 40°C or 45°C for a desired time. Activity was then measured using a 51x solution of 20 mM Tris-HCl (pH 7.0). The activity value of the sample before heat treatment was taken as the initial activity, and the inactivation rate per unit time (h) due to heat treatment was calculated, from which the half-life (h) was calculated. The relative stability was determined by dividing the half-life (h) of each mutant by the half-life (h) of the parent polypeptide. The results are shown in Table 5. All mutants were shown to have improved stability compared to the parent polypeptide. Note that positions 210 of SspLip_m, 210 of EtLip_m, and 209 of CspLipA ​​correspond to positions 208 of CnLipAm, respectively.

[0124]

[0125] (8) SDS-PAGE of Lipase Variants. Culture supernatants containing SspLip_m, EtLip_m, CspLipA, and SspLip_m L210C, EtLip_m F210C, and CspLipA ​​L209C mutants prepared by the method described in (2) were mixed with equal volumes of Laemmli Sample Buffer (BIO-RAD) containing or not containing 100 mM DTT as a reducing agent, and the mixture was then heat-treated at 99°C for 5 minutes to prepare samples. Mini-PROTEIN TGX Stain-Free gel (BIO-RAD) was used. 5 μL of sample was applied to each well and electrophoresed at 210 V for 25 minutes. Precision Plus protein unstained standard (BIO-RAD) was used as a molecular weight marker. Protein band intensity was calculated using the ChemiDoc MP Imaging System. The monomer ratio (%) of each mutant was calculated by dividing the band intensity under non-reducing conditions by the band intensity under reducing conditions (Table 6). The monomer ratio was less than 100% for the mutants, demonstrating that a complex was formed in the culture supernatant via disulfide formation.

[0126]

[0127] Example 2: Evaluation of Stability of Combination Mutants 4-Nitrophenyl butyrate (SIGMA) was used as the substrate. Lipase activity can be determined by measuring the rate of increase in absorbance associated with the release of 4-nitrophenol by the action of lipase. 4-Nitrophenyl butyrate was added to 20 mM Tris-HCl (pH 7.0) at a final concentration of 2 mM and mixed to prepare the substrate solution. Lipase solution was added to the model cleaning solution shown in Table 2, incubated at 40°C for 3 hours, and then diluted 51 times with 20 mM Tris-HCl (pH 7.0) for activity measurement. The activity value of the sample before 40°C treatment was used as the initial activity, and the inactivation rate per unit time (h) due to 40°C treatment was calculated, from which the half-life (h) was calculated. Relative stability was determined by dividing the half-life (h) of each mutant by the half-life (h) of the parent polypeptide. The results are shown in Table 7. All mutants demonstrated improved stability compared to the parent polypeptide.

[0128]

[0129]

[0130] Reference Example 1: Comparison of stability between model cleaning solution 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 Example 1(2) was added to the model cleaning solution shown in Table 2 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 taken as the initial activity, and the ratio of the activity after treatment to the initial activity was taken as the residual activity (%). The results are shown in Table 8. In the model cleaning solution shown in Table 2 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.

[0131]

[0132] Reference Example 2: Evaluation of Lipase Variant Stability 4-Nitrophenyl butyrate (SIGMA) was used as a substrate. Lipase activity can be determined by measuring the rate of increase in absorbance accompanying the release of 4-nitrophenol by the action of lipase. A mixture of 20 mM Tris-HCl (pH 7.0) and 4-nitrophenyl butyrate at a final concentration of 2 mM was used as the substrate solution. Plasmid pHY-PvLip (encoding the polynucleotide sequence of SEQ ID NO: 11 and the amino acid sequence of SEQ ID NO: 12), synthesized artificially, was constructed by in-fusion reaction using plasmid pHY-S237 (described in Example 7 of WO 2006 / 068148 A1) as a template. Additional mutations were introduced into the lipase using site-directed mutagenesis by PCR using complementary primer pairs. Culture supernatant containing PvLip and the PvLip A205C mutant prepared by the method described in Example 1(2) was added to the model washing solution shown in Table 2, incubated at 45°C for 50 minutes, and then diluted 51 times with 20 mM Tris-HCl (pH 7.0) and activity was measured. The activity value of the sample before heat treatment was used as the initial activity, and the inactivation rate per unit time (h) due to heat treatment was calculated, from which the half-life (h) was calculated. Relative stability was determined by dividing the half-life (h) of each mutant by the half-life (h) of the parent polypeptide. The results are shown in Table 9. It was demonstrated that the PvLip A205C mutant did not have improved stability compared to the parent polypeptide. Note that position 205 of PvLip corresponds to position 208 of CnLipAm.

[0133]

[0134] Reference Example 3: SDS-PAGE of lipase mutants Equal volumes of culture supernatant containing PvLip and the PvLip A205C mutant prepared by the method described in Example 1(2) were mixed with Laemmli Sample Buffer (BIO-RAD) containing or not containing 100 mM DTT as a reducing agent, and the mixture was heat-treated at 99°C for 5 minutes to prepare a sample. Mini-PROTEIN TGX Stain-Free (BIO-RAD) was used as the gel. 5 μL of sample was applied to each well and electrophoresed at 210 V for 25 minutes. Precision Plus protein unstained standard (BIO-RAD) was used as a molecular weight marker. Protein band intensities were calculated using the ChemiDoc MP Imaging System. The monomer ratio (%) of each mutant was calculated by dividing the band intensity under non-reducing conditions by the band intensity under reducing conditions (Table 10). The PvLip A205C mutant had a monomer ratio of 100%, demonstrating that no disulfide-mediated complexes were formed in the culture supernatant.

[0135]

[0136] Reference Example 4: Evaluation of lipase mutant stability 4-Nitrophenyl butyrate (SIGMA) was used as a substrate. Lipase activity can be determined by measuring the rate of increase in absorbance accompanying 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. Culture supernatants containing CnLipAm (parent enzyme), CnLipAm P207C mutant, or CnLipAm H209C mutant prepared by the method described in Example 1(2) were added to the model washing solutions shown in Table 2 and incubated at 40°C for 1 hour. Activity was then measured using a solution diluted 51 times with 20 mM Tris-HCl (pH 7.0). The activity value of the sample before heat treatment was used as the initial activity, and the inactivation rate per unit time (h) due to heat treatment 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 Table 11. It was confirmed that none of the mutants had improved stability compared to the parent polypeptide.

[0137]

Claims

1. A lipase complex comprising two or more polypeptide molecules, the two or more polypeptide molecules being linked via disulfide bonds, and at least one of the two or more polypeptide molecules being a polypeptide having lipase activity consisting of an amino acid sequence having at least 70% identity to the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 14, 16, 18 or 20.

2. The lipase complex according to claim 1, wherein the polypeptide having lipase activity has an amino acid sequence which has at least 70% identity with the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 14, 16, 18 or 20 and in which the amino acid residue at one or more positions selected from positions corresponding to 208 and 272 in the numbering of SEQ ID NO: 4 is cysteine.

3. A lipase complex according to claim 1, which is composed of two polypeptide molecules linked together via a disulfide bond, and each of the two polypeptide molecules is independently a polypeptide having lipase activity, which has an amino acid sequence that has at least 70% identity with the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 14, 16, 18 or 20 and in which the amino acid residue at one or more positions selected from positions corresponding to 208 and 272 in the numbering of SEQ ID NO: 4 is cysteine.

4. A method for producing a lipase complex, comprising a step of linking, via a disulfide bond, two or more polypeptide molecules, including at least one polypeptide molecule having lipase activity, the polypeptide molecule having an amino acid sequence that has at least 70% identity to the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 14, 16, 18 or 20 and in which the amino acid residues at one or more positions selected from positions corresponding to 208 and 272 in the numbering of SEQ ID NO: 4 are cysteine.

5. The method according to claim 4, further comprising, prior to the binding step, a step of providing a polypeptide having lipase activity consisting of an amino acid sequence having at least 70% identity with the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 14, 16, 18 or 20 and in which the amino acid residue at one or more positions selected from positions corresponding to 208 and 272 in the numbering of SEQ ID NO: 4 is cysteine.

6. A method for improving the stability of lipase, comprising the step of linking, via a disulfide bond, two or more polypeptide molecules, including at least one polypeptide molecule having lipase activity, the polypeptide molecule having an amino acid sequence that has at least 70% identity to the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 14, 16, 18 or 20 and in which the amino acid residues at one or more positions selected from positions corresponding to 208 and 272 in the numbering of SEQ ID NO: 4 are cysteine.

7. The method according to claim 6, further comprising, prior to the binding step, a step of providing a polypeptide having lipase activity consisting of an amino acid sequence having at least 70% identity with the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 14, 16, 18 or 20, and in which the amino acid residue at one or more positions selected from positions corresponding to 208 and 272 in the numbering of SEQ ID NO: 4 is cysteine.

8. A polypeptide having lipase activity, which consists of an amino acid sequence having at least 70% identity to the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 14, 16, 18 or 20 and in which the amino acid residue at one or more positions selected from positions corresponding to 208 and 272 in the numbering of SEQ ID NO: 4 is cysteine.

9. A polynucleotide encoding the polypeptide of claim 8.

10. A vector or DNA fragment comprising the polynucleotide of claim 9.

11. A transformed cell containing the vector or DNA fragment according to claim 10.

12. The transformed cell according to claim 11, which is a microorganism.

13. A detergent composition comprising either or both of the lipase complex according to any one of claims 1 to 3 and the polypeptide according to claim 8.

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