Lipase stability improvement method

Abstract

To provide a lipase stability improvement method.SOLUTION: 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 of complexing a lipase.SELECTED DRAWING: None

Description

Technical Field

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

Background Art

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

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

[0004] On the other hand, the amino acid sequence may be 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 environmental pH and redox potential, and the structure and function may change due to a significant change in the properties of amino acids site-specifically caused by a change in the oxidation state. Further, in Non-Patent Document 2, the introduction of cysteine residues is avoided during protein design. Since the introduction of cysteine residues may inhibit the maintenance of the structure and function of proteins, it is often treated as an unfavorable mutation.

Prior Art Documents

Patent Documents

[0005] [Patent Document 1] Japanese Unexamined Patent Application Publication No. 2021-17508 [Patent Document 2] Japanese Patent No. 4776997 [Non-Patent Document]

[0006] [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 [Summary of the Invention] [Problems to be Solved by the Invention]

[0007] Compared with general detergents, in an environment harsh for enzymes in a detergent composition having a large amount of water and containing more solvent than surfactant, the stability of the incorporated lipase was unknown. However, as a result of studies by the present inventors, it has been clarified that the stability is significantly reduced compared with when incorporated into general detergents. Therefore, the present invention relates to a method for improving the stability of lipase and a lipase exhibiting excellent stability. [Means for Solving the Problems]

[0008] When the present inventors substituted the amino acid residue at a specific position in the amino acid sequence of the parent lipase with cysteine, the obtained lipase mutant was able to form a complex containing the lipase mutant. In particular, in the form of the complex, compared with the parent lipase, it has improved stability in the presence of a large amount of water and / or a large amount of solvent more than surfactant. That is, it has been found that the stability of lipase can be improved by complex formation of lipase.

[0009] That is, the present invention relates to the following 1) to 8). 1) A lipase complex comprising two or more polypeptides, wherein the two or more polypeptides are linked via disulfide bonds, and at least one of the two or more polypeptides is a polypeptide having lipase activity consisting of an amino acid sequence having at least 70% identity with the amino acid sequence represented by SEQ ID NO: 4, 6, 8, 10, 14, 16, 18 or 20. 2) A method for producing a lipase complex, comprising a step of linking two or more polypeptides, wherein at least one of the two or more polypeptides is a polypeptide having lipase activity consisting of an amino acid sequence having at least 70% identity with the amino acid sequence represented by SEQ ID NO: 4, 6, 8, 10, 14, 16, 18 or 20 and having cysteine as an amino acid residue at one or more positions selected from the positions corresponding to positions 208 and 272 in the numbering of SEQ ID NO: 4, via disulfide bonds. 3) A method for improving the stability of lipase, comprising a step of linking two or more polypeptides, wherein at least one of the two or more polypeptides is a polypeptide having lipase activity consisting of an amino acid sequence having at least 70% identity with the amino acid sequence represented by SEQ ID NO: 4, 6, 8, 10, 14, 16, 18 or 20 and having cysteine as an amino acid residue at one or more positions selected from the positions corresponding to positions 208 and 272 in the numbering of SEQ ID NO: 4, via disulfide bonds. 4) A polypeptide having lipase activity, consisting of an amino acid sequence having at least 70% identity with the amino acid sequence represented by SEQ ID NO: 4, 6, 8, 10, 14, 16, 18 or 20 and having cysteine as an amino acid residue at one or more positions selected from the positions corresponding to positions 208 and 272 in the numbering of SEQ ID NO: 4. 5) A polynucleotide encoding the polypeptide according to 4). 6) A vector or DNA fragment containing 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 according to 1) and the polypeptide according to 4).

Advantages of the Invention

[0010] According to the present invention, the stability of lipase can be improved by complexing the lipase. A polypeptide having lipase activity capable of forming a complex of lipase exhibits excellent stability particularly in the form of a complex, and can be suitably formulated in a detergent composition containing a larger amount of solvent than a large amount of water and / or surfactant.

Brief Description of Drawings

[0011]

Figure 1

Figure 2

Embodiments for Carrying Out the Invention

[0012] All patent documents, non-patent documents, and other publications cited herein are hereby incorporated by reference in their entirety.

[0013] As used herein, "lipase" refers to triacylglycerol lipase (EC 3.1.1.3) and means a group of enzymes having lipase activity that hydrolyzes an ester bond in a lipid to produce a fatty acid. Lipase activity can be determined by measuring the rate of increase in absorbance accompanying the release of 4-nitrophenol by hydrolysis of 4-nitrophenyl butyrate. The specific procedure for measuring lipase activity is described in detail in the examples below.

[0014] As used herein, the identity of an amino acid sequence or nucleotide sequence is calculated by the Lipman-Pearson method (Science, 1985, 227: 1435-1441). Specifically, it is calculated by performing an analysis with the homology analysis (Search homology) program of the genetic information processing software GENETYX Ver. 12 with Unit size to compare (ktup) being 2.

[0015] As used herein, "at least 70% identity" with respect to an amino acid sequence or a nucleotide sequence means an identity of 70% or more, preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, still more preferably 93% or more, still more preferably 94% or more, still more preferably 95% or more, still more preferably 96% or more, still more preferably 97% or more, still more preferably 98% or more, still more preferably 99% or more, still more preferably 99.5% or more.

[0016] As used herein, the "corresponding position" on an amino acid sequence or a nucleotide sequence can be determined by aligning the target sequence and a reference sequence (for example, the amino acid sequence shown in SEQ ID NO: 4) so as to give the maximum homology. Alignment of amino acid sequences or nucleotide sequences can be performed using known algorithms, and the procedures are known to those skilled in the art. For example, alignment can be performed by using the Clustal W multiple alignment program (Thompson, J.D. et al, 1994, Nucleic Acids Res. 22: 4673-4680) with default settings. Alternatively, Clustal W2 or Clustal omega, which is a revised version of Clustal W, can also be used. Clustal W, Clustal W2, and Clustal omega can be used, for example, on the websites of Clustal run by University College Dublin [www.clustal.org], the European Bioinformatics Institute (EBI [www.ebi.ac.uk / index.html]), or the DNA Data Bank of Japan (DDBJ [www.ddbj.nig.ac.jp / searches-j.html]) run by the National Institute of Genetics. The position of the target sequence aligned with any position of the reference sequence by the above alignment is regarded as the "corresponding position" to the arbitrary position.

[0017] A person skilled in the art can further fine-tune the alignment of the amino acid sequences obtained above so as to optimize it. Such an optimal alignment is preferably determined in consideration of the similarity of the amino acid sequences, the frequency of inserted gaps, etc. Here, the similarity of amino acid sequences refers to the ratio (%) of the number of positions where identical or similar amino acid residues exist in both sequences when two amino acid sequences are aligned to the total number of amino acid residues in the full length. Similar amino acid residues mean amino acid residues that have properties similar to each other in terms of polarity and charge among the 20 amino acids constituting proteins and cause so-called conservative substitutions. Groups consisting of such similar amino acid residues are well known to those skilled in the art. For example, arginine and lysine or glutamine; glutamic acid and aspartic acid or glutamine; serine and threonine or alanine; glutamine and asparagine or arginine; leucine and isoleucine, etc. can be mentioned respectively, but are not limited thereto.

[0018] In this specification, "amino acid residue" means the 20 amino acid residues constituting proteins, alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0019] In this specification, the description of the position of amino acids and variants is represented as follows using the recognized one-letter amino acid abbreviations of IUPAC. The amino acid at a predetermined position is represented as [amino acid, position]. For example, leucine at position 208 is shown as "L208". Regarding the "substitution" of amino acids, it is represented by [original amino acid, position, substituted amino acid]. For example, the substitution of leucine at position 208 with cysteine is denoted as "L208C". Variants containing multiple modifications are represented by the plus sign ("+"). For example, "L208C+N272C" represents the substitution of leucine at position 208 with cysteine and the substitution of asparagine at position 272 with cysteine, respectively. When different modifications can be introduced at one position, the different modifications are separated by a slash (" / "). For example, "G22C / M" represents the substitution of glycine at position 22 with cysteine or methionine.

[0020] As used herein, the "operable linkage" between a control region such as a promoter and a gene means that the gene and the control region are linked such that the gene can be expressed under the control of the control region. The procedures for "operable linkage" between a gene and a control region are well known to those skilled in the art.

[0021] As used herein, "upstream" and "downstream" with respect to a gene refer to upstream and downstream in the transcription direction of the gene. For example, a "gene located downstream of a promoter" means that the gene is present on the 3' side of the promoter in the DNA sense strand, and upstream of the gene means the region on the 5' side of the gene in the DNA sense strand.

[0022] As used herein, the "parent" polypeptide of a given mutant polypeptide refers to the polypeptide that becomes the mutant polypeptide by making a predetermined mutation to its amino acid residues. In other words, the "parent" polypeptide is the polypeptide before the mutation is added to the mutant polypeptide.

[0023] As used herein, the "lipase complex" refers to a complex in which two or more polypeptides, including at least one polypeptide (lipase) having lipase activity, are integrated via disulfide bonds. The individual polypeptides constituting the lipase complex are not particularly limited with respect to the presence or absence of lipase activity in other polypeptides as long as at least one polypeptide is a lipase, and they may be of the same or different types, preferably lipases of the same or different types, and more preferably lipases of the same type. Also, the number of polypeptides constituting the complex is not particularly limited, but is preferably 2. Therefore, examples of the lipase complex preferably include a dimer composed of two lipases of the same or different types, i.e., a lipase dimer, and more preferably a lipase dimer composed of two lipases of the same type. Hereinafter, the lipase complex may be simply referred to as a complex.

[0024] <1. Polypeptide having lipase activity> The polypeptide having lipase activity capable of forming the lipase complex of the present invention (referred to as "the polypeptide of the present invention") is a polypeptide having lipase activity, which consists of an amino acid sequence having at least 70% identity with the amino acid sequence represented by SEQ ID NO: 4, 6, 8, 10, 14, 16, 18 or 20. The polypeptide of the present invention has a cysteine involved in a disulfide bond in the amino acid sequence. The polypeptide of the present invention preferably consists of an amino acid sequence having at least 70% identity with the amino acid sequence represented by SEQ ID NO: 4, 6, 8, 10, 14, 16, 18 or 20 and having an amino acid residue at one or more positions selected from the positions corresponding to positions 208 and 272 in the numbering based on SEQ ID NO: 4 being cysteine, and is a polypeptide having lipase activity.

[0025] In one embodiment, the polypeptide of the present invention is a natural (wild-type) polypeptide having lipase activity, which consists of an amino acid sequence having at least 70% identity with the amino acid sequence represented by SEQ ID NO: 4, 6, 8, 10, 14, 16, 18 or 20. Such a natural polypeptide 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 with the amino acid sequence represented by SEQ ID NO: 4, 6, 8, 10, 14, 16, 18 or 20 (the mutant polypeptide among the polypeptides of the present invention may be particularly referred to as "the mutant polypeptide of the present invention"). Such a mutant polypeptide can have good stability in the form of a complex formed via disulfide bonds. Here, the stability more specifically refers to the stability in the presence of a larger amount of solvent than a large amount of water and / or surfactant.

[0026] In a preferred embodiment, the polypeptide of the present invention has at least 70% identity with the amino acid sequence represented by SEQ ID NO: 4, 6, 8, 10, 14, 16, 18 or 20, and is a natural (wild-type) polypeptide having lipase activity, which consists of an amino acid sequence in which at least one amino acid residue selected from the positions corresponding to positions 208 and 272 in the numbering based on SEQ ID NO: 4 is cysteine. The cysteine at the above-mentioned predetermined position contributes to the complex formation and stability of the lipase. Therefore, such a natural polypeptide can form a complex, has good stability in the monomer form, and particularly can have good stability in the complex form. 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 at least one amino acid residue selected from the positions corresponding to positions 208 and 272 in the numbering based on SEQ ID NO: 4 in the amino acid sequence of the parent lipase is substituted with cysteine, and has at least 70% identity with the amino acid sequence represented by SEQ ID NO: 4, 6, 8, 10, 14, 16, 18 or 20, and is a mutant polypeptide having lipase activity, which consists of an amino acid sequence in which at least one amino acid residue selected from the positions corresponding to positions 208 and 272 in the numbering based on SEQ ID NO: 4 is cysteine. The substitution of the amino acid residue at the above-mentioned predetermined position with cysteine contributes to the complex formation of the lipase and is a modification for improving the stability of the lipase. Therefore, the polypeptide after the substitution of the amino acid residue at the above-mentioned predetermined position in the amino acid sequence of the parent lipase, that is, the mutant polypeptide having lipase activity consisting of the amino acid sequence after the substitution of the amino acid residue, can form a complex, has improved stability compared to the parent lipase in the monomer form, and particularly has much improved stability compared to the parent lipase in the complex form.Generally, the introduction of cysteine into a protein is often treated as an unfavorable mutation because it may inhibit the maintenance of the protein's structure and function. Also, since the effect of lipase complexation on stability was not known, it is quite unexpected that the complexation of lipase, particularly the complexation of lipase based on substituting an amino acid residue at a specific position in the amino acid sequence of lipase with cysteine, improves stability. Here, the stability more specifically refers to the stability in the presence of a larger amount of solvent than a large amount of water and / or surfactant.

[0027] In the present invention, the parent lipase is a polypeptide having an amino acid sequence having at least 70% identity with the amino acid sequence represented by SEQ ID NO: 4, 6, 8, 10, 14, 16, 18 or 20 and having lipase activity. The parent lipase can be a natural (wild-type) polypeptide or a variant thereof.

[0028] Examples of the parent lipase include polypeptides 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. Here, the lipase consisting of the amino acid sequence shown in SEQ ID NO: 4 is a lipase CnLip (NCBI Accession No. WP_061278013.1) derived from Cedecea neteri, in which amino acid residues at positions I44T, L120S, S130A, A134T, A135V, L136M, and S137N are substituted (CnLipA having excellent detergency described in Japanese Patent Application No. 2023-025141), and further, amino acid residues at positions F16L, I47L, A91V, E97D, and A149E are substituted, and it is the CnLipAm shown in the following Examples. The above CnLip and CnLipA can also be the parent lipase in the present invention. The parent lipase, which is 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, preferably has leucine at a position corresponding to position 208 in the numbering of SEQ ID NO: 4 and asparagine at a position corresponding to position 272 in the numbering of SEQ ID NO: 4, and more preferably has leucine at a position corresponding to position 208 in the numbering of SEQ ID NO: 4 and asparagine at a position corresponding to position 272 in the numbering of SEQ ID NO: 4.

[0029] Another example of a parent lipase includes a polypeptide consisting of an amino acid sequence having at least 70% identity with the amino acid sequence shown in SEQ ID NO: 6 and having lipase activity. Here, the lipase consisting of the amino acid sequence shown in SEQ ID NO: 6 is a lipase in which the amino acid residue substitution of E118D is made in the amino acid sequence constituting lipase SspLip (NCBI Accession No. WP_025122441.1) derived from Serratia sp., which is a variant SspLip_m with excellent productivity described in Japanese Patent Application No. 2022-149382. The parent lipase, which is a polypeptide consisting of an amino acid sequence having at least 70% identity with the amino acid sequence shown in SEQ ID NO: 6 and having lipase activity, preferably has leucine at the position corresponding to position 208 in the numbering of SEQ ID NO: 4 and proline at the position corresponding to position 272 in the numbering of SEQ ID NO: 4, and more preferably has leucine at the position corresponding to position 208 and proline at the position corresponding to 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.

[0030] As another example of the parent lipase, there is a polypeptide comprising an amino acid sequence having at least 70% identity with the amino acid sequence represented by SEQ ID NO: 8 and having lipase activity. Here, the lipase consisting of the amino acid sequence represented by SEQ ID NO: 8 is a lipase in which the amino acid residue substitution of I222G is made in the amino acid sequence constituting the lipase EtLip (NCBI Accession No. WP_115457195.1) derived from Enterobacillus tribolii (mutant EtLip_m with excellent productivity described in Japanese Patent Application No. 2022-149382). The parent lipase, which is a polypeptide comprising an amino acid sequence having at least 70% identity with the amino acid sequence represented by SEQ ID NO: 8 and having lipase activity, preferably has phenylalanine at the position corresponding to position 208 in the numbering of SEQ ID NO: 4 and threonine at the position corresponding to position 272 in the numbering of SEQ ID NO: 4, and more preferably has phenylalanine at the position corresponding to position 208 in the numbering of SEQ ID NO: 4 and threonine at the position corresponding to 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.

[0031] Another example of a parent lipase is a polypeptide consisting of an amino acid sequence having at least 70% identity with the amino acid sequence shown in SEQ ID NO: 10 and having lipase activity. Here, the lipase consisting of the amino acid sequence shown in SEQ ID NO: 10 is a lipase in which substitutions of amino acid residues of I45T, L121S, S131A, A135T, A136V and S138N are made in the amino acid sequence constituting lipase CspLip (NCBI Accession No. WP_016537805.1) derived from Cedecea sp. (CspLipA having excellent detergency described in Japanese Patent Application No. 2023-025141). The parent lipase, which is a polypeptide consisting of an amino acid sequence having at least 70% identity with the amino acid sequence shown in SEQ ID NO: 10 and having lipase activity, preferably has leucine at the position corresponding to position 208 in the numbering of SEQ ID NO: 4 and asparagine at the position corresponding to position 272 in the numbering of SEQ ID NO: 4, and more preferably has leucine at the position corresponding to position 208 and asparagine at the position corresponding to 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.

[0032] Another example of a parent lipase includes a polypeptide consisting of an amino acid sequence having at least 70% identity with the amino acid sequence shown in SEQ ID NO: 14 and having lipase activity. Here, the lipase consisting of the amino acid sequence shown in SEQ ID NO: 14 is KAL (Japanese Patent Application Laid-Open No. 2023-156914). The parent lipase, which is a polypeptide consisting of an amino acid sequence having at least 70% identity with the amino acid sequence shown in SEQ ID NO: 14 and having lipase activity, preferably has leucine at a position corresponding to position 208 in the numbering of SEQ ID NO: 4 and serine at a position corresponding to position 272 in the numbering of SEQ ID NO: 4, and more preferably has leucine at a position corresponding to position 208 and serine at a position corresponding to 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.

[0033] As another example of a parent lipase, there is a polypeptide comprising an amino acid sequence having at least 70% identity with the amino acid sequence shown in SEQ ID NO: 16 and having lipase activity. Here, the lipase consisting of the amino acid sequence shown in SEQ ID NO: 16 is the ancestral lipase AncLip10 (Japanese Patent Application Laid-Open No. 2022-66571). Here, the ancestral lipase refers to a lipase consisting of an amino acid sequence that is presumed 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 the evolution of proteins. The amino acid sequence of the ancestral lipase can be determined by the ancestral sequence reconstruction (ASR) method based on the existing sequences of lipases. The parent lipase, which is a polypeptide comprising an amino acid sequence having at least 70% identity with the amino acid sequence shown in SEQ ID NO: 16 and having lipase activity, preferably has leucine at a position corresponding to position 208 in the numbering of SEQ ID NO: 4 and threonine at a position corresponding to position 272 in the numbering of SEQ ID NO: 4, and more preferably has leucine at a position corresponding to position 208 and threonine at a position corresponding to 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.

[0034] As another example of the parental lipase, there is a polypeptide comprising an amino acid sequence having at least 70% identity with the amino acid sequence shown in SEQ ID NO: 18 and having lipase activity. Here, the lipase consisting of the amino acid sequence shown in SEQ ID NO: 18 is a lipase derived from Enterobacteriaceae (NCBI Accession No. WP_045783583.1). The parental lipase, which is a polypeptide comprising an amino acid sequence having at least 70% identity with the amino acid sequence shown in SEQ ID NO: 18 and having lipase activity, preferably has leucine at the position corresponding to position 208 in the numbering of SEQ ID NO: 4 and serine at the position corresponding to position 272 in the numbering of SEQ ID NO: 4, and more preferably has leucine at the position corresponding to position 208 and serine at the position corresponding to 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.

[0035] As another example of the parental lipase, there is a polypeptide comprising an amino acid sequence having at least 70% identity with the amino acid sequence shown in SEQ ID NO: 20 and having lipase activity. Here, the lipase consisting of the amino acid sequence shown in SEQ ID NO: 20 is a lipase derived from Chania multitudinisentens (NCBI Accession No. WP_037407093.1). The parental lipase, which is a polypeptide comprising an amino acid sequence having at least 70% identity with the amino acid sequence shown in SEQ ID NO: 20 and having lipase activity, preferably has isoleucine at the position corresponding to position 208 in the numbering of SEQ ID NO: 4 and glycine at the position corresponding to position 272 in the numbering of SEQ ID NO: 4, and more preferably has isoleucine at the position corresponding to position 208 and glycine at the position corresponding to 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.

[0036] The amino acid sequences represented by SEQ ID NO: 4, 6, 8, 10, 14, 16, 18 or 20 have at least 70% identity with at least one amino acid sequence excluding itself among the amino acid sequences represented by SEQ ID NO: 4, 6, 8, 10, 14, 16, 18 and 20 (Figure 2). For example, each of the amino acid sequences represented by SEQ ID NO: 6, 8, 10, 14, 16, 18 or 20 has 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 NO: 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 following examples.

[0037] From the viewpoint of stability, the polypeptide of the present invention preferably has lipase activity and consists of an amino acid sequence having at least 70% identity with the amino acid sequence represented by SEQ ID NO: 4 and having at least one amino acid residue selected from the positions corresponding to positions 208 and 272 in the numbering of SEQ ID NO: 4 being cysteine, and a polypeptide having lipase activity and consisting of an amino acid sequence having at least 70% identity with the amino acid sequence represented by SEQ ID NO: 6, 8, 10, 14, 16, 18 or 20 and having the amino acid residue at the position corresponding to position 208 in the numbering of SEQ ID NO: 4 being cysteine. More preferably, it is a polypeptide having lipase activity and consisting of an amino acid sequence having at least 70% identity with the amino acid sequence represented by SEQ ID NO: 4 and having at least one amino acid residue selected from the positions corresponding to positions 208 and 272 in the numbering of SEQ ID NO: 4 being cysteine, and a polypeptide having lipase activity and consisting of an amino acid sequence having at least 70% identity with the amino acid sequence represented by SEQ ID NO: 6, 8 or 10 and having the amino acid residue at the position corresponding to position 208 in the numbering of SEQ ID NO: 4 being cysteine.

[0038] The polypeptide of the present invention may have one or more, preferably one, amino acid residues selected from phenylalanine at the position corresponding to position 16 numbered in SEQ ID NO: 4, cysteine or methionine at the position corresponding to position 22, phenylalanine at the position corresponding to position 29, isoleucine or valine at the position corresponding to position 50, alanine or glutamine at the position corresponding to position 120, isoleucine or threonine at the position corresponding to position 123, glutamic acid at the position corresponding to position 126, phenylalanine at the position corresponding to position 142, alanine at the position corresponding to position 156, lysine at the position corresponding to position 181, glutamic acid at the position corresponding to position 186, glutamine at the position corresponding to position 188, valine at the position corresponding to position 191, histidine at the position corresponding to position 220, histidine at the position corresponding to position 221, glutamine at the position corresponding to position 224, threonine at the position corresponding to position 233, asparagine at the position corresponding to position 256, alanine, cysteine or leucine at the position corresponding to position 260, isoleucine at the position corresponding to position 269, and glutamine at the position corresponding to position 274, as long as the formation of the complex is not hindered.

[0039] <2. Polynucleotide encoding the polypeptide of the present invention> The polypeptide of the present invention can be produced by expressing the polypeptide from the polynucleotide encoding the polypeptide. The polynucleotide can be prepared by extracting genomic DNA from a microorganism that produces the lipase of interest by a conventional method, or by extracting RNA and synthesizing cDNA by reverse transcription. Alternatively, based on the amino acid sequence of the lipase of interest, the corresponding nucleotide sequence can be chemically synthesized and used as the polynucleotide encoding the polypeptide of the present invention.

[0040] Alternatively, the polypeptide of the present invention can be produced using various mutagenesis techniques known in the art. For example, a polynucleotide encoding the amino acid residue to be mutated within the parental lipase gene (reference lipase gene) encoding its reference amino acid sequence is mutated into a polynucleotide encoding the amino acid residue after mutation, and then a mutant polypeptide is expressed from the mutant gene, whereby it can be produced.

[0041] In the present invention, as a means for mutating the amino acid residue of the parental lipase, various mutagenesis techniques known in the art can be used. For example, in a polynucleotide encoding the amino acid sequence of the parental lipase (hereinafter also referred to as the parental gene), the nucleotide sequence encoding the amino acid residue to be mutated is mutated into the nucleotide sequence encoding the amino acid residue after mutation, whereby a polynucleotide encoding the mutant polypeptide of the present invention can be obtained.

[0042] The introduction of the desired mutation into the parental gene can basically be carried out using various site-directed mutagenesis methods well-known to those skilled in the art. The site-directed mutagenesis method can be carried out by any method such as, for example, the inverse PCR method or the annealing method. It is also possible to use a commercially available site-directed mutagenesis kit (for example, QuickChange II Site-Directed Mutagenesis Kit or QuickChange Multi Site-Directed Mutagenesis Kit from Stratagene).

[0043] Site-specific mutagenesis into a parental gene can most commonly be carried out using a mutagenic primer containing the nucleotide mutation to be introduced. The mutagenic primer is designed to anneal to a region containing the nucleotide sequence encoding the amino acid residue to be mutated in the parental gene and to contain a nucleotide sequence (codon) encoding the amino acid residue after mutation instead of the nucleotide sequence (codon) encoding the amino acid residue to be mutated. The nucleotide sequences (codons) encoding the amino acid residues before and after mutation can be appropriately recognized and selected by those skilled in the art based on ordinary textbooks and the like. Alternatively, site-specific mutagenesis can also be carried out using a method in which two complementary primers containing the nucleotide mutation to be introduced are separately used to amplify the DNA fragments on the upstream and downstream sides of the mutation site, respectively, and then ligated together by SOE (splicing by overlap extension)-PCR (Gene, 1989, 77(1): p61-68).

[0044] The template DNA containing the parental gene can be prepared by extracting genomic DNA from the microorganism producing the above-described parental lipase by a conventional method, or by extracting RNA and synthesizing cDNA by reverse transcription. Alternatively, based on the amino acid sequence of the parental lipase, the corresponding nucleotide sequence can be chemically synthesized and used as the template DNA. The DNA sequences containing the base sequences encoding the lipases consisting of the amino acid sequences shown in SEQ ID NOs: 4, 6, 8, 10, 14, 16, 18, and 20 are shown in SEQ ID NOs: 3, 5, 7, 9, 13, 15, 17, and 19, respectively.

[0045] The 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 carried out using, for example, a commercially available oligonucleotide synthesizer (such as those manufactured by ABI). By using a primer set containing the mutation primers and performing site-directed mutagenesis as described above with the parental gene as the template DNA, a polynucleotide encoding the mutant polypeptide of the present invention having the desired mutation can be obtained.

[0046] The polynucleotide encoding the polypeptide of the present invention may include single-stranded or double-stranded DNA, cDNA, RNA, or other artificial nucleic acids. The DNA, cDNA, and RNA may be chemically synthesized. Further, the polynucleotide may include the nucleotide sequence of the untranslated region (UTR) in addition to the open reading frame (ORF). Also, the polynucleotide may be codon-optimized according to the species of the transformant for producing the polypeptide of the present invention. Information on the codons used by various organisms is available from the Codon Usage Database ([www.kazusa.or.jp / codon / ]).

[0047] <3. Vector or DNA Fragment> The polynucleotide encoding the polypeptide of the present invention obtained can be incorporated into a vector. The type of vector containing the polynucleotide is not particularly limited, and any vector such as a plasmid, phage, phagemid, cosmid, virus, YAC vector, shuttle vector, etc. may be used. Further, 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 its mutant strain), and more preferably an expression vector that can induce the expression of the transgene in Bacillus bacteria. Among them, a shuttle vector that can replicate in both Bacillus bacteria and other organisms can be preferably used for the recombinant production of the polypeptide of the present invention. Examples of preferred vectors include, but are not limited to, pHA3040SP64, pHSP64R or pASP64 (Patent No. 3492935), pHY300PLK (an expression vector capable of transforming both Escherichia coli and Bacillus subtilis; Jpn J Genet, 1985, 60:235-243), pAC3 (Nucleic Acids Res, 1988, 16:8732), etc. shuttle vectors; pUB110 (J Bacteriol, 1978, 134:318-329), pTA10607 (Plasmid, 1987, 18:8-15), etc. plasmid vectors available for the transformation of Bacillus bacteria, and the like. Also, plasmid vectors derived from Escherichia coli (e.g., pET22b(+), pBR322, pBR325, pUC57, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.) can be used.

[0048] The above vector may contain a DNA region including a DNA replication origin region or an origin of replication. Alternatively, in the above vector, a control sequence such as a promoter region for initiating the transcription of the gene, a terminator region, or a secretion signal region for secreting the expressed protein extracellularly may be operably linked upstream of the polynucleotide encoding the polypeptide of the present invention.

[0049] The types of control sequences such as the above-mentioned promoter region, terminator region, and secretion signal region are not particularly limited, and depending on the host to be introduced, commonly used promoters and secretion signal sequences can be appropriately selected and used. For example, preferred examples of control sequences that can be incorporated into a vector include the promoter of the cellulase gene of Bacillus sp. KSM-S237 strain, the secretion signal sequence, and the like.

[0050] Alternatively, the vector of the present invention may further incorporate a marker gene (for example, a drug resistance gene such as ampicillin, neomycin, kanamycin, chloramphenicol, etc.) for selecting a host into which the vector has been appropriately introduced. Alternatively, when a auxotrophic strain is used as the host, a gene encoding the synthetic enzyme of the required nutrient may be incorporated into the vector as a marker gene. Alternatively, when a selective medium that requires a specific metabolism for growth is used, a gene related to the metabolism may be incorporated into the vector as a marker gene. An example of such a metabolism-related gene is the acetamidase gene for utilizing acetamide as a nitrogen source.

[0051] The ligation of the polynucleotide encoding the polypeptide of the present invention with the control sequence and the marker gene can be carried out by a method known in the art such as the SOE (splicing by overlap extension)-PCR method (Gene, 1989, 77: 61-68). The procedure for introducing the ligated fragment into the vector is well known in the art.

[0052] <4. Transformed Cells> The transformed cells of the present invention can be obtained by introducing a vector containing the polynucleotide encoding the polypeptide of the present invention into a host, or by introducing a DNA fragment containing the polynucleotide encoding the polypeptide of the present invention into the genome of the host.

[0053] Examples of host cells include microorganisms such as bacteria and filamentous fungi. Examples of bacteria include bacteria belonging to Escherichia coli, Staphylococcus, Enterococcus, Listeria, and Bacillus. Among these, Escherichia coli and Bacillus bacteria are preferred, Bacillus bacteria are more preferred, and Bacillus subtilis (for example, Bacillus subtilis Marburg No. 168 (Bacillus subtilis 168 strain) or its mutant strain) is even more preferred. Examples of Bacillus mutant strains include the protease nonuple-deficient strain KA8AX described in J. Biosci. Bioeng., 2007, 104(2): 135-143, and the D8PA strain described in Biotechnol. Lett., 2011, 33(9): 1847-1852, in which the protein folding efficiency was improved in the protease octuple-deficient strain. Examples of filamentous fungi include Trichoderma, Aspergillus, Rhizopus, and the like.

[0054] As a method for introducing a vector into a host, methods commonly used in the art such as the protoplast method and the electroporation method can be used. By selecting a strain into which the introduction has been appropriately performed using the expression of a marker gene, auxotrophy, etc. as indicators, a target transformant into which the vector has been introduced can be obtained.

[0055] Alternatively, a fragment obtained by ligating a polynucleotide encoding the polypeptide of the present invention, a control sequence, and a marker gene can also be directly introduced into the genome of the host. For example, by the SOE-PCR method or the like, a DNA fragment having sequences complementary to the genome of the host added to both ends of the above ligation fragment is constructed, and this is introduced into the host to cause homologous recombination between the host genome and the DNA fragment, whereby the polynucleotide encoding the polypeptide of the present invention is introduced into the genome of the host.

[0056] When the transformant introduced with the polynucleotide encoding the polypeptide of the present invention obtained in this manner or a vector containing the same 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 for culturing the transformant can be appropriately selected by those skilled in the art according to the type of microorganism of the transformant.

[0057] Alternatively, the polypeptide of the present invention may be expressed from the polynucleotide encoding the polypeptide of the present invention or its transcription product using a cell-free translation system. The "cell-free translation system" is a suspension obtained by mechanically disrupting host cells, to which reagents such as amino acids necessary for protein translation are added to constitute an in vitro transcription / translation system or an in vitro translation system.

[0058] <5. Lipase complex> The polypeptide of the present invention can form a complex of lipase by binding via a disulfide bond, preferably by binding via a disulfide bond under non-reducing conditions. The complex of lipase 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 a complex include a complex in which the same or different polypeptides of the present invention are integrated via a disulfide bond, and a complex in which the polypeptide of the present invention and a component other than the polypeptide of the present invention are integrated via a disulfide bond. The component other than the polypeptide of the present invention that constitutes the complex is a polypeptide capable of forming a disulfide bond with the cysteine of the polypeptide of the present invention, and examples thereof include a polypeptide containing cysteine that can be produced by a host (for example, Bacillus subtilis) when the polypeptide of the present invention is expressed. As the complex of lipase formed by the polypeptide of the present invention, a complex containing two or more polypeptide molecules containing at least one molecule of the polypeptide of the present invention is preferable, a lipase dimer composed of two molecules of the same or different polypeptides of the present invention is more preferable, and a lipase dimer composed of two molecules of the same polypeptide of the present invention is even more preferable.That is, as the lipase complex formed by the polypeptide of the present invention, it contains two or more polypeptides, the two or more polypeptides are linked by disulfide bonds, and at least one of the two or more polypeptides has a lipase activity consisting of an amino acid sequence having at least 70% identity with the amino acid sequence represented by SEQ ID NO: 4, 6, 8, 10, 14, 16, 18 or 20. A complex is preferred. It contains two or more polypeptides, the two or more polypeptides are linked by disulfide bonds, and at least one of the two or more polypeptides has an amino acid sequence having at least 70% identity with the amino acid sequence represented by SEQ ID NO: 4, 6, 8, 10, 14, 16, 18 or 20 and has an amino acid residue at one or more positions selected from the positions corresponding to positions 208 and 272 in the numbering of SEQ ID NO: 4 being cysteine. A complex which is a polypeptide having lipase activity is more preferred. It consists of two polypeptides, the two polypeptides are linked by disulfide bonds, and the two polypeptides each independently have an amino acid sequence having at least 70% identity with the amino acid sequence represented by SEQ ID NO: 4, 6, 8, 10, 14, 16, 18 or 20 and have an amino acid residue at one or more positions selected from the positions corresponding to positions 208 and 272 in the numbering of SEQ ID NO: 4 being cysteine. A lipase dimer which is a polypeptide having lipase activity is even more preferred. It consists of two identical polypeptides, the two identical polypeptides are linked by disulfide bonds, and the polypeptide has an amino acid sequence having at least 70% identity with the amino acid sequence represented by SEQ ID NO: 4, 6, 8, 10, 14, 16, 18 or 20 and has an amino acid residue at one or more positions selected from the positions corresponding to positions 208 and 272 in the numbering of SEQ ID NO: 4 being cysteine. A lipase dimer which is a polypeptide having lipase activity is even more preferred.

[0059] <6. Method for producing lipase complex> The present invention also provides a method for producing a complex of lipase. The method for producing a complex of lipase of the present invention includes a step of binding two or more polypeptides containing at least one molecule of the polypeptide of the present invention via disulfide bonds. The formation of a complex by disulfide bonds between cysteines in the two or more polypeptides can be carried out, for example, by expressing the polypeptide of the present invention under non-reducing conditions using the transformed cells of the present invention or by retaining the polypeptide of the present invention under non-reducing conditions. Here, the "non-reducing conditions" refer to an environment in which an oxidizing power for forming disulfide bonds exists, and examples thereof include an environment in which a reducing agent such as dithiothreitol (DTT) does not exist in an amount that hinders the formation of disulfide bonds. Since the cytoplasm is generally known to be kept in a reduced state, it is preferable to express the polypeptide of the present invention under conditions such that it is transferred to an oxidizing environment such as the endoplasmic reticulum or periplasm using the transformed cells of the present invention or cultured under conditions such that it is released extracellularly by secretion or lysis after culture. Alternatively, it is preferable to express the polypeptide of the present invention or retain the polypeptide of the present invention using the transformed cells of the present invention in an aerated and agitated state in order to promote air oxidation. Further, the complex formation can be carried out by expressing the polypeptide of the present invention under non-reducing conditions using a cell-free translation system or by retaining the polypeptide of the present invention under non-reducing conditions. In order to enhance the oxidizing power in the cell-free translation system, it is preferable to add chaperones such as glutathione, disulfide bond isomerase (DsbC), and protein disulfide isomerase (PDI) to express the polypeptide of the present invention using the cell-free translation system. Alternatively, it is preferable to express the polypeptide of the present invention or retain the polypeptide of the present invention using the cell-free translation system in an aerated and agitated state in order to promote air oxidation.

[0060] The method for producing the complex of the lipase of the present invention may further include a step of providing the polypeptide of the present invention prior to the above-described binding step. In a preferred embodiment, the step includes a step of substituting the amino acid residue at a specific position of the amino acid sequence contributing to the integration of the polypeptide with cysteine in at least one parent lipase. In a more preferred embodiment, the step includes substituting the amino acid residue at one or more positions selected from the positions corresponding to positions 208 and 272 in the numbering based on SEQ ID NO: 4 with cysteine in a polypeptide consisting of an amino acid sequence having at least 70% identity with the amino acid sequence represented by at least one of SEQ ID NO: 4, 6, 8, 10, 14, 16, 18 or 20 and having lipase activity.

[0061] The substitution of the amino acid residue at such a predetermined position with cysteine is a modification contributing to the formation of the lipase complex. The substitution site of the amino acid residue is preferably at one or more positions selected from the positions corresponding to 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 represented by SEQ ID NO: 4 and having lipase activity, and is 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 represented by SEQ ID NO: 6, 8, 10, 14, 16, 18 or 20 and having lipase activity, from the viewpoint of improving stability.

[0062] In the step of providing the polypeptide of the present invention, as long as the formation of the complex is not hindered, in addition to the substitution of the above amino acid residues, one or more positions selected from the 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, preferably one amino acid residue substitution may be performed. As preferred embodiments of each substitution, substitution of the amino acid residue at the position corresponding to position 16 in the numbering of SEQ ID NO: 4 with phenylalanine, substitution of the amino acid residue at the position corresponding to position 22 with cysteine or methionine, substitution of the amino acid residue at the position corresponding to position 29 with phenylalanine, substitution of the amino acid residue at the position corresponding to position 50 with isoleucine or valine, substitution of the amino acid residue at the position corresponding to position 120 with alanine or glutamine, substitution of the amino acid residue at the position corresponding to position 123 with isoleucine or threonine, substitution of the amino acid residue at the position corresponding to position 126 with glutamic acid, substitution of the amino acid residue at the position corresponding to position 142 with phenylalanine, substitution of the amino acid residue at the position corresponding to position 156 with alanine, substitution of the amino acid residue at the position corresponding to position 181 with lysine, substitution of the amino acid residue at the position corresponding to position 186 with glutamic acid, substitution of the amino acid residue at the position corresponding to position 188 with glutamine, substitution of the amino acid residue at the position corresponding to position 191 with valine, substitution of the amino acid residue at the position corresponding to position 220 with histidine, substitution of the amino acid residue at the position corresponding to position 221 with histidine, substitution of the amino acid residue at the position corresponding to position 224 with glutamine, substitution of the amino acid residue at the position corresponding to position 233 with threonine, substitution of the amino acid residue at the position corresponding to position 256 with asparagine, substitution of the amino acid residue at the position corresponding to position 260 with alanine, cysteine or leucine, substitution of the amino acid residue at the position corresponding to position 269 with isoleucine and substitution of the amino acid residue at the position corresponding to position 274 with glutamine can be mentioned. Also, as long as the formation of the complex is not hindered, any number of conservative amino acid substitutions may be performed.

[0063] The polypeptide of the present invention can be isolated or purified in the form of a single entity or a complex by using general methods used for protein purification, such as centrifugation, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc., either alone or in appropriate combination. The protein recovered from the culture may be further purified by known means.

[0064] <7. Method for improving the stability of lipase> 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 single entity. More specifically, it has improved stability in the form of a complex compared to the form of a single entity in the presence of a large amount of solvent compared to a large amount of water and / or surfactant. "Stability" means the ability to maintain lipase activity. "Improved stability in the form of a complex compared to the form of a single entity" means the ability to maintain the lipase activity of the complex, which is improved compared to that of the single entity. "Improved stability in the form of a complex compared to the form of a single entity in the presence of a large amount of solvent compared to a large amount of water and / or surfactant" means the ability to maintain the lipase activity of the complex, which is improved compared to that of the single entity in the presence of a large amount of solvent compared to a large amount of water and / or surfactant.

[0065] The mutant polypeptide of the present invention has improved stability in the form of a single entity compared to the parent lipase without impairing the detergency of the parent lipase, and further has much improved stability in the form of a complex compared to the parent lipase. More specifically, it has improved stability in the presence of a large amount of solvent compared to a large amount of water and / or surfactant. "Stability" means the ability to maintain lipase activity. "Improved stability compared to the parent lipase" means the ability to maintain the lipase activity, which is improved compared to that of the parent lipase. "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" means the ability to maintain the lipase activity, which is improved compared to that of the parent lipase in the presence of a large amount of solvent compared to a large amount of water and / or surfactant.

[0066] Here, the stability in the presence of a large amount of moisture means the ability to maintain lipase activity when stored in a detergent composition with a water content of 60 to 99% by mass, and the stability in the presence of a solvent in an amount larger than that of the surfactant means the ability to maintain lipase activity in a detergent composition in which the solvent content is higher than the surfactant content or in a cleaning solution in which the solvent content is higher than the surfactant content prepared by dissolving or diluting the detergent composition in water. Examples of the surfactant and the solvent include the surfactants and solvents described below.

[0067] Such lipase stability can be evaluated using methods well known in the art. For example, lipase stability is evaluated by adding lipase to an appropriate detergent composition or a cleaning solution prepared by dissolving or diluting the detergent composition in water, measuring the lipase activity before and after treatment for a predetermined time, taking the activity value of the sample before treatment as the initial activity, and calculating the half-life of the activity based on the inactivation rate per unit time by the treatment. The value obtained by dividing the half-life of the mutant polypeptide by the half-life of the parent lipase indicates the relative stability of the mutant polypeptide, and when it exceeds 1, the mutant polypeptide is evaluated to have improved stability compared to the parent lipase. Examples of the cleaning solution include, but are not limited to, the model cleaning solution having the composition shown in Table 2 of the following Examples. The stability of the lipase complex (lipase dimer) containing the polypeptide of the present invention is preferably 1.3 or more, more preferably 1.5 or more, as the relative stability with respect to the polypeptide alone of the present invention when treated at 45°C for 1 hour. The stability of the mutant polypeptide of the present invention is preferably 1.5 or more, more preferably 1.7 or more, as the relative stability with respect to the parent lipase when treated at 45°C for 1 hour. The stability of the lipase complex (lipase dimer) containing the mutant polypeptide of the present invention is preferably 2 or more, more preferably 3 or more, as the relative stability with respect to the parent lipase when treated at 45°C for 1 hour.

[0068] The present invention also provides a method for improving the stability of lipase. The method for improving the stability of lipase according to the present invention includes a step of complexing lipase. The complexing of lipase is a step of preparing a complex of lipase by integrating two or more polypeptides including at least one polypeptide having lipase activity through disulfide bonds between cysteines in the two or more polypeptides. Specifically, the method for improving the stability of lipase according to the present invention includes a step of binding two or more polypeptides including at least one molecule of the polypeptide of the present invention through disulfide bonds. The formation of a complex by disulfide bonds between cysteines in the two or more polypeptides can be carried out, for example, by expressing the polypeptide of the present invention under non-reducing conditions using the transformed cells of the present invention or by retaining the polypeptide of the present invention under non-reducing conditions. The non-reducing conditions and preferred embodiments thereof are as described above.

[0069] The method for improving the stability of lipase according to the present invention may further include a step of providing the polypeptide of the present invention prior to the above binding step. In a preferred embodiment, the step includes a step of substituting amino acid residues at specific positions of an amino acid sequence contributing to the integration of the polypeptide with cysteine in at least one parent lipase. In a more preferred embodiment, the step includes a step of substituting amino acid residues at one or more positions selected from positions corresponding to positions 208 and 272 in the numbering based on SEQ ID NO: 4 with cysteine in a polypeptide consisting of an amino acid sequence having at least 70% identity with the amino acid sequence represented by at least one of SEQ ID NO: 4, 6, 8, 10, 14, 16, 18 or 20 and having lipase activity.

[0070] Substitution of the amino acid residue at the predetermined position with cysteine contributes to the complex formation of lipase and is a modification for improving the stability of lipase. Therefore, the polypeptide after substitution of the amino acid residue at the predetermined position in the amino acid sequence of the parent lipase, that is, the mutant polypeptide having lipase activity consisting of the amino acid sequence after the substitution of the amino acid residue, can form a complex and has improved stability compared to the parent lipase in the form of a monomer, and particularly has much improved stability compared to the parent lipase in the form of a complex. The substitution site of the amino acid residue is preferably at one or more positions selected from the positions corresponding to 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, from the viewpoint of improving stability, and is 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.

[0071] In the step of providing the polypeptide of the present invention, unless the formation of the complex is hindered, in addition to the substitution of the above amino acid residues, one or more positions selected from the 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, preferably one amino acid residue substitution may be performed. Preferred embodiments of each substitution include substitution of the amino acid residue at the position corresponding to position 16 in the numbering of SEQ ID NO: 4 with phenylalanine, substitution of the amino acid residue at the position corresponding to position 22 with cysteine or methionine, substitution of the amino acid residue at the position corresponding to position 29 with phenylalanine, substitution of the amino acid residue at the position corresponding to position 50 with isoleucine or valine, substitution of the amino acid residue at the position corresponding to position 120 with alanine or glutamine, substitution of the amino acid residue at the position corresponding to position 123 with isoleucine or threonine, substitution of the amino acid residue at the position corresponding to position 126 with glutamic acid, substitution of the amino acid residue at the position corresponding to position 142 with phenylalanine, substitution of the amino acid residue at the position corresponding to position 156 with alanine, substitution of the amino acid residue at the position corresponding to position 181 with lysine, substitution of the amino acid residue at the position corresponding to position 186 with glutamic acid, substitution of the amino acid residue at the position corresponding to position 188 with glutamine, substitution of the amino acid residue at the position corresponding to position 191 with valine, substitution of the amino acid residue at the position corresponding to position 220 with histidine, substitution of the amino acid residue at the position corresponding to position 221 with histidine, substitution of the amino acid residue at the position corresponding to position 224 with glutamine, substitution of the amino acid residue at the position corresponding to position 233 with threonine, substitution of the amino acid residue at the position corresponding to position 256 with asparagine, substitution of the amino acid residue at the position corresponding to position 260 with alanine, cysteine or leucine, substitution of the amino acid residue at the position corresponding to position 269 with isoleucine and substitution of the amino acid residue at the position corresponding to position 274 with glutamine. Also, any number of conservative amino acid substitutions may be performed unless the formation of the complex is hindered.

[0072] The polypeptide of the present invention can be isolated or purified in the form of a single entity or a complex by using common methods used in protein purification, such as centrifugation, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc., either alone or in appropriate combination. The protein recovered from the culture may be further purified by known means.

[0073] <8. Detergent Composition> The complex of the polypeptide of the present invention and lipase is useful as an enzyme for formulating various detergent compositions, and particularly useful as an enzyme for formulating detergent compositions suitable for low-temperature washing. From the viewpoint of stability, it is preferable to use either one or both of the polypeptide of the present invention and the complex of lipase, and it is more preferable to use at least the complex of lipase of the present invention. Hereinafter, the complex of the polypeptide of the present invention and lipase is collectively referred to as the polypeptide of the present invention, etc. Here, examples of "low temperature" include 40°C or lower, 35°C or lower, 30°C or lower, 25°C or lower, and also 5°C or higher, 10°C or higher, 15°C or higher. Also, 5 to 40°C, 10 to 35°C, 15 to 30°C, 15 to 25°C can be mentioned.

[0074] The amount of the polypeptide of the present invention, etc. formulated in the detergent composition is not particularly limited as long as the polypeptide, etc. exhibits activity. For example, it is preferably 0.1 mg or more, more preferably 1 mg or more, still more preferably 5 mg or more per 1 kg of the detergent composition, and preferably 5000 mg or less, more preferably 1000 mg or less, still more preferably 500 mg or less. Also, it is preferably 0.1 to 5000 mg, more preferably 1 to 1000 mg, and still more preferably 5 to 500 mg.

[0075] The cleaning composition preferably contains, in addition to the polypeptide of the present invention and the like, a sulfosuccinic acid ester or a salt thereof, which is a surfactant. The sulfosuccinic acid ester or a salt thereof is known as a component to be blended in the cleaning composition (for example, JP-A-2019-182911). As the sulfosuccinic acid ester or a salt thereof, a sulfosuccinic acid branched alkyl ester having a branched alkyl group having 8 to 12 carbon atoms or a salt thereof is preferable, a sulfosuccinic acid branched alkyl ester having a branched alkyl group having 9 or 10 carbon atoms or a salt thereof is more preferable, and a sulfosuccinic acid branched alkyl ester having a branched alkyl group having 10 carbon atoms or a salt thereof is still more preferable. Further, the sulfosuccinic acid ester or a salt thereof is a sulfosuccinic acid dibranched alkyl ester or a salt thereof, and a sulfosuccinic acid dibranched alkyl ester or a salt thereof in which two branched alkyl groups are each a branched alkyl group having 8 to 12 carbon atoms is preferable, a sulfosuccinic acid dibranched alkyl ester or a salt thereof in which two branched alkyl groups are each a branched alkyl group having 9 or 10 carbon atoms is more preferable, a sulfosuccinic acid dibranched alkyl ester or a salt thereof in which two branched alkyl groups are each a branched alkyl group having 10 carbon atoms is still more preferable, and bis-(2-propylheptyl)sulfosuccinic acid or a salt thereof is still more preferable.

[0076] Examples of the salt include an alkali metal salt, an alkanolamine salt, etc., an alkali metal salt or an alkanolamine salt is preferable, and a salt selected from a sodium salt, a potassium salt, a triethanolamine salt, a diethanolamine salt, and a monoethanolamine salt is more preferable, and a sodium salt is still more preferable.

[0077] Examples of the sulfosuccinic acid ester or a salt thereof include a compound represented by the following formula 1.

[0078]

Chemical formula

[0079] 〔In formula 1, R 1 , R 2is a branched alkyl group having 8 to 12 carbon atoms, respectively, and A 1 O, A 2 O are each 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 or more and 10 or less, and M is a cation. ]]

[0080] 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.

[0081] In Formula 1, A 1 O, A 2 O are each an alkyleneoxy group having 2 to 4 carbon atoms, preferably an alkyleneoxy group having 2 or 3 carbon atoms from the viewpoint of lubricity with respect to water. In Formula 1, x1 and x2 represent the average number of moles added of A 1 O, A 2 O, and are each a number of 0 or more and 10 or less, preferably 6 or less, more preferably 4 or less, still more preferably 2 or less, and even more preferably 0 from the viewpoint of lubricity with respect to water.

[0082] 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 sodium ion, potassium ion, triethanolammonium ion, diethanolammonium ion, or monoethanolammonium ion, and still more preferably sodium ion.

[0083] The sulfosuccinic acid ester or its salt is preferably a compound represented by the following formula 1-1. The compound of formula 1-1 is a compound in which x1 and x2 in formula 1 are each 0.

[0084]

Chemical formula

[0085] 〔In formula 1-1, R 1 , R 2 are each a branched alkyl group having 8 to 12 carbon atoms, and M is a cation.〕 Specific examples and preferred examples of R 1 , R 2 , and M in formula 1-1 are the same as those in formula 1. In a preferred embodiment, the sulfosuccinic acid ester or its salt is bis-(2-propylheptyl)sulfosuccinic acid or its salt.

[0086] The blending amount of the sulfosuccinic acid ester or its salt in the detergent composition is preferably 0.01% by mass or more, more preferably 0.1% by mass or more, and preferably 30.0% by mass or less, more preferably 10.0% by mass or less, and still more preferably 2.0% by mass or less. Also, it is preferably 0.01 to 30.0% by mass, more preferably 0.1 to 10.0% by mass, and still more preferably 0.1 to 2.0% by mass.

[0087] In addition to the polypeptide of the present invention, the detergent composition can also be used in combination with various enzymes. For example, hydrolases, oxidases, reductases, transferases, lyases, isomerases, ligases, synthetases, etc. Among these, lipases, amylases, proteases, cellulases, keratinases, esterases, cutinases, pullulanases, pectinases, mannanases, glucosidases, glucanases, cholesterol oxidases, peroxidases, laccases, etc., which are different from the lipase mutants of the present invention, are preferred, and particularly proteases, cellulases, amylases, and lipases different from the polypeptide of the present invention are preferred. Examples of proteases include commercially available Alcalase, Esperase, Everlase, Savinase, Kannase, Progress Uno (registered trademark; Novozymes), PREFERENZ, EFFECTENZ, EXCELLENZ (registered trademark; DuPont), Lavergy (registered trademark; BASF), and KAP (Kao), etc. Alternatively, examples include each variant of the KP43 protease described in JP-A No. 2020-145938 and Japanese Patent Application No. 2022-118096. Examples of cellulases include Celluclean, Carezyme (registered trademark; Novozymes), KAC, the alkaline cellulase produced by Bacillus sp. KSM-S237 described in JP-A No. 10-313859, and the mutant alkaline cellulase described in JP-A No. 2003-313592 (both are from Kao), etc. Examples of amylases include Termamyl, Duramyl, Stainzyme, Stainzyme Plus, Amplify Prime (registered trademark; Novozymes), PREFERENZ, EFFECTENZ (registered trademark; DuPont), and KAM (Kao), etc. Alternatively, examples include each variant of the YR288 amylase described in JP-A No. 2022-60158 and WO2023 / 176970. Alternatively, examples include each variant of the amylase described in JP-T No. 2019-500058 and JP-T No. 2018-516553. Examples of lipases include Lipolase, Lipex (registered trademark; Novozymes), etc. In the detergent composition, in addition to the polypeptide of the present invention and the like, one or more of the above enzymes can be used in combination. Examples of the combined use mode include, but are not limited to, the combined use of the polypeptide of the present invention and the like and protease, the combined use of the polypeptide of the present invention and the like and amylase, the combined use of the polypeptide of the present invention and the like, protease and amylase, the combined use of the polypeptide of the present invention and the like, the combined use of a lipase different from the polypeptide of the present invention and protease, the combined use of the polypeptide of the present invention and the like, the combined use of a lipase different from the polypeptide of the present invention and amylase, the combined use of the polypeptide of the present invention and the like, a lipase different from the polypeptide of the present invention, protease and amylase, etc. Among them, it is preferable to use the polypeptide of the present invention and the like, protease and amylase in combination.

[0088] Known detergent components can be incorporated into the detergent composition, and examples of such known detergent components include the following.

[0089] (1) Surfactant The surfactant is incorporated in the detergent composition in an amount of 0.5 to 90% by mass, and particularly preferably in an amount of 10 to 45% by mass for the powdery detergent composition and 20 to 90% by mass for the liquid detergent composition. When the detergent composition is a laundry detergent for clothes or a detergent for an automatic dishwashing machine, the surfactant is generally incorporated in an amount of 0.5 to 20% by mass, preferably 1 to 15% by mass, more preferably 1.5 to 10% by mass, and still more preferably 2 to 8% by mass.

[0090] Examples of the surfactant used in the detergent composition include one or a combination of an anionic surfactant, a nonionic surfactant, an amphoteric surfactant, and a cationic surfactant other than the above-mentioned sulfosuccinate ester or its salt, and an amphoteric surfactant is preferable.

[0091] As the amphoteric surfactant, an amine oxide type surfactant or a betaine type surfactant is preferred, and a tertiary amine oxide type surfactant, a sulfobetaine type surfactant or a carbobetaine type surfactant is more preferred. As the tertiary amine oxide type surfactant, an alkyl group having 8 or more and 18 or less carbon atoms, preferably 8 or more and 16 or less carbon atoms, more preferably 8 or more and 14 or less carbon atoms, in which one of the groups bonded to the nitrogen atom may be interrupted by an amide group or an ester group, and the remainder is an alkyl group having 1 or more and 3 or less carbon atoms, preferably a methyl group, can be mentioned. As the sulfobetaine type surfactant, a compound having one alkyl group having 10 or more and 18 or less carbon atoms, preferably 16 or less carbon atoms, more preferably 14 or less carbon atoms, two alkyl groups having 1 or more and 3 or less carbon atoms, preferably methyl groups, and a 3-sulfopropyl group or a 2-hydroxy-3-sulfopropyl group is suitable. Further, as the carbobetaine type surfactant, a carbobetaine type surfactant having one alkyl group having 10 or more and 18 or less carbon atoms, preferably 16 or less carbon atoms, 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 carbon atoms, preferably methyl groups, and one carboxyalkyl group, preferably a carboxymethyl group is preferred.

[0092] (2) Divalent metal ion capturer The divalent metal ion capturer is incorporated in an amount of 0.01 to 50% by mass, preferably 5 to 40% by mass. Examples of the divalent metal ion capturer used in the detergent composition include condensed phosphates such as tripolyphosphate, pyrophosphate, and orthophosphate, aluminosilicates such as zeolite, synthetic layered crystalline silicate, nitrilotriacetate, ethylenediaminetetraacetate, citrate, isocitrate, polyacetal carboxylate, and the like. Among these, crystalline aluminosilicate (synthetic zeolite) is particularly preferred, and among A-type, X-type, and P-type zeolites, A-type is particularly preferred. Synthetic zeolite having an average primary particle size of 0.1 to 10 μm, particularly 0.1 to 5 μm is preferably used.

[0093] (3) Alkaline agent The alkali agent is formulated in an amount of 0.01 to 80% by mass, preferably 1 to 40% by mass. In the case of a powder detergent, examples of the alkali agent include alkali metal carbonates such as sodium carbonate, which is collectively referred to as dense ash or light ash, and amorphous alkali metal silicates such as JIS No. 1, No. 2, and No. 3. These inorganic alkali agents are effective in forming the particle skeleton during detergent drying, and a detergent that is relatively hard and has excellent fluidity can be obtained. Examples of other alkalis include sesquicarbonate and sodium hydrogen carbonate, and phosphates such as tripolyphosphate also have the action of an alkali agent. In addition, as the alkali agent used in a liquid detergent, in addition to the above alkali agents, sodium hydroxide, and mono-, di- or triethanolamine can be used, and they can also be used as counterions of the surfactant.

[0094] (4) Anti-redeposition agent The anti-redeposition agent is formulated in an amount of 0.001 to 10% by mass, preferably 1 to 5% by mass. Examples of the anti-redeposition agent used in the detergent composition include polyethylene glycol, carboxylic acid polymers, polyvinyl alcohol, polyvinyl pyrrolidone, and the like. Among these, the carboxylic acid polymer has, in addition to the anti-redeposition ability, a function of capturing metal ions and an action of dispersing solid particle stains from the clothing into the washing bath. The carboxylic acid polymer is a homopolymer or copolymer of acrylic acid, methacrylic acid, itaconic acid, etc. As the copolymer, those copolymerized with the above monomers and maleic acid are preferred, and those having a molecular weight of several thousand to 100,000 are preferred. In addition to the above carboxylic acid polymers, polymers such as polyglycidylates, cellulose derivatives such as carboxymethyl cellulose, and aminocarboxylic acid polymers such as polyaspartic acid are also preferred because they have the functions of a metal ion capturer, a dispersant, and anti-redeposition ability.

[0095] (5) Bleaching agent For example, it is preferable to formulate 1 to 10% by mass of a bleaching agent such as hydrogen peroxide and percarbonate. When using a bleaching agent, 0.01 to 10% by mass of a bleach activator (activator) such as tetraacetylethylenediamine (TAED) or described in JP-A-6-316700 can be formulated.

[0096] (6) Fluorescent agent Examples of the fluorescent agent used in the detergent composition include biphenyl type fluorescent agents (such as Tinopal CBS-X, etc.) and stilbene type fluorescent agents (such as DM type fluorescent dyes, etc.). It is preferable to blend the fluorescent agent in an amount of 0.001 to 2% by mass.

[0097] (7) Chelating agent The chelating agent is blended, for example, for easy cleaning of dirt and reduction of water hardness during washing. Examples of such chelating agents include aminocarboxylic acid-based chelating agents, phosphonic acid-based chelating agents, hydroxycarboxylic acid-based chelating agents, polycarboxylic acid-based chelating agents, etc. Examples of aminocarboxylic acid-based chelating agents include ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), hydroxyethylethylenediaminetriacetic acid (HEDTA), diethylenetriaminepentaacetic acid (DTPA), methylglycine diacetic acid (MGDA), triethylenetetraminehexaacetic acid (TTHA), glutamic acid diacetic acid (GLDA), hydroxyethyliminodiacetic acid (HIDA), dihydroxyethylglycine (DHEG), aspartic acid diacetic acid (ASDA), ethylenediamine succinic acid (EDDS) and their salts, etc. Examples of phosphonic acid-based chelating agents include hydroxyethylidene diphosphonic acid (HEDP), nitrilotris(methylene phosphonic acid) (NTMP), phosphonobutane tricarboxylic acid (PBTC), ethylenediamine tetramethylene phosphonic acid (EDTMP) and their salts, etc. Examples of hydroxycarboxylic acid-based chelating agents include citric acid, malic acid, tartaric acid, gluconic acid, lactic acid and their salts, etc. Examples of polycarboxylic acid-based chelating agents include succinic acid, oxalic acid, glutaric acid, adipic acid, fumaric acid, malonic acid and their salts, etc.

[0098] (8) Other components The detergent composition may contain solvents, builders, softeners, reducing agents (such as sulfites), antifoaming agents (such as silicone), fragrances, antibacterial and antifungal agents (such as Proxel [trade name], benzoic acid), and other additives known in the field of detergents.

[0099] Examples of the solvent include monohydric alcohols having 1 to 3 carbon atoms; polyhydric alcohols having 2 to 4 carbon atoms; di- or trialkylene glycols having 2 to 4 carbon atoms in the alkylene glycol unit; monoalkoxy (methoxy, ethoxy, propoxy, butoxy), phenoxy or benzooxy ethers of di- to tetraalkylene glycols having 2 to 4 carbon atoms in the alkylene glycol unit. As the solvent, water-soluble organic solvents having 2 or more carbon atoms, preferably 3 or more carbon atoms, and 10 or less carbon atoms, preferably 8 or less carbon atoms are preferred. Here, the water-soluble organic solvent refers to a solvent having an octanol / water partition coefficient (LogPow) of 3.5 or less. Specifically, ethanol, isopropyl alcohol; ethylene glycol, propylene glycol, glycerin, isoprene glycol; diethylene glycol, dipropylene glycol; propylene glycol monomethyl ether, propylene glycol monoethyl ether, diethylene glycol monobutyl ether (also referred to as butyl diglycol, etc.), phenoxyethanol, phenoxy triethylene glycol, phenoxyisopropanol can be mentioned. As the solvent, a solvent selected from ethanol, propylene glycol, dipropylene glycol, diethylene glycol monobutyl ether, phenoxyethanol, phenyl glycol, and phenoxyisopropanol is preferred. The solvent preferably has an alkoxy group, and more preferably contains one or more selected from the above-mentioned monoalkoxy, phenoxy or benzooxy ethers of di- to tetraalkylene glycols having 2 to 4 carbon atoms in the alkylene glycol unit. It is more preferred to contain diethylene glycol monobutyl ether as the solvent.

[0100] As the detergent composition, those having a water content of 60 to 99% by mass and / or having a higher solvent content than the surfactant content are preferred, and those having a water content of 60 to 99% by mass and a higher solvent content than the surfactant content are more preferred.

[0101] The detergent composition can be produced according to a conventional method by combining the polypeptide of the present invention obtained by the above method and the above-mentioned known cleaning components. The form of the detergent can be selected according to the use, and can be, for example, liquid, powder, granule, paste, solid, etc.

[0102] The thus obtained detergent composition can be used as a laundry detergent, dishwashing detergent, bleaching agent, hard surface cleaning detergent, drain pipe cleaning detergent, denture cleaning detergent, sterilizing detergent for medical instruments, etc. Preferably, laundry detergents and dishwashing detergents are mentioned, and more preferably, laundry detergents for laundry (laundry detergents for washing), dishwashing detergents for hand washing, and detergents for automatic dishwashers are mentioned. In addition, the detergent composition is suitable for use at 40°C or lower, 35°C or lower, 30°C or lower, 25°C or lower, and 5°C or higher, 10°C or higher, 15°C or higher. It is also suitable for use at 5 to 40°C, 10 to 35°C, 15 to 30°C, 15 to 25°C. Preferred usage modes include use in low-temperature (15 to 30°C) washing in laundry, use in low-temperature (15 to 30°C) in dishwashing by hand, and use in low-temperature (15 to 30°C) washing by an automatic dishwasher.

[0103] By using the detergent composition of the present invention, it is possible to wash an object to be washed that requires removal of dirt (for example, clothes, tableware, hard surfaces, drain pipes, dentures, medical instruments, etc.), that is, to remove dirt. Such a cleaning method includes bringing the object to be washed that requires removal of dirt into contact with the detergent composition of the present invention. Preferably, the dirt is dirt containing oil, and examples include oil stains and composite stains containing proteins and carbohydrates in addition to oil.

[0104] In the cleaning method of the present invention, in order to bring the object to be cleaned into contact with the cleaning composition, the object to be cleaned may be immersed in water in which the cleaning composition is dissolved, or the cleaning composition may be directly applied to the object to be cleaned. In the method of the present invention, the object to be cleaned after the immersion or application of the detergent composition may be further hand-washed, scrubbed with a sponge, washed in a washing machine, etc., but this is not necessarily required.

[0105] Regarding the above-described embodiments, the following aspects are further disclosed in the present invention. <1>A lipase complex comprising two or more polypeptides, wherein the two or more polypeptides are linked via disulfide bonds, and at least one of the two or more polypeptides has a lipase activity consisting of an amino acid sequence having at least 70% identity with the amino acid sequence represented by 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 has at least 70% identity with the amino acid sequence represented by SEQ ID NO: 4, 6, 8, 10, 14, 16, 18 or 20 and the amino acid residues at one or more positions selected from the positions corresponding to positions 208 and 272 in the numbering of SEQ ID NO: 4 are cysteine. <3>The lipase complex according to <1> or <2>, comprising two polypeptides, wherein the two polypeptides are linked via disulfide bonds, and each of the two polypeptides independently has at least 70% identity with the amino acid sequence represented by SEQ ID NO: 4, 6, 8, 10, 14, 16, 18 or 20 and the amino acid residues at one or more positions selected from the positions corresponding to positions 208 and 272 in the numbering of SEQ ID NO: 4 are cysteine, and has lipase activity. <4>It consists of two identical polypeptides, and the two polypeptides are linked via a disulfide bond. The polypeptide 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 has lipase activity consisting of an amino acid sequence in which at least one amino acid residue at a position selected from the positions corresponding to positions 208 and 272 in the numbering of SEQ ID NO: 4 is cysteine. A complex of the lipase according to <1> or <2>.

[0106] <5>A method for producing a complex of lipase, comprising the step of linking two or more polypeptides containing at least one 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, 12, 14, 16 or 18 via a disulfide bond. <6>The method according to <5>, wherein the polypeptide having lipase activity 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 consists of an amino acid sequence in which at least one amino acid residue at a position selected from the positions corresponding to positions 208 and 272 in the numbering of SEQ ID NO: 4 is cysteine. <7>The binding step is to bind two polypeptides via a disulfide bond. The two polypeptides are each independently 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 having at least one amino acid residue at a position selected from the positions corresponding to positions 208 and 272 in the numbering of SEQ ID NO: 4 as cysteine. The method according to <5> or <6>. <8>The method according to <5> or <6>, wherein the binding step binds two molecules of the same polypeptide via a disulfide bond, and the polypeptide has at least 70% identity with the amino acid sequence represented by SEQ ID NO: 4, 6, 8, 10, 14, 16, 18 or 20 and has lipase activity consisting of an amino acid sequence in which at least one amino acid residue at a position selected from the positions corresponding to positions 208 and 272 in the numbering of SEQ ID NO: 4 is cysteine. <9>The method according to any one of <5> to <8>, further comprising, before the binding step, providing a polypeptide having lipase activity consisting of an amino acid sequence having at least 70% identity with the amino acid sequence represented by SEQ ID NO: 4, 6, 8, 10, 12, 14, 16 or 18 and having at least one amino acid residue at a position selected from the positions corresponding to positions 208 and 272 in the numbering of SEQ ID NO: 4 being cysteine. <10>In a polypeptide consisting of an amino acid sequence having at least 70% identity with the amino acid sequence represented by SEQ ID NO: 4, 6, 8, 10, 12, 14, 16 or 18 and having lipase activity, the step of substituting at least one amino acid residue at a position selected from the positions corresponding to positions 208 and 272 in the numbering of SEQ ID NO: 4 with cysteine, and The step of binding two or more polypeptides containing at least one molecule of the polypeptide after amino acid residue substitution via a disulfide bond. A method for producing a complex of lipase, comprising: <11>The method according to <10>, wherein the binding step binds 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 each independently have at least 70% identity with the amino acid sequence represented by SEQ ID NO: 4, 6, 8, 10, 14, 16, 18 or 20 and have lipase activity consisting of an amino acid sequence in which at least one amino acid residue at a position selected from the positions corresponding to positions 208 and 272 in the numbering of SEQ ID NO: 4 is cysteine. <12>The method according to <10>, wherein the binding step binds two polypeptides after amino acid residue substitution through 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, the binding step is performed by expressing or retaining a polypeptide under non-reducing conditions.

[0107] <14>A method for improving the stability of lipase, comprising a step of complexing lipase. <15>The method according to <14>, comprising a step of binding two or more polypeptides containing at least one molecule of a polypeptide having lipase activity and consisting of an amino acid sequence having at least 70% identity with the amino acid sequence represented by SEQ ID NO: 4, 6, 8, 10, 12, 14, 16 or 18 through a disulfide bond. <16>The method according to <15>, wherein the polypeptide having lipase activity consists of an amino acid sequence having at least 70% identity with the amino acid sequence represented by SEQ ID NO: 4, 6, 8, 10, 12, 14, 16 or 18 and having cysteine as the amino acid residue at one or more positions selected from the positions corresponding to positions 208 and 272 in the numbering of SEQ ID NO: 4. <17>The method according to <15> or <16>, wherein the binding step binds two polypeptides through a disulfide bond, and the two polypeptides each independently have at least 70% identity with the amino acid sequence represented by SEQ ID NO: 4, 6, 8, 10, 14, 16, 18 or 20 and have lipase activity consisting of an amino acid sequence having cysteine as the amino acid residue at one or more positions selected from the positions corresponding to positions 208 and 272 in the numbering of SEQ ID NO: 4. <18>The method according to <15> or <16>, wherein the binding step binds two molecules of the same polypeptide via a disulfide bond, and the polypeptide has at least 70% identity with the amino acid sequence represented by SEQ ID NO: 4, 6, 8, 10, 14, 16, 18 or 20 and has lipase activity consisting of an amino acid sequence in which at least one amino acid residue at a position selected from the positions corresponding to positions 208 and 272 in the numbering of SEQ ID NO: 4 is cysteine. <19>The method according to any one of <15> to <18>, further comprising, before the binding step, providing a polypeptide having lipase activity consisting of an amino acid sequence having at least 70% identity with the amino acid sequence represented by SEQ ID NO: 4, 6, 8, 10, 12, 14, 16 or 18 and in which at least one amino acid residue at a position selected from the positions corresponding to positions 208 and 272 in the numbering of SEQ ID NO: 4 is cysteine. <20>In a polypeptide consisting of an amino acid sequence having at least 70% identity with the amino acid sequence represented by SEQ ID NO: 4, 6, 8, 10, 12, 14, 16 or 18 and having lipase activity, substituting at least one amino acid residue at a position selected from the positions corresponding to positions 208 and 272 in the numbering of SEQ ID NO: 4 with cysteine, and binding two or more polypeptides containing at least one molecule of the polypeptide after amino acid residue substitution via a disulfide bond, A method for improving the stability of lipase, comprising the steps of. <21>The method according to <20>, wherein the binding step binds 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 each independently have at least 70% identity with the amino acid sequence represented by SEQ ID NO: 4, 6, 8, 10, 14, 16, 18 or 20 and have lipase activity consisting of an amino acid sequence in which at least one amino acid residue at a position selected from the positions corresponding to positions 208 and 272 in the numbering of SEQ ID NO: 4 is cysteine. <22>The method according to <20>, wherein the binding step binds the same polypeptide after substitution of two amino acid residues via a disulfide bond. <23>The method according to any one of <15> to <22>, wherein the binding step is carried out under non-reducing conditions, preferably, the binding step is carried out by expressing or retaining the polypeptide under non-reducing conditions.

[0108] <24>A lipase-active polypeptide comprising 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 having cysteine as the amino acid residue at one or more positions selected from the positions corresponding to positions 208 and 272 in the numbering of SEQ ID NO: 4. <25>The complex according to any one of <1> to <4> or the polypeptide according to <24>, having one or more amino acid residues selected from phenylalanine at the position corresponding to position 16 in the numbering of SEQ ID NO: 4, cysteine or methionine at the position corresponding to position 22, phenylalanine at the position corresponding to position 29, isoleucine or valine at the position corresponding to position 50, alanine or glutamine at the position corresponding to position 120, isoleucine or threonine at the position corresponding to position 123, glutamic acid at the position corresponding to position 126, phenylalanine at the position corresponding to position 142, alanine at the position corresponding to position 156, lysine at the position corresponding to position 181, glutamic acid at the position corresponding to position 186, glutamine at the position corresponding to position 188, valine at the position corresponding to position 191, histidine at the position corresponding to position 220, histidine at the position corresponding to position 221, glutamine at the position corresponding to position 224, threonine at the position corresponding to position 233, asparagine at the position corresponding to position 256, alanine, cysteine or leucine at the position corresponding to position 260, isoleucine at the position corresponding to position 269, and glutamine at the position corresponding to position 274. <26>The complex of lipase 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.

[0109] <27>A polynucleotide encoding the polypeptide according to any one of <24> to <26>. <28>A vector or DNA fragment containing the polynucleotide according to <27>. <29>A transformed cell containing 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 bacterium belonging to the genus Bacillus, preferably a bacterium belonging to the genus Bacillus, more preferably Bacillus subtilis. <32>A method for producing a polypeptide having lipase activity, comprising the step of culturing the transformed cell according to any one of <29> to <31>.

[0110] <33>Either one or both of the complex of lipase according to any one of <1> to <4>, <25> and <26> and the polypeptide according to any one of <24> to <26>, preferably the complex of lipase according to any one of <1> to <4>, <25> and <26>, or the complex of lipase according to any one of <1> to <4>, <25> and <26> and the polypeptide according to any one of <24> to <26>, a detergent composition containing the same. <34>The detergent composition according to <33>, further containing a sulfosuccinate ester or a salt thereof, preferably a branched alkyl sulfosuccinate ester having a branched alkyl group having 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, still more preferably a branched alkyl sulfosuccinate ester having a branched alkyl group having 10 carbon atoms or a salt thereof. <35>A diester of sulfosuccinic acid or a salt thereof, preferably a di-branched alkyl ester of sulfosuccinic acid or a salt thereof in which two branched alkyl groups are each a branched alkyl group having 8 to 12 carbon atoms, more preferably a di-branched alkyl ester of sulfosuccinic acid or a salt thereof in which two branched alkyl groups are each a branched alkyl group having 9 or 10 carbon atoms, still more preferably a di-branched alkyl ester of sulfosuccinic acid or a salt thereof in which two branched alkyl groups are each a branched alkyl group having 10 carbon atoms, still more preferably bis-(2-propylheptyl) sulfosuccinic acid or a salt thereof, and further containing the detergent composition according to <33>. <36>The detergent composition according to any one of <33> to <35>, wherein the water content is 60 to 99% by mass and / or the solvent content is higher than the surfactant content, preferably, the water content is 60 to 99% by mass and the solvent content is higher 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 in powder or liquid form.

[0111] <39>A method for cleaning dirt, using the detergent composition according to any one of <33> to <38>. <40>The method according to <39>, which includes contacting an object to be cleaned with the detergent composition according to any one of <33> to <38>. <41>Either one or both of the complex of lipase 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, preferably, the complex of lipase according to any one of <1> to <4>, <25> and <26>, or the complex of lipase according to any one of <1> to <4>, <25> and <26> and the polypeptide according to any one of <24> to <26>. <42>The use of any 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> for cleaning dirt, 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>.

Example

[0112] Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited thereto.

[0113] Example 1 (1) Construction of lipase expression plasmid Using the plasmid pHY-S237 described in Example 7 of WO2006 / 068148A1 as a template, the ORF full length of the alkaline cellulase gene was replaced with CnLip I44T L120S S130A A134T A135V L136M S137N (hereinafter referred to as CnLipA, encoding the polynucleotide of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2) described in Japanese Patent Application No. 2023-025141 synthesized by artificial gene synthesis by In-Fusion reaction to construct the plasmid pHY-CnLipA. Using pHY-CnLipA as a template, five mutations were introduced by site-directed mutagenesis by PCR using a complementary primer pair (Zheng, Lei, Ulrich Baumann, and Jean-Louis Reymond. Nucleic Acids Research 32.14 (2004): e115.) to create a plasmid pHY-CnLipAm expressing CnLipAm (encoding the polynucleotide of SEQ ID NO: 3 and the amino acid sequence of SEQ ID NO: 4). Using the plasmid pHY-S237 described in Example 7 of WO2006 / 068148A1 as a template, the entire ORF of the alkaline cellulase gene was synthetically replaced with the 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 plasmids pHY-SspLip_m, pHY-EtLip_m, and pHY-CspLipA. For further mutagenesis of the lipase, a site-directed mutagenesis method by PCR using a complementary primer pair was used.

[0114] (2) Preparation of lipase solution The lipase expression plasmid was introduced into the Bacillus subtilis strain 168ΔsigF strain described in JP 2003-47490 A by the protoplast method, and cultured in a 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. Then, the culture supernatant containing lipase was collected by centrifugation.

[0115] (3) SDS-PAGE of lipase mutants The culture supernatant containing CnLipAm (parent enzyme), CnLipAm L208C mutant or CnLipAm N272C mutant prepared by the method described in (2) and Laemmli Sample Buffer (BIO-RAD) with or without 100 mM DTT as a reducing agent were mixed in equal amounts, and then heat-treated at 99 °C for 5 minutes to prepare samples. The gel used was Mini-PROTEIN TGX Stain-Free (BIO-RAD). 5 μL of the sample was applied to each well and electrophoresed at 210 V for 25 minutes. Precision Plus protein Unstained standard (BIO-RAD) was used as the molecular weight marker. The protein band intensity was calculated using the ChemiDoc MP Imaging System. The monomer ratio (%) was determined by dividing the band intensity of each mutant in the non-reduced state by the band intensity in the reduced state (Table 1). Since the monomer ratio decreased in the mutants, it was revealed that complexes were formed via disulfide bond formation in the culture supernatant.

[0116]

Table 1

[0117] (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 applied to Amicon Ultra 10K (Merck Millipore), and buffer-exchanged into 20 mM Tris-HCl (pH 7.0), 2 mM CaCl2, 0.01% Triton-X100, 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 fractionated samples. (Conditions) Apparatus: AKTA pure (GE healthcare) Column: HiLoad 26 / 600 Superdex 200pg (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

[0118] For the measurement of lipase activity in the fraction samples, 4-nitrophenyl butyrate (SIGMA) was used as a substrate. The lipase activity can be determined by measuring the rate of increase in absorbance associated with the release of 4-nitrophenol due to the action of lipase. A substrate solution was prepared by adding 4-nitrophenyl butyrate to 20 mM Tris-HCl (pH 7.0) at a final concentration of 2 mM and mixing. Activity measurements were performed using a fraction sample diluted 51-fold with 20 mM Tris-HCl (pH 7.0). Fractions containing the 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 were identified based on lipase activity. The fraction solution containing the lipase monomer or complex was subjected to SDS-PAGE by the method described in (3) (Figure 1). A distinct band was confirmed at a molecular weight twice the estimated molecular weight for CnLipAm L208C complex 1, indicating that it contains a homodimer. A band at a molecular weight twice the estimated molecular weight was confirmed for CnLipAm N272C complex 2, indicating that it contains a homodimer, and the presence of a complex via a disulfide bond with a component contained in the culture supernatant was also confirmed. It was confirmed that CnLipAm L208C complex 2 and CnLipAm N272C complex 1 form complexes via disulfide bonds with components contained in the culture supernatant.

[0119] (5) Stability evaluation in the model cleaning solution 4-Nitrophenyl butyrate (SIGMA) was used as the substrate. The lipase activity can be determined by measuring the rate of increase in absorbance associated with the release of 4-nitrophenol due to the action of lipase. A substrate solution was prepared by adding 4-nitrophenyl butyrate to 20 mM Tris-HCl (pH 7.0) at a final concentration of 2 mM and mixing them. A fraction solution containing lipase monomer or complex was added to the model cleaning solution shown in Table 2, incubated at 45 °C for 1 hour, and then the activity was measured using a solution diluted 51-fold with 20 mM Tris-HCl (pH 7.0). The activity value of the sample before the 45 °C treatment was taken as the initial activity, the inactivation rate per unit time (h) due to the 45 °C treatment was calculated, and the half-life (h) was calculated from it. The relative stability was determined by dividing the half-life (h) of each mutant by the half-life (h) of the parental polypeptide. The results are shown in Table 3. It was revealed that the CnLipAm L208C and CnLipAm N272C mutants were significantly stabilized compared to the parental enzyme by forming complexes.

[0120] [Table 2]

[0121] [Table 3]

[0122] (6) Evaluation of detergency in model cleaning solution The ability to remove triglycerides on a hard surface was evaluated as detergency. A mixture of beef tallow (SIGMA, 03-0660) and rapeseed oil (SIGMA, 23-0450) at a weight ratio of 9:1 was dissolved in 3 volumes of chloroform and then colored with 0.2 wt% Sudan III to prepare a model stain. 10 μL of the model stain was dropped onto the bottom of each well of a 96-well deep well plate made of polypropylene, and the chloroform was evaporated and dried to obtain a stained plate. The lipase solution used was prepared by diluting the lipase concentration obtained by gel filtration chromatography in (4) to 200 mg / L with 20 mM Tris-HCl (pH 7.0), 2 mM CaCl2, 0.01% Triton-X100, and 150 mM NaCl in the model cleaning solution described in Table 2, and 1 / 25 volume of this was added to the model cleaning solution. 300 μL of the cleaning solution was gently added to the soiled plate and left to stand at room temperature (about 22 °C) for 20 minutes for immersion cleaning. 100 μL of the cleaning solution was aliquoted without touching the dirt at the bottom and transferred to a new 96-well plate. To quantify Sudan III in the model dirt solubilized in the cleaning solution by immersion cleaning, the absorbance at 500 nm (A500) was measured. A500 corresponds to the amount of oil released into the cleaning solution and can be used as an indicator of detergency. The enzymatic effect on detergency (ΔA500) was determined by subtracting the A500 of the cleaning solution added with 20 mM Tris-HCl (pH 7.0), 2 mM CaCl2, 0.01% Triton-X100, and 150 mM NaCl instead of lipase from the A500 of the cleaning solution containing each lipase. The cleaning results for 20 minutes are shown in Table 4. It was revealed that the CnLipAm L208C and CnLipAm N272C mutants maintained their detergency even by complex formation.

[0123]

Table 4

[0124] (7) Stability evaluation of lipase mutants Butyric acid 4-nitrophenyl (SIGMA) was used as the substrate. The activity of lipase can be determined by measuring the rate of increase in absorbance accompanying the release of 4-nitrophenol due to the action of lipase. A substrate solution was prepared by adding butyric acid 4-nitrophenyl to 20 mM Tris-HCl (pH 7.0) at a final concentration of 2 mM and mixing. The culture supernatants containing SspLip_m, EtLip_m, CspLipA and SspLip_m L210C, EtLip_m F210C, CspLipA L209C mutants prepared by the method described in (2) were added to the model cleaning solution shown in Table 2, incubated at 40 °C or 45 °C for an arbitrary time, and then the activity was measured using a 51-fold dilution with 20 mM Tris-HCl (pH 7.0). Using the activity value of the sample before heat treatment as the initial activity, the inactivation rate per unit time (h) due to heat treatment was calculated, and the half-life (h) was calculated from that. 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. It was shown that the stability of all mutants was improved compared to the parent polypeptide. The 210th position of SspLip_m, the 210th position of EtLip_m, and the 209th position of CspLipA correspond to the 208th position of CnLipAm, respectively.

[0125] [Table 5]

[0126] (8) SDS-PAGE of Lipase Mutants The culture supernatants containing SspLip_m, EtLip_m, CspLipA and SspLip_m L210C, EtLip_m F210C, CspLipA L209C mutants prepared by the method described in (2) were mixed in equal amounts with Laemmli Sample Buffer (BIO-RAD) containing or not containing 100 mM DTT as a reducing agent, and then heat-treated at 99 °C for 5 minutes to prepare samples. The gel used was Mini-PROTEIN TGX Stain-Free (BIO-RAD). 5 μL of the sample was applied to each well and electrophoresed at 210 V for 25 minutes. Precision Plus protein Unstained standard (BIO-RAD) was used as the molecular weight marker. The protein band intensity was calculated using the ChemiDoc MP Imaging System. The monomer ratio (%) was determined by dividing the band intensity of each mutant in the non-reduced state by the band intensity in the reduced state (Table 6). It was revealed that the monomer ratio was less than 100% in the mutants, and complexes were formed via disulfide formation in the culture supernatant.

[0127]

Table 6

[0128] Example 2: Evaluation of the stability of the combined mutants 4-Nitrophenyl butyrate (SIGMA) was used as the substrate. The lipase activity can be determined by measuring the rate of increase in absorbance accompanying the release of 4-nitrophenol due to the action of lipase. A substrate solution was prepared by adding 4-nitrophenyl butyrate to 20 mM Tris-HCl (pH 7.0) at a final concentration of 2 mM and mixing. A lipase solution was added to the model cleaning solution shown in Table 2, incubated at 40 °C for 3 hours, and then the activity was measured using a solution diluted 51-fold with 20 mM Tris-HCl (pH 7.0). Using the activity value of the sample before the 40 °C treatment as the initial activity, the inactivation rate per unit time (h) due to the 40 °C treatment was calculated, and the half-life (h) was calculated from that. 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 7. It was shown that the stability of all mutants was improved compared to the parent polypeptide.

[0129]

Table 7-1

[0130]

Table 7-2

[0131] Reference Example 1: Comparison of stability in model cleaning solution and other formulations A commercially available liquid detergent (Kao Corporation, Attack ZERO (hereinafter referred to as detergent 1)) was allowed 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, incubated at 45 °C for 1 hour, and then the activity was measured using a solution diluted 51-fold with 20 mM Tris-HCl (pH 7.0). Using the activity value of the sample before the 45 °C treatment as the initial activity, the ratio of the activity value after the treatment to the initial activity was defined as the residual activity (%). The results are shown in Table 8. In the model cleaning solution described in Table 2, for which suitability for cleaning is disclosed in Patent Document 1, it was confirmed that lipase was significantly destabilized compared to other formulations containing a protease that degrades proteins.

[0132]

Table 8

[0133] Reference Example 2: Evaluation of the Stability of Lipase Variants Butyric acid 4-nitrophenyl (SIGMA) was used as a substrate. The activity of lipase can be determined by measuring the rate of increase in absorbance accompanying the release of 4-nitrophenol due to the action of lipase. A substrate solution was prepared by adding butyric acid 4-nitrophenyl to 20 mM Tris-HCl (pH 7.0) at a final concentration of 2 mM and mixing them. Using the plasmid pHY-S237 described in Example 7 of WO2006 / 068148A1 as a template, the entire ORF of the alkaline cellulase gene was replaced with the artificially gene-synthesized PvLip (encoding the polynucleotide of SEQ ID NO: 11 and the amino acid sequence of SEQ ID NO: 12) by In-Fusion reaction to construct the plasmid pHY-PvLip. For further mutagenesis of lipase, a site-directed mutagenesis method by PCR using a complementary primer pair was used. The culture supernatants containing PvLip and PvLip A205C variant prepared by the method described in Example 1(2) were added to the model cleaning solutions shown in Table 2, incubated at 45 °C for 50 minutes, and then the activity measurement was performed using a solution diluted 51-fold with 20 mM Tris-HCl (pH 7.0). Using the activity value of the sample before heat treatment as the initial activity, the inactivation rate per unit time (h) due to heat treatment was calculated, and the half-life (h) was calculated therefrom. The relative stability was determined by dividing the half-life (h) of each variant by the half-life (h) of the parental polypeptide. The results are shown in Table 9. It was shown that the stability of the PvLip A205C variant was not improved compared to the parental polypeptide. Note that position 205 of PvLip corresponds to position 208 of CnLipAm.

[0134] [Table 9]

[0135] Reference Example 3: SDS-PAGE of Lipase Variants The culture supernatant containing PvLip and the PvLip A205C mutant prepared by the method described in Example 1(2) was mixed in equal amounts with Laemmli Sample Buffer (BIO-RAD) containing or not containing 100 mM DTT as a reducing agent, and then heat-treated at 99 °C for 5 minutes to prepare samples. The gel used was Mini-PROTEIN TGX Stain-Free (BIO-RAD). 5 μL of the sample was applied to each well and electrophoresed at 210 V for 25 minutes. Precision Plus protein Unstained standard (BIO-RAD) was used. The protein band intensity was calculated using the ChemiDoc MP Imaging System. The monomer ratio (%) was determined by dividing the band intensity of each mutant in the non-reduced state by the band intensity in the reduced state (Table 10). It was revealed that the monomer ratio of the PvLip A205C mutant was 100%, and no complex was formed through disulfide formation in the culture supernatant.

[0136]

Table 10

[0137] Reference Example 4: Evaluation of the Stability of Lipase Mutants 4-Nitrophenyl butyrate (SIGMA) was used as a substrate. The activity of lipase can be determined by measuring the rate of increase in absorbance accompanying the release of 4-nitrophenol due to the action of lipase. A substrate solution was prepared by adding 4-nitrophenyl butyrate to 20 mM Tris-HCl (pH 7.0) at a final concentration of 2 mM and mixing. The culture supernatant containing CnLipAm (parent enzyme), CnLipAm P207C mutant, or CnLipAm H209C mutant prepared by the method described in Example 1(2) was added to the model cleaning solution shown in Table 2, incubated at 40 °C for 1 hour, and then the activity was measured using a 51-fold dilution with 20 mM Tris-HCl (pH 7.0). Using the activity value of the sample before heat treatment as the initial activity, the inactivation rate per unit time (h) due to heat treatment was calculated, and the half-life (h) was calculated therefrom. The relative stability was determined by dividing the half-life (h) of each mutant by the half-life (h) of the parent polypeptide. The results are shown in Table 11. It was confirmed that the stability of none of the mutants was improved compared to the parent polypeptide.

[0138]

Table 11

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. The lipase complex according to claim 1, which is composed of two polypeptide molecules bonded 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 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.

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 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.

6. A method for improving the stability of lipase, comprising a step of linking two or more polypeptide molecules via a disulfide bond, the polypeptide including at least one molecule 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, 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 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.

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 of 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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