Lipase variant

Abstract

To provide a lipase variant having improved heterologous expression properties.SOLUTION: This lipase variant comprises an amino acid sequence in which, in comparison to the amino acid sequence of the parent lipase, an amino acid residue that is at a prescribed position having numbering based on a specific amino acid sequence is substituted with a prescribed amino acid residue. The parent lipase may be a lipase belonging to the same clade (Proteus / Yersinia clade) that includes lipases derived from Proteus or Yersinia bacteria.SELECTED DRAWING: None

Description

[Technical field]

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

[0002] Lipases contribute to the removal of oily dirt by hydrolyzing ester bonds in lipids to generate fatty acids, and are useful in cleaning applications. As a lipase useful for cleaning, lipase derived from Thermomyces lanuginosus (hereinafter referred to as TLL) is sold under the trade name LIPOLASE (registered trademark). Patent Document 1 discloses that lipase Lipr139 derived from Cedecea sp-16640 strain has superior cleaning performance compared to TLL. Patent Document 2 discloses a variant of lipase derived from Proteus bacteria (hereinafter referred to as PvLip) having improved cleaning performance when compared to one or more reference lipolytic enzymes. Patent Document 3 discloses that lipase Lipr138 derived from metagenomics has superior cleaning performance compared to TLL. Lipr139, PvLip, and Lipr138 are lipases belonging to the same clade (Proteus / Yersinia clade) that includes lipases derived from Proteus bacteria and Yersinia bacteria.

[0003] Many lipases derived from Gram-negative bacteria require lipase-specific chaperones for folding into an active form (Non-Patent Document 1), which poses a challenge to inexpensive production by heterologous expression. It has been reported that heterologous expression can be improved by co-expression of specific chaperones (Non-Patent Document 2), but the productivity is low, and inexpensive production by heterologous expression remains a major challenge. On the other hand, bacterial lipases of the Proteus / Yersinia clade do not require specific chaperones and have the advantage of inexpensive production by heterologous expression (Non-Patent Documents 3 and 4). As described above, bacterial lipases of the Proteus / Yersinia clade have excellent potential as cleaning enzymes in terms of both their suitability for cleaning and their low-cost production potential.

[0004] Lipases catalyze various reactions, such as ester synthesis, transesterification, and acidolysis, in addition to ester hydrolysis. Taking advantage of this reactivity, lipases are used in various applications, such as cleaning, optical resolution, biofuel production, oil and fat processing, wastewater treatment, pulp production, and leather production. Non-Patent Documents 5 and 6 show the excellent performance of LipC12 (same sequence as Lipr138) in ester synthesis applications, and bacterial lipases of the Proteus / Yersinia clade are expected to be useful not only in cleaning but also in various industrial applications.

[0005] Substrate specificity, such as the positional specificity of the ester bond in acylglycerol, fatty acid chain length specificity, and enantioselectivity in optical resolution, varies greatly depending on the lipase species. Differences in substrate specificity are observed even between lipases with closely related sequences, and screening of lipases for each reaction system is necessary to perform optimal reactions. Therefore, there is a need to expand the sequence diversity of lipases that can be used industrially. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Special Publication No. 2015-523078 [Patent Document 2] International Publication No. 2020 / 046613 [Patent Document 3] Special Publication No. 2015-525248 [Non-patent literature]

[0007] [Non-Patent Document 1] Hobson, Audrey H., et al. Proceedings of the National Academy of Sciences 90.12 (1993): 5682-5686. [Non-Patent Document 2] Quyen, ThiDinh, ChiHai Vu, and GiangThi Thu Le. Microbial cell factories 11.1 (2012): 1-12. [Non-Patent Document 3] Lee, Hong-Weon, et al. Biotechnology letters 22.19 (2000): 1543-1547. [Non-Patent Document 4] Glogauer, Arnaldo, et al. Microbial cell factories 10.1 (2011): 1-15. [Non-Patent Document 5] Madalozzo, Aline Dutra, et al. Journal of molecular catalysis b: enzymatic 116 (2015): 45-51. [Non-Patent Document 6] Madalozzo, Aline Dutra, et al. Biocatalysis and Agricultural Biotechnology 8 (2016): 294-300. Summary of the Invention [Problem to be solved by the invention]

[0008] The present inventors evaluated the heterologous expression potential in Bacillus subtilis for 16 diverse lipases belonging to the Proteus / Yersinia clade, which includes lipases derived from bacteria of the genus Proteus and Yersinia on a rooted phylogenetic tree representing protein evolution, and found for the first time a problem that, despite reports that lipases from the Proteus / Yersinia clade have advantages in heterologous expression, only three lipases were clearly expressed, meaning that the diversity of sequences that can be heterologously expressed in lipases from the Proteus / Yersinia clade is actually small. Thus, the present invention relates to providing a lipase variant with improved heterologous expression potential. [Means for solving the problem]

[0009] In view of these problems, the present inventors conducted intensive research and found mutations that improve the heterologous expression of lipases using eight of the above 16 types of lipases and one type of lipase closely related to these.

[0010] That is, the present invention relates to the following 1) to 11). 1) Any lipase variant selected from the following (a) to (v): (a) a lipase variant consisting of an amino acid sequence having at least 82% identity to the amino acid sequence of SEQ ID NO:2, wherein the lipase variant has D at the position corresponding to position 118 of the amino acid sequence of SEQ ID NO:2; (b) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:4, and having a Y at the position corresponding to position 31 of the amino acid sequence of SEQ ID NO:2; (c) a lipase variant consisting of an amino acid sequence having at least 91% identity to the amino acid sequence of SEQ ID NO:4, and having a G at the position corresponding to position 32 of the amino acid sequence of SEQ ID NO:2; (d) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:4 and having a C at the position corresponding to position 90 of the amino acid sequence of SEQ ID NO:2; (e) a lipase variant consisting of an amino acid sequence having at least 94% identity to the amino acid sequence of SEQ ID NO:4, and having an L at the position corresponding to position 181 of the amino acid sequence of SEQ ID NO:2; (f) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:6, and having A, T, H or G at the position corresponding to position 222 of the amino acid sequence of SEQ ID NO:2; (g) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8, and having a K at the position corresponding to position 39 of the amino acid sequence of SEQ ID NO:2; (h) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8, and having a T at the position corresponding to position 125 of the amino acid sequence of SEQ ID NO:2; (i) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8, and having a P at the position corresponding to position 126 of the amino acid sequence of SEQ ID NO:2; (j) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8, and having G at the position corresponding to position 293 of the amino acid sequence of SEQ ID NO:2; (k) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 10, and having a Y at the position corresponding to position 29 of the amino acid sequence of SEQ ID NO: 2; (l) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 10, and having a Y at the position corresponding to position 31 of the amino acid sequence of SEQ ID NO: 2; (m) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 10, and having G at the position corresponding to position 32 of the amino acid sequence of SEQ ID NO: 2; (n) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 10, and having G at the position corresponding to position 186 of the amino acid sequence of SEQ ID NO: 2; (o) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 12, and having a Q at a position corresponding to position 206 of the amino acid sequence of SEQ ID NO: 2; (p) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 14, and having a Y at the position corresponding to position 31 of the amino acid sequence of SEQ ID NO: 2; (q) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 14, and having G at the position corresponding to position 32 of the amino acid sequence of SEQ ID NO: 2; (r) a lipase variant consisting of an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 14, and having a K at the position corresponding to position 77 of the amino acid sequence of SEQ ID NO: 2; (s) a lipase variant consisting of an amino acid sequence having at least 83% identity to the amino acid sequence of SEQ ID NO: 14, and having an L at a position corresponding to position 181 of the amino acid sequence of SEQ ID NO: 2; (t) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 16 and having a C at the position corresponding to position 90 of the amino acid sequence of SEQ ID NO: 2; (u) a lipase variant consisting of an amino acid sequence having at least 81% identity to the amino acid sequence of SEQ ID NO: 16, and having an L at a position corresponding to position 181 of the amino acid sequence of SEQ ID NO: 2; (v) A lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 34, and having a Y at the position corresponding to position 29 of the amino acid sequence of SEQ ID NO: 2. 2) A lipase variant selected from the following (aa) to (ae): (aa) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 4, and having at least two amino acid residues selected from Y at the position corresponding to position 31, G at the position corresponding to position 32, C at the position corresponding to position 90, and L at the position corresponding to position 181 of the amino acid sequence of SEQ ID NO: 2; (ab) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8, and having at least two amino acid residues selected from K at the position corresponding to position 39, T at the position corresponding to position 125, P at the position corresponding to position 126, and G at the position corresponding to position 293 of the amino acid sequence of SEQ ID NO:2; (ac) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 10, and having at least two amino acid residues selected from Y at the position corresponding to position 29, Y at the position corresponding to position 31, G at the position corresponding to position 32, and G at the position corresponding to position 186 of the amino acid sequence of SEQ ID NO: 2; (ad) a lipase variant consisting of an amino acid sequence having at least 83% identity to the amino acid sequence of SEQ ID NO: 14, and having at least two amino acid residues selected from Y at the position corresponding to position 31, G at the position corresponding to position 32, K at the position corresponding to position 77, and L at the position corresponding to position 181 of the amino acid sequence of SEQ ID NO: 2; (ae) A lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 16, and having a C at the position corresponding to position 90 and an L at the position corresponding to position 181 of the amino acid sequence of SEQ ID NO: 2. 3) An enzyme composition comprising the lipase variant according to 1) or 2). 4) A polynucleotide encoding the lipase variant according to 1) or 2). 5) A vector or a DNA fragment comprising the polynucleotide according to 4). 6) A transformed cell containing the vector or DNA fragment described in 5). 7) A method for producing a lipase variant, comprising the step of culturing the transformed cell according to 6). 8) A method for producing a lipase variant, comprising any one of the steps selected from the following (i) to (xxii): (i) substituting an amino acid residue at a position corresponding to position 118 of the amino acid sequence of SEQ ID NO: 2 with D in a polypeptide having lipase activity and consisting of an amino acid sequence having at least 82% identity to the amino acid sequence of SEQ ID NO: 2; (ii) substituting an amino acid residue at a position corresponding to position 31 of the amino acid sequence of SEQ ID NO:2 with Y in a polypeptide having lipase activity and consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO:4; (iii) substituting, in a polypeptide having lipase activity and consisting of an amino acid sequence having at least 91% identity with the amino acid sequence of SEQ ID NO:4, an amino acid residue at a position corresponding to position 32 of the amino acid sequence of SEQ ID NO:2 with G; (iv) substituting, in a polypeptide having lipase activity and consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO:4, an amino acid residue at a position corresponding to position 90 of the amino acid sequence of SEQ ID NO:2 with C; (v) substituting an amino acid residue at a position corresponding to position 181 of the amino acid sequence of SEQ ID NO:2 with L in a polypeptide having lipase activity and consisting of an amino acid sequence having at least 94% identity with the amino acid sequence of SEQ ID NO:4; (vi) substituting an amino acid residue at a position corresponding to position 222 of the amino acid sequence of SEQ ID NO:2 with A, T, H, or G in a polypeptide having lipase activity and consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO:6; (vii) substituting an amino acid residue at a position corresponding to position 39 of the amino acid sequence of SEQ ID NO:2 with K in a polypeptide having lipase activity, the polypeptide having an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8; (viii) substituting, with T, an amino acid residue at a position corresponding to position 125 of the amino acid sequence of SEQ ID NO:2 in a polypeptide having lipase activity and consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO:8; (ix) substituting an amino acid residue at a position corresponding to position 126 of the amino acid sequence of SEQ ID NO:2 with P in a polypeptide having lipase activity, the polypeptide having an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8; (x) substituting, with G, an amino acid residue at a position corresponding to position 293 of the amino acid sequence of SEQ ID NO:2 in a polypeptide having lipase activity and consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8; (xi) substituting an amino acid residue at a position corresponding to position 29 of the amino acid sequence of SEQ ID NO:2 with Y in a polypeptide having lipase activity, the polypeptide having an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:10; (xii) substituting an amino acid residue at a position corresponding to position 31 of the amino acid sequence of SEQ ID NO:2 with Y in a polypeptide having lipase activity, the polypeptide having an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:10; (xiii) substituting, in a polypeptide having lipase activity, an amino acid residue at a position corresponding to position 32 of the amino acid sequence of SEQ ID NO:2 with G, the polypeptide having an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:10; (xiv) substituting, with G, an amino acid residue at a position corresponding to position 186 of the amino acid sequence of SEQ ID NO:2 in a polypeptide having lipase activity and consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:10; (xv) substituting an amino acid residue at a position corresponding to position 206 of the amino acid sequence of SEQ ID NO:2 with Q in a polypeptide having lipase activity, the polypeptide having an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:12; (xvi) substituting an amino acid residue at a position corresponding to position 31 of the amino acid sequence of SEQ ID NO: 2 with Y in a polypeptide having lipase activity, the polypeptide having an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 14; (xvii) substituting, with G, an amino acid residue at a position corresponding to position 32 of the amino acid sequence of SEQ ID NO:2 in a polypeptide having lipase activity and consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:14; (xviii) substituting an amino acid residue at a position corresponding to position 77 of the amino acid sequence of SEQ ID NO: 2 with K in a polypeptide having lipase activity, the polypeptide having an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 14; (xix) substituting an amino acid residue at a position corresponding to position 181 of the amino acid sequence of SEQ ID NO:2 with L in a polypeptide having lipase activity, the polypeptide having an amino acid sequence having at least 83% identity to the amino acid sequence of SEQ ID NO:14; (xx) substituting an amino acid residue at a position corresponding to position 90 of the amino acid sequence of SEQ ID NO:2 with C in a polypeptide having lipase activity, the polypeptide having an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:16; (xxi) substituting an amino acid residue at a position corresponding to position 181 of the amino acid sequence of SEQ ID NO: 2 with L in a polypeptide having lipase activity, the polypeptide having an amino acid sequence having at least 81% identity to the amino acid sequence of SEQ ID NO: 16; (xxii) replacing the amino acid residue at the position corresponding to position 29 of the amino acid sequence of SEQ ID NO: 2 with Y in a polypeptide having lipase activity and consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 34. 9) A method for producing a lipase variant, comprising any one of the steps selected from the following (xxiii) to (xxvii): (xxiii) substituting amino acid residues at at least two positions selected from positions 31, 32, 90 and 181 of the amino acid sequence of SEQ ID NO:2 with Y at the position corresponding to position 31, G at the position corresponding to position 32, C at the position corresponding to position 90 and L at the position corresponding to position 181 in a polypeptide having an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:4 and having lipase activity; (xxiv) substituting amino acid residues at at least two positions selected from positions 39, 125, 126, and 293 of the amino acid sequence of SEQ ID NO:2 in a polypeptide having lipase activity, the polypeptide having an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8, with K at the position corresponding to position 39, with T at the position corresponding to position 125, with P at the position corresponding to position 126, and with G at the position corresponding to position 293; (xxv) substituting amino acid residues at at least two positions selected from positions 29, 31, 32, and 186 of the amino acid sequence of SEQ ID NO:2 with Y at the position corresponding to position 29, Y at the position corresponding to position 31, G at the position corresponding to position 32, and G at the position corresponding to position 186 in a polypeptide having an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:10 and having lipase activity; (xxvi) substituting amino acid residues at at least two positions selected from positions 31, 32, 77, and 181 of the amino acid sequence of SEQ ID NO:2 with Y at the position corresponding to position 31, G at the position corresponding to position 32, K at the position corresponding to position 77, and L at the position corresponding to position 181 in a polypeptide having a lipase activity, the polypeptide having an amino acid sequence having at least 83% identity to the amino acid sequence of SEQ ID NO:14; (xxvii) substituting an amino acid residue at a position corresponding to position 90 in the amino acid sequence of SEQ ID NO:2 with C and substituting an amino acid residue at a position corresponding to position 181 in the amino acid sequence of SEQ ID NO:2 with L in a polypeptide having lipase activity, the polypeptide having an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:16. 10) A method for improving heterologous expression of lipase, comprising any one of the steps selected from (i) to (xxii) above. 11) A method for improving heterologous expression of lipase, comprising any one of the steps selected from (xxiii) to (xxvii) above. Effect of the Invention

[0011] The lipase variants of the present invention have improved heterologous expression properties compared to the parent lipase, and can be produced inexpensively through heterologous expression, and can be used not only for cleaning applications but also for general industrial applications such as optical resolution, biofuel production, oil and fat processing, wastewater treatment, pulp production, and leather production. [Brief description of the drawings]

[0012] [Figure 1]Esterase activity of each lipase. pHY300PLK shows the result of an empty vector that does not encode lipase. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] All patents, non-patent publications, and other publications cited herein are hereby incorporated by reference in their entirety.

[0014] In this specification, "lipase" refers to triacylglycerol lipase (EC3.1.1.3), and means a group of enzymes having the activity of hydrolyzing ester bonds in lipids to produce fatty acids. Lipase activity is typically esterase activity, and can be determined by measuring the rate of increase in absorbance accompanying the release of 4-nitrophenol by hydrolysis of 4-nitrophenyl octanoate. Specific procedures for measuring lipase activity are described in detail in the Examples below.

[0015] As used herein, the term "Proteus / Yersinia clade" refers to a clade that includes lipases derived from bacteria of the genus Proteus and Yersinia on a rooted phylogenetic tree that represents the evolution of proteins. For example, the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, and 32 are all amino acid sequences of lipases that belong to the Proteus / Yersinia clade.

[0016] In the present specification, the identity of an amino acid sequence or a nucleotide sequence is calculated by the Lipman-Pearson method (Science, 1985, 227:1435-1441). Specifically, it is calculated by performing an analysis using the search homology program of the genetic information processing software GENETYX Ver. 12 with the unit size to compare (ktup) set to 2.

[0017] As used herein, "at least 80% identity" with respect to amino acid sequences and nucleotide sequences refers to 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 92% or more, even more preferably 94% or more, even more preferably 95% or more, even more preferably 96% or more, even more preferably 98% or more, even more preferably 99% or more, and even more preferably 99.5% or more identity. "At least 81% identity" refers to 81% or more, preferably 85% or more, more preferably 90% or more, even more preferably 92% or more, even more preferably 94% or more, even more preferably 95% or more, even more preferably 96% or more, even more preferably 98% or more, even more preferably 99% or more, and even more preferably 99.5% or more identity. "At least 83% identity" refers to 83% or more, preferably 85% or more, more preferably 90% or more, even more preferably 92% or more, even more preferably 94% or more, even more preferably 95% or more, even more preferably 96% or more, even more preferably 98% or more, even more preferably 99% or more, and even more preferably 99.5% or more identity. "At least 91% identity" refers to 91% or more, preferably 92% or more, more preferably 94% or more, even more preferably 95% or more, even more preferably 96% or more, even more preferably 98% or more, even more preferably 99% or more, and even more preferably 99.5% or more identity. "At least 94% identity" refers to 94% or more, preferably 95% or more, more preferably 96% or more, even more preferably 98% or more, even more preferably 99% or more, and even more preferably 99.5% or more identity. "At least 97% identity" refers to 97% or more, more preferably 98% or more, even more preferably 99% or more, and even more preferably 99.5% or more identity.

[0018] In the present specification, the "corresponding position" on an amino acid sequence or a nucleotide sequence can be determined by aligning a target sequence with a reference sequence (e.g., the amino acid sequence shown in SEQ ID NO: 2) so as to give maximum homology. Alignment of amino acid sequences or nucleotide sequences can be performed using known algorithms, and the procedures are known to those skilled in the art. For example, alignment can be performed using the Clustal W multiple alignment program (Thompson, J. D. et al., 1994, Nucleic Acids Res. 22: 4673-4680) with default settings. Alternatively, Clustal W2 or Clustal omega, which are revised versions of Clustal W, can be used. Clustal W, Clustal W2 and Clustal omega are available, for example, on the Clustal website operated by University College Dublin [www.clustal.org], the European Bioinformatics Institute (EBI [www.ebi.ac.uk / index.html]), and the website of the DNA Data Bank of Japan operated by the National Institute of Genetics (DDBJ [www.ddbj.nig.ac.jp / searches-j.html]). The position of the target sequence aligned to any position of the reference sequence by the above-mentioned alignment is considered to be a "position corresponding to" the any position.

[0019] Those skilled in the art can further fine-tune the alignment of the amino acid sequences obtained above to optimize it. Such an optimal alignment is preferably determined taking into consideration the similarity of the amino acid sequences, the frequency of gaps to be inserted, and the like. Here, the similarity of the amino acid sequences refers to the ratio (%) of the number of positions at which identical or similar amino acid residues exist in both sequences when the two amino acid sequences are aligned to the total number of amino acid residues. The similar amino acid residues refer to amino acid residues that have similar properties in terms of polarity and charge among the 20 types of amino acids that constitute proteins, and that cause so-called conservative substitution. Such groups of similar amino acid residues are well known to those skilled in the art, and examples thereof include, but are not limited to, arginine and lysine or glutamine; glutamic acid and aspartic acid or glutamine; serine and threonine or alanine; glutamine and asparagine or arginine; leucine and isoleucine.

[0020] As used herein, the term "amino acid residue" refers to the 20 types of amino acid residues constituting proteins: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0021] In this specification, amino acid positions and variants are described using the accepted IUPAC one-letter amino acid abbreviations as follows: The amino acid at a given position is designated as [amino acid, position], e.g., phenylalanine at position 31 is designated "F31". Amino acid "substitutions" are denoted as [original amino acid, position, substituted amino acid]. For example, the substitution of phenylalanine at position 31 with tyrosine is designated "F31Y". When different modifications can be introduced at one position, the different modifications are separated by a slash (" / "), for example, "I222A / T / H / G" represents a substitution of the isoleucine at position 222 with alanine, threonine, histidine or glycine.

[0022] As used herein, the term "operably linked" between a control region such as a promoter and a gene means that the gene and the control region are linked so that the gene can be expressed under the control of the control region. The procedure for "operably linked" between a gene and a control region is well known to those skilled in the art.

[0023] In this specification, "upstream" and "downstream" of a gene refer to the upstream and downstream of the transcription direction of the gene. For example, "a gene located downstream of a promoter" means that the gene is present on the 3' side of the promoter in the DNA sense strand, and "upstream" of a gene means the 5' region of the gene in the DNA sense strand.

[0024] As used herein, a "parent" polypeptide of a given mutant polypeptide refers to a polypeptide in which a given amino acid residue is mutated to give the mutant polypeptide. In other words, a "parent" polypeptide is a polypeptide before the mutant polypeptide is mutated. Such a parent polypeptide may be a naturally occurring (wild-type) polypeptide or a mutant thereof.

[0025] 1. Lipase variants and methods for producing the same The present invention provides a lipase variant with improved heterologous expression properties and a method for producing the same.

[0026] In one aspect, the invention provides a method for producing a lipase variant, the method comprising substituting an amino acid residue at a predetermined position, numbered based on SEQ ID NO:2, in a parent lipase with a predetermined amino acid residue.

[0027] An example of the parent lipase of the lipase variant of the present invention is a polypeptide having lipase activity and consisting of an amino acid sequence having at least 82% identity with the amino acid sequence of SEQ ID NO: 2. Here, the polypeptide having lipase activity and consisting of the amino acid sequence of SEQ ID NO: 2 is lipase SspLip (NCBI Accession No. WP_025122441.1) derived from Serratia sp. The parent lipase, which is a polypeptide having lipase activity and consisting of an amino acid sequence having at least 82% identity with the amino acid sequence of SEQ ID NO: 2, preferably has E at the position corresponding to position 118 of the amino acid sequence of SEQ ID NO: 2.

[0028] When the lipase variant of the present invention is produced from the parent lipase, the amino acid residue at the position corresponding to position 118 in the amino acid sequence of SEQ ID NO:2 in the parent lipase is substituted with D.

[0029] More specifically, when the lipase variant of the present invention is produced from the parent lipase, E at the position corresponding to position 118 of the amino acid sequence of SEQ ID NO:2 in the parent lipase is replaced with D.

[0030] Another example of a parent lipase is a polypeptide having lipase activity and consisting of an amino acid sequence having at least 80, 91 or 94% identity to the amino acid sequence of SEQ ID NO: 4. Here, the polypeptide having lipase activity and consisting of the amino acid sequence of SEQ ID NO: 4 is lipase PfLip derived from Pseudomonas frederiksbergensis (NCBI Accession No. WP_123598507.1). The parent lipase, which is a polypeptide having lipase activity and which consists of an amino acid sequence having at least the above-mentioned predetermined identity with the amino acid sequence of SEQ ID NO:4, is preferably one which has F at a position corresponding to position 31 of the amino acid sequence of SEQ ID NO:2, one which has E at a position corresponding to position 32 of the amino acid sequence of SEQ ID NO:2, one which has A at a position corresponding to position 90 of the amino acid sequence of SEQ ID NO:2, or one which has V at a position corresponding to position 181 of the amino acid sequence of SEQ ID NO:2, more preferably one which has F and E at positions 31 and 32, respectively, of the amino acid sequence of SEQ ID NO:2, one which has A and V at positions 90 and 181, respectively, of the amino acid sequence of SEQ ID NO:2, or one which has E and A at positions 32 and 90, respectively, of the amino acid sequence of SEQ ID NO:2, and even more preferably one which has F, E, A and V at positions 31, 32, 90 and 181, respectively, of the amino acid sequence of SEQ ID NO:2.

[0031] When producing a lipase variant of the present invention from the parent lipase, preferably, in the parent lipase, the amino acid residue at the position corresponding to position 31 of the amino acid sequence of SEQ ID NO:2 is substituted with Y, the amino acid residue at the position corresponding to position 32 of the amino acid sequence of SEQ ID NO:2 is substituted with G, the amino acid residue at the position corresponding to position 90 of the amino acid sequence of SEQ ID NO:2 is substituted with C, the amino acid residue at the position corresponding to position 181 of the amino acid sequence of SEQ ID NO:2 is substituted with L, or at least two (preferably three, more preferably four) amino acid residues selected from positions corresponding to positions 31, 32, 90 and 181 of the amino acid sequence of SEQ ID NO:2 are substituted with Y at the position corresponding to position 31, G at the position corresponding to position 32, C at the position corresponding to position 90 and L at the position corresponding to position 181. More preferably, in the parent lipase, the amino acid residue at position 32 of the amino acid sequence of SEQ ID NO:2 is substituted with G, the amino acid residue at position 90 of the amino acid sequence of SEQ ID NO:2 is substituted with C, the amino acid residue at position 181 of the amino acid sequence of SEQ ID NO:2 is substituted with L, the amino acid residues at positions 31 and 32 of the amino acid sequence of SEQ ID NO:2 are substituted with Y and G, respectively, the amino acid residues at positions 90 and 181 of the amino acid sequence of SEQ ID NO:2 are substituted with C and L, respectively, the amino acid residues at positions 32 and 90 of the amino acid sequence of SEQ ID NO:2 are substituted with G and C, respectively, or the amino acid residues at positions 31, 32, 90 and 181 of the amino acid sequence of SEQ ID NO:2 are substituted with Y, G, C and L, respectively.

[0032] More specifically, when producing the lipase variant of the present invention from the parent lipase, preferably, in the parent lipase, F at the position corresponding to position 31 of the amino acid sequence of SEQ ID NO:2 is replaced with Y, E at the position corresponding to position 32 of the amino acid sequence of SEQ ID NO:2 is replaced with G, A at the position corresponding to position 90 of the amino acid sequence of SEQ ID NO:2 is replaced with C, V at the position corresponding to position 181 of the amino acid sequence of SEQ ID NO:2 is replaced with L, or at least two (preferably three, more preferably four) amino acid residues selected from F at the position corresponding to position 31, E at the position corresponding to position 32, A at the position corresponding to position 90, and V at the position corresponding to position 181 of the amino acid sequence of SEQ ID NO:2 are replaced with Y at the position corresponding to position 31, G at the position corresponding to position 32, C at the position corresponding to position 90, and L at the position corresponding to position 181. More preferably, in the parent lipase, E at position corresponding to position 32 of the amino acid sequence of SEQ ID NO:2 is replaced with G, A at position corresponding to position 90 of the amino acid sequence of SEQ ID NO:2 is replaced with C, V at position corresponding to position 181 of the amino acid sequence of SEQ ID NO:2 is replaced with L, F and E at positions corresponding to positions 31 and 32 of the amino acid sequence of SEQ ID NO:2 are replaced with Y and G, respectively, A and V at positions corresponding to positions 90 and 181 of the amino acid sequence of SEQ ID NO:2 are replaced with C and L, respectively, or E and A at positions corresponding to positions 32 and 90 of the amino acid sequence of SEQ ID NO:2 are replaced with G and C, respectively, or F, E, A and V at positions corresponding to positions 31, 32, 90 and 181 of the amino acid sequence of SEQ ID NO:2 are replaced with Y, G, C and L, respectively.

[0033] Another example of the parent lipase is a polypeptide having lipase activity and consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 6. Here, the polypeptide having lipase activity and consisting of the amino acid sequence of SEQ ID NO: 6 is lipase EtLip (NCBI Accession No. WP_115457195.1) derived from Enterobacillus tribolii. The parent lipase, which is a polypeptide having lipase activity and consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 6, preferably has I at the position corresponding to position 222 of the amino acid sequence of SEQ ID NO: 2.

[0034] When the lipase variant of the present invention is produced from the parent lipase, the amino acid residue at the position corresponding to position 222 of the amino acid sequence of SEQ ID NO:2 in the parent lipase is substituted with A, T, H or G.

[0035] More specifically, when producing the lipase variant of the present invention from the parent lipase, I at the position corresponding to position 222 of the amino acid sequence of SEQ ID NO:2 in the parent lipase is replaced with A, T, H or G.

[0036] Another example of the parent lipase is a polypeptide having lipase activity and consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 8. Here, the polypeptide having lipase activity and consisting of the amino acid sequence of SEQ ID NO: 8 is lipase YeLip (NCBI Accession No. WP_064517898.1) derived from Yersinia entomophaga. The parent lipase, which is a polypeptide having lipase activity and consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 8, preferably has V at the position corresponding to position 39 of the amino acid sequence of SEQ ID NO: 2, V at the position corresponding to position 125 of the amino acid sequence of SEQ ID NO: 2, E at the position corresponding to position 126 of the amino acid sequence of SEQ ID NO: 2, or S at the position corresponding to position 293 of the amino acid sequence of SEQ ID NO: 2, and more preferably has V and E at the positions corresponding to positions 125 and 126 of the amino acid sequence of SEQ ID NO: 2, or S at the positions corresponding to positions 39 and 293 of the amino acid sequence of SEQ ID NO: 2. and 293 of the amino acid sequence of SEQ ID NO:2 are more preferred, those having V, E, and S at positions 125, 126, and 293, respectively, those having V, V, and E at positions 39, 125, and 126, respectively, of the amino acid sequence of SEQ ID NO:2 are even more preferred, and those having V, E, and S at positions 39, 126, and 293, respectively, of the amino acid sequence of SEQ ID NO:2 are even more preferred, and those having V, V, E, and S at positions 39, 125, 126, and 293, respectively, of the amino acid sequence of SEQ ID NO:2 are even more preferred.

[0037] When producing a lipase variant of the present invention from the parent lipase, preferably, in the parent lipase, the amino acid residue at the position corresponding to position 39 of the amino acid sequence of SEQ ID NO:2 is substituted with K, the amino acid residue at the position corresponding to position 125 of the amino acid sequence of SEQ ID NO:2 is substituted with T, the amino acid residue at the position corresponding to position 126 of the amino acid sequence of SEQ ID NO:2 is substituted with P, the amino acid residue at the position corresponding to position 293 of the amino acid sequence of SEQ ID NO:2 is substituted with G, or at least two (preferably three, more preferably four) amino acid residues selected from positions corresponding to positions 39, 125, 126 and 293 of the amino acid sequence of SEQ ID NO:2 are substituted with K at the position corresponding to position 39, T at the position corresponding to position 125, P at the position corresponding to position 126 and G at the position corresponding to position 293. More preferably, in the parent lipase, the amino acid residue at position 39 of the amino acid sequence of SEQ ID NO:2 is substituted with K, the amino acid residue at position 126 of the amino acid sequence of SEQ ID NO:2 is substituted with P, the amino acid residue at position 293 of the amino acid sequence of SEQ ID NO:2 is substituted with G, the amino acid residues at positions 125 and 126 of the amino acid sequence of SEQ ID NO:2 are substituted with T and P, respectively, or the amino acid residues at positions 39 and 293 of the amino acid sequence of SEQ ID NO:2 are substituted with K and G, respectively; The amino acid residues at positions corresponding to 125, 126 and 293 of the amino acid sequence of SEQ ID NO:2 are substituted with T, P and G, respectively; the amino acid residues at positions corresponding to 39, 125 and 126 of the amino acid sequence of SEQ ID NO:2 are substituted with K, T and P, respectively; the amino acid residues at positions corresponding to 39, 126 and 293 of the amino acid sequence of SEQ ID NO:2 are substituted with K, P and G, respectively; or the amino acid residues at positions corresponding to 39, 125, 126 and 293 of the amino acid sequence of SEQ ID NO:2 are substituted with K, T, P and G, respectively.

[0038] More specifically, when producing a lipase variant of the present invention from the parent lipase, preferably, in the parent lipase, V at the position corresponding to position 39 of the amino acid sequence of SEQ ID NO:2 is replaced with K, V at the position corresponding to position 125 of the amino acid sequence of SEQ ID NO:2 is replaced with T, E at the position corresponding to position 126 of the amino acid sequence of SEQ ID NO:2 is replaced with P, or S at the position corresponding to position 293 of the amino acid sequence of SEQ ID NO:2 is replaced with G, or at least two (preferably three, more preferably four) amino acid residues selected from V at the position corresponding to position 39, V at the position corresponding to position 125, E at the position corresponding to position 126, and S at the position corresponding to position 293 of the amino acid sequence of SEQ ID NO:2 are replaced with K at the position corresponding to position 39, T at the position corresponding to position 125, P at the position corresponding to position 126, and G at the position corresponding to position 293. More preferably, in the parent lipase, V at position corresponding to position 39 of the amino acid sequence of SEQ ID NO:2 is replaced by K, E at position corresponding to position 126 of the amino acid sequence of SEQ ID NO:2 is replaced by P, S at position corresponding to position 293 of the amino acid sequence of SEQ ID NO:2 is replaced by G, V and E at positions corresponding to positions 125 and 126 of the amino acid sequence of SEQ ID NO:2 are replaced by T and P, respectively, V and S at positions corresponding to positions 39 and 293 of the amino acid sequence of SEQ ID NO:2 are replaced by K and G, respectively, or the parent lipase is replaced by a 5'-amino acid sequence of SEQ ID NO:2. or the V, E, and S at positions corresponding to positions 39, 125, and 126 of the amino acid sequence of SEQ ID NO:2 are replaced with K, T, and P, respectively; the V, E, and S at positions corresponding to positions 39, 126, and 293 of the amino acid sequence of SEQ ID NO:2 are replaced with K, P, and G, respectively; or the V, E, and S at positions corresponding to positions 39, 125, 126, and 293 of the amino acid sequence of SEQ ID NO:2 are replaced with K, P, and G, respectively; or the V, V, E, and S at positions corresponding to positions 39, 125, 126, and 293 of the amino acid sequence of SEQ ID NO:2 are replaced with K, T, P, and G, respectively.

[0039] Another example of the parent lipase is a polypeptide having lipase activity and consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 10. Here, the polypeptide having lipase activity and consisting of the amino acid sequence of SEQ ID NO: 10 is lipase AnLip (NCBI Accession No. WP_026822497.1) derived from Arsenophonus nasoniae. The parent lipase, which is a polypeptide having lipase activity and which consists of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO:10, preferably has C at the position corresponding to position 29 of the amino acid sequence of SEQ ID NO:2, F at the position corresponding to position 31 of the amino acid sequence of SEQ ID NO:2, A at the position corresponding to position 32 of the amino acid sequence of SEQ ID NO:2, or R at the position corresponding to position 186 of the amino acid sequence of SEQ ID NO:2, more preferably has C and R at the positions corresponding to positions 29 and 186 of the amino acid sequence of SEQ ID NO:2, and even more preferably has C, F, A and R at the positions corresponding to positions 29, 31, 32 and 186 of the amino acid sequence of SEQ ID NO:2, respectively.

[0040] When producing a lipase variant of the present invention from the parent lipase, preferably, in the parent lipase, the amino acid residue at the position corresponding to position 29 of the amino acid sequence of SEQ ID NO:2 is substituted with Y, the amino acid residue at the position corresponding to position 31 of the amino acid sequence of SEQ ID NO:2 is substituted with Y, the amino acid residue at the position corresponding to position 32 of the amino acid sequence of SEQ ID NO:2 is substituted with G, the amino acid residue at the position corresponding to position 186 of the amino acid sequence of SEQ ID NO:2 is substituted with G, or the amino acid residue at at least two (preferably three, more preferably four) positions selected from the positions corresponding to positions 29, 31, 32 and 186 of the amino acid sequence of SEQ ID NO:2 is substituted with Y at the position corresponding to position 29, Y at the position corresponding to position 31, G at the position corresponding to position 32 and G at the position corresponding to position 186. More preferably, in the parent lipase, the amino acid residue at position corresponding to position 29 of the amino acid sequence of SEQ ID NO:2 is substituted with Y, the amino acid residue at position corresponding to position 186 of the amino acid sequence of SEQ ID NO:2 is substituted with G, the amino acid residues at positions corresponding to positions 29 and 186 of the amino acid sequence of SEQ ID NO:2 are substituted with Y and G, respectively, or the amino acid residues at positions corresponding to positions 29, 31, 32 and 186 of the amino acid sequence of SEQ ID NO:2 are substituted with Y, Y, G and G, respectively.

[0041] More specifically, when producing a lipase variant of the present invention from the parent lipase, preferably, in the parent lipase, C at position corresponding to position 29 of the amino acid sequence of SEQ ID NO:2 is replaced with Y, F at position corresponding to position 31 of the amino acid sequence of SEQ ID NO:2 is replaced with Y, A at position corresponding to position 32 of the amino acid sequence of SEQ ID NO:2 is replaced with G, or R at position corresponding to position 186 of the amino acid sequence of SEQ ID NO:2 is replaced with G, or at least two (preferably three, more preferably four) amino acid residues selected from C at position corresponding to position 29, F at position corresponding to position 31, A at position corresponding to position 32, and R at position corresponding to position 186 of the amino acid sequence of SEQ ID NO:2 are replaced with Y at position corresponding to position 29, Y at position corresponding to position 31, G at position corresponding to position 32, and G at position corresponding to position 186. More preferably, in the parent lipase, C at position corresponding to position 29 of the amino acid sequence of SEQ ID NO:2 is replaced with Y, R at position corresponding to position 186 of the amino acid sequence of SEQ ID NO:2 is replaced with G, C and R at positions corresponding to positions 29 and 186 of the amino acid sequence of SEQ ID NO:2 are replaced with Y and G, respectively, or C, F, A and R at positions corresponding to positions 29, 31, 32 and 186 of the amino acid sequence of SEQ ID NO:2 are replaced with Y, Y, G and G, respectively.

[0042] An example of the parent lipase of the lipase variant of the present invention is a polypeptide having lipase activity and consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 12. Here, the polypeptide having lipase activity and consisting of the amino acid sequence of SEQ ID NO: 12 is lipase YfLip (NCBI Accession No. WP_145530745.1) derived from Yersinia frederiksenii. The parent lipase, which is a polypeptide having lipase activity and consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 12, preferably has G at the position corresponding to position 206 of the amino acid sequence of SEQ ID NO: 2.

[0043] When the lipase variant of the present invention is produced from the parent lipase, the amino acid residue at the position corresponding to position 206 in the amino acid sequence of SEQ ID NO:2 in the parent lipase is substituted with Q.

[0044] More specifically, when producing the lipase variant of the present invention from the parent lipase, G at the position corresponding to position 206 of the amino acid sequence of SEQ ID NO:2 in the parent lipase is substituted with Q.

[0045] Another example of a parent lipase is a polypeptide having lipase activity and consisting of an amino acid sequence having at least 80, 83 or 97% identity to the amino acid sequence of SEQ ID NO: 14. Here, the polypeptide having lipase activity and consisting of the amino acid sequence of SEQ ID NO: 14 is lipase PspLip derived from Pseudomonas sp. (NCBI Accession No. WP_056840927.1). The parent lipase, which is a polypeptide having lipase activity and which consists of an amino acid sequence having at least the above-mentioned predetermined identity with the amino acid sequence of SEQ ID NO:14, is preferably one having F at a position corresponding to position 31 of the amino acid sequence of SEQ ID NO:2, one having D at a position corresponding to position 32 of the amino acid sequence of SEQ ID NO:2, one having R at a position corresponding to position 77 of the amino acid sequence of SEQ ID NO:2, or one having M at a position corresponding to position 181 of the amino acid sequence of SEQ ID NO:2, more preferably one having F and D at positions corresponding to positions 31 and 32 of the amino acid sequence of SEQ ID NO:2, or one having R and M at positions corresponding to positions 77 and 181 of the amino acid sequence of SEQ ID NO:2, even more preferably one having F, D and M at positions corresponding to positions 31, 32 and 181 of the amino acid sequence of SEQ ID NO:2, and even more preferably one having F, D, R and M at positions corresponding to positions 31, 32, 77 and 181 of the amino acid sequence of SEQ ID NO:2,

[0046] When producing a lipase variant of the present invention from the parent lipase, preferably, in the parent lipase, the amino acid residue at the position corresponding to position 31 of the amino acid sequence of SEQ ID NO:2 is substituted with Y, the amino acid residue at the position corresponding to position 32 of the amino acid sequence of SEQ ID NO:2 is substituted with G, the amino acid residue at the position corresponding to position 77 of the amino acid sequence of SEQ ID NO:2 is substituted with K, the amino acid residue at the position corresponding to position 181 of the amino acid sequence of SEQ ID NO:2 is substituted with L, or at least two (preferably three, more preferably four) amino acid residues selected from positions corresponding to positions 31, 32, 77, and 181 of the amino acid sequence of SEQ ID NO:2 are substituted with Y at the position corresponding to position 31, G at the position corresponding to position 32, K at the position corresponding to position 77, and L at the position corresponding to position 181. More preferably, in the parent lipase, the amino acid residue at position 77 of the amino acid sequence of SEQ ID NO:2 is substituted with K; the amino acid residues at positions 31 and 32 of the amino acid sequence of SEQ ID NO:2 are substituted with Y and G, respectively; the amino acid residues at positions 77 and 181 of the amino acid sequence of SEQ ID NO:2 are substituted with K and L, respectively; the amino acid residues at positions 31, 32 and 181 of the amino acid sequence of SEQ ID NO:2 are substituted with Y, G and L, respectively; or the amino acid residues at positions 31, 32, 77 and 181 of the amino acid sequence of SEQ ID NO:2 are substituted with Y, G, K and L, respectively.

[0047] More specifically, when producing the lipase variant of the present invention from the parent lipase, preferably, in the parent lipase, F at the position corresponding to position 31 of the amino acid sequence of SEQ ID NO:2 is replaced with Y, D at the position corresponding to position 32 of the amino acid sequence of SEQ ID NO:2 is replaced with G, R at the position corresponding to position 77 of the amino acid sequence of SEQ ID NO:2 is replaced with K, or M at the position corresponding to position 181 of the amino acid sequence of SEQ ID NO:2 is replaced with L, or at least two (preferably three, more preferably four) amino acid residues selected from F at the position corresponding to position 31, D at the position corresponding to position 32, R at the position corresponding to position 77, and M at the position corresponding to position 181 of the amino acid sequence of SEQ ID NO:2 are replaced with Y at the position corresponding to position 31, G at the position corresponding to position 32, K at the position corresponding to position 77, and L at the position corresponding to position 181. More preferably, in the parent lipase, R at position corresponding to position 77 of the amino acid sequence of SEQ ID NO:2 is replaced with K, F and D at positions corresponding to positions 31 and 32 of the amino acid sequence of SEQ ID NO:2 are replaced with Y and G, respectively, R and M at positions corresponding to positions 77 and 181 of the amino acid sequence of SEQ ID NO:2 are replaced with K and L, respectively, F, D and M at positions corresponding to positions 31, 32 and 181 of the amino acid sequence of SEQ ID NO:2 are replaced with Y, G and L, respectively, or F, D, R and M at positions corresponding to positions 31, 32, 77 and 181 of the amino acid sequence of SEQ ID NO:2 are replaced with Y, G, K and L, respectively.

[0048] Another example of the parent lipase is a polypeptide having lipase activity and consisting of an amino acid sequence having at least 80 or 81% identity with the amino acid sequence of SEQ ID NO: 16. Here, the polypeptide having lipase activity and consisting of the amino acid sequence of SEQ ID NO: 16 is lipase PspLip2 (NCBI Accession No. WP_088424928.1) derived from Pseudomonas sp. The parent lipase, which is a polypeptide having lipase activity and consisting of an amino acid sequence having at least the above-mentioned predetermined identity with the amino acid sequence of SEQ ID NO: 16, preferably has A at the position corresponding to position 90 of the amino acid sequence of SEQ ID NO: 2, or has V at the position corresponding to position 181 of the amino acid sequence of SEQ ID NO: 2, and more preferably has A and V at the positions corresponding to positions 90 and 181 of the amino acid sequence of SEQ ID NO: 2, respectively.

[0049] When producing a lipase variant of the present invention from the parent lipase, preferably, in the parent lipase, the amino acid residue at the position corresponding to position 90 in the amino acid sequence of SEQ ID NO:2 is substituted with C, the amino acid residue at the position corresponding to position 181 in the amino acid sequence of SEQ ID NO:2 is substituted with L, or the amino acid residues at positions corresponding to positions 90 and 181 in the amino acid sequence of SEQ ID NO:2 are substituted with C and L, respectively.

[0050] More specifically, when producing the lipase variant of the present invention from the parent lipase, preferably, in the parent lipase, A at the position corresponding to position 90 of the amino acid sequence of SEQ ID NO:2 is replaced with C, V at the position corresponding to position 181 of the amino acid sequence of SEQ ID NO:2 is replaced with L, or A and V at the positions corresponding to positions 90 and 181 of the amino acid sequence of SEQ ID NO:2 are replaced with C and L, respectively.

[0051] Another example of the parent lipase is a polypeptide having lipase activity and consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 34. Here, the polypeptide having lipase activity and consisting of the amino acid sequence of SEQ ID NO: 16 is lipase PaLip (NCBI Accession No. WP_062381422.1) derived from Pseudomonas abietaniphila. The parent lipase, which is a polypeptide having lipase activity and consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 16, preferably has M at the position corresponding to position 29 of the amino acid sequence of SEQ ID NO: 2.

[0052] When the lipase variant of the present invention is produced from the parent lipase, the amino acid residue at the position corresponding to position 29 in the amino acid sequence of SEQ ID NO:2 in the parent lipase is substituted with Y.

[0053] More specifically, when the lipase variant of the present invention is produced from the parent lipase, M at the position corresponding to position 29 of the amino acid sequence of SEQ ID NO:2 in the parent lipase is replaced with Y.

[0054] The present invention also provides a lipase variant, which is a polypeptide having lipase activity and consisting of an amino acid sequence in which an amino acid residue at a predetermined position, as numbered based on SEQ ID NO: 2, in the amino acid sequence of a parent lipase is substituted with a predetermined amino acid residue.

[0055] Specifically, the lipase variant of the present invention is any one of lipase variants selected from the following (a) to (v) and (aa) to (ae): (a) a lipase variant consisting of an amino acid sequence having at least 82% identity to the amino acid sequence of SEQ ID NO:2, wherein the lipase variant has D at the position corresponding to position 118 of the amino acid sequence of SEQ ID NO:2; (b) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:4, and having a Y at the position corresponding to position 31 of the amino acid sequence of SEQ ID NO:2; (c) a lipase variant consisting of an amino acid sequence having at least 91% identity to the amino acid sequence of SEQ ID NO:4, and having a G at the position corresponding to position 32 of the amino acid sequence of SEQ ID NO:2; (d) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:4 and having a C at the position corresponding to position 90 of the amino acid sequence of SEQ ID NO:2; (e) a lipase variant consisting of an amino acid sequence having at least 94% identity to the amino acid sequence of SEQ ID NO:4, and having an L at the position corresponding to position 181 of the amino acid sequence of SEQ ID NO:2; (f) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:6, and having A, T, H or G at the position corresponding to position 222 of the amino acid sequence of SEQ ID NO:2; (g) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8, and having a K at the position corresponding to position 39 of the amino acid sequence of SEQ ID NO:2; (h) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8, and having a T at the position corresponding to position 125 of the amino acid sequence of SEQ ID NO:2; (i) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8, and having a P at the position corresponding to position 126 of the amino acid sequence of SEQ ID NO:2; (j) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8, and having G at the position corresponding to position 293 of the amino acid sequence of SEQ ID NO:2; (k) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 10, and having a Y at the position corresponding to position 29 of the amino acid sequence of SEQ ID NO: 2; (l) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 10, and having a Y at the position corresponding to position 31 of the amino acid sequence of SEQ ID NO: 2; (m) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 10, and having G at the position corresponding to position 32 of the amino acid sequence of SEQ ID NO: 2; (n) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 10, and having G at the position corresponding to position 186 of the amino acid sequence of SEQ ID NO: 2; (o) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 12, and having a Q at a position corresponding to position 206 of the amino acid sequence of SEQ ID NO: 2; (p) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 14, and having a Y at the position corresponding to position 31 of the amino acid sequence of SEQ ID NO: 2; (q) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 14, and having G at the position corresponding to position 32 of the amino acid sequence of SEQ ID NO: 2; (r) a lipase variant consisting of an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 14, and having a K at the position corresponding to position 77 of the amino acid sequence of SEQ ID NO: 2; (s) a lipase variant consisting of an amino acid sequence having at least 83% identity to the amino acid sequence of SEQ ID NO: 14, and having an L at a position corresponding to position 181 of the amino acid sequence of SEQ ID NO: 2; (t) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 16 and having a C at the position corresponding to position 90 of the amino acid sequence of SEQ ID NO: 2; (u) a lipase variant consisting of an amino acid sequence having at least 81% identity to the amino acid sequence of SEQ ID NO: 16, and having an L at a position corresponding to position 181 of the amino acid sequence of SEQ ID NO: 2; (v) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 34, and having a Y at the position corresponding to position 29 of the amino acid sequence of SEQ ID NO: 2; (aa) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 4, and having at least two amino acid residues selected from Y at the position corresponding to position 31, G at the position corresponding to position 32, C at the position corresponding to position 90, and L at the position corresponding to position 181 of the amino acid sequence of SEQ ID NO: 2; (ab) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8, and having at least two amino acid residues selected from K at the position corresponding to position 39, T at the position corresponding to position 125, P at the position corresponding to position 126, and G at the position corresponding to position 293 of the amino acid sequence of SEQ ID NO:2; (ac) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 10, and having at least two amino acid residues selected from Y at the position corresponding to position 29, Y at the position corresponding to position 31, G at the position corresponding to position 32, and G at the position corresponding to position 186 of the amino acid sequence of SEQ ID NO: 2; (ad) a lipase variant consisting of an amino acid sequence having at least 83% identity to the amino acid sequence of SEQ ID NO: 14, and having at least two amino acid residues selected from Y at the position corresponding to position 31, G at the position corresponding to position 32, K at the position corresponding to position 77, and L at the position corresponding to position 181 of the amino acid sequence of SEQ ID NO: 2; (ae) A lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 16, and having a C at the position corresponding to position 90 and an L at the position corresponding to position 181 of the amino acid sequence of SEQ ID NO: 2.

[0056] Among the lipase variants (a) to (v), preferred lipase variants are the lipase variants (a), (c) to (g), (i) to (k), (n), (o), (r) and (t) to (v).

[0057] The lipase variant (aa) is preferably the lipase variants (aa-1) to (aa-4) shown below, the lipase variant (ab) is preferably the lipase variants (ab-1) to (ab-6) shown below, the lipase variant (ac) is preferably the lipase variants (ac-1) to (ac-2) shown below, and the lipase variant (ad) is preferably the lipase variants (ad-1) to (ad-4) shown below. (aa-1) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 4, and having a Y at the position corresponding to position 31 and a G at the position corresponding to position 32 of the amino acid sequence of SEQ ID NO: 2; (aa-2) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 4, and having C at the position corresponding to position 90 and L at the position corresponding to position 181 of the amino acid sequence of SEQ ID NO: 2; (aa-3) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 4, and having G at the position corresponding to position 32 and C at the position corresponding to position 90 of the amino acid sequence of SEQ ID NO: 2; (aa-4) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:4, and having Y at the position corresponding to position 31, G at the position corresponding to position 32, C at the position corresponding to position 90, and L at the position corresponding to position 181 of the amino acid sequence of SEQ ID NO:2; (ab-1) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8, and having a T at the position corresponding to position 125 and a P at the position corresponding to position 126 of the amino acid sequence of SEQ ID NO:2; (ab-2) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8, and having K at the position corresponding to position 39 and G at the position corresponding to position 293 of the amino acid sequence of SEQ ID NO:2; (ab-3) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8, and having a T at a position corresponding to position 125, a P at a position corresponding to position 126, and a G at a position corresponding to position 293 of the amino acid sequence of SEQ ID NO:2; (ab-4) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8, and having K at the position corresponding to position 39, T at the position corresponding to position 125, and P at the position corresponding to position 126 of the amino acid sequence of SEQ ID NO:2; (ab-5) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8, and having K at a position corresponding to position 39, P at a position corresponding to position 126, and G at a position corresponding to position 293 of the amino acid sequence of SEQ ID NO:2; (ab-6) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8, and having K at the position corresponding to position 39, T at the position corresponding to position 125, P at the position corresponding to position 126, and G at the position corresponding to position 293 of the amino acid sequence of SEQ ID NO:2; (ac-1) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 10, and having a Y at a position corresponding to position 29 and a G at a position corresponding to position 186 of the amino acid sequence of SEQ ID NO: 2; (ac-2) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 10, or having a Y at a position corresponding to position 29, a Y at a position corresponding to position 31, a G at a position corresponding to position 32, and a G at a position corresponding to position 186 of the amino acid sequence of SEQ ID NO: 2; (ad-1) a lipase variant consisting of an amino acid sequence having at least 83% identity with the amino acid sequence of SEQ ID NO: 14, and having a Y at the position corresponding to position 31 and a G at the position corresponding to position 32 of the amino acid sequence of SEQ ID NO: 2; (ad-2) a lipase variant consisting of an amino acid sequence having at least 83% identity to the amino acid sequence of SEQ ID NO: 14, and having K at a position corresponding to position 77 and L at a position corresponding to position 181 of the amino acid sequence of SEQ ID NO: 2; (ad-3) a lipase variant consisting of an amino acid sequence having at least 83% identity to the amino acid sequence of SEQ ID NO: 14, and having Y at the position corresponding to position 31, G at the position corresponding to position 32, and L at the position corresponding to position 181 of the amino acid sequence of SEQ ID NO: 2; (ad-4) A lipase variant consisting of an amino acid sequence having at least 83% identity to the amino acid sequence of SEQ ID NO:14, and having Y at the position corresponding to position 31, G at the position corresponding to position 32, K at the position corresponding to position 77, and L at the position corresponding to position 181 of the amino acid sequence of SEQ ID NO:2.

[0058] The substitution of amino acid residues at the above-mentioned specific positions is a substitution for improving the heterologous expressibility of the lipase, and therefore, the lipase variant of the present invention has improved expressibility in heterologous species compared to the parent lipase, i.e., the lipase before the substitution of the amino acid residues at the specific positions.

[0059] In the lipase variants of the present invention, the substitution sites (mutation positions) of amino acid residues are numbered based on SEQ ID NO: 2. In Table 1 below, the substitution sites of amino acid residues for each parent lipase are numbered based on the amino acid sequence of each parent lipase and based on the amino acid sequence of SEQ ID NO: 2.

[0060] [Table 1]

[0061] As shown in Table 1, the substitution of the amino acid residue at position 29 of the amino acid sequence of SEQ ID NO:2 with Y is a common substitution when the parent lipase is a polypeptide having lipase activity and has an amino acid sequence having at least the above-mentioned predetermined identity with SEQ ID NO:10 or 34. The substitution of the amino acid residue at position 31 of the amino acid sequence of SEQ ID NO:2 with Y is a common substitution when the parent lipase is a polypeptide having lipase activity and has an amino acid sequence having at least the above-mentioned predetermined identity with SEQ ID NO:4, 10 or 14. The substitution of the amino acid residue at position 32 of the amino acid sequence of SEQ ID NO:2 with G is a common substitution when the parent lipase is a polypeptide having lipase activity and has an amino acid sequence having at least the above-mentioned predetermined identity with SEQ ID NO:4, 10 or 14. The substitution of the amino acid residue at position 90 of the amino acid sequence of SEQ ID NO:2 with C is a common substitution when the parent lipase is a polypeptide having lipase activity and has an amino acid sequence having at least the above-mentioned predetermined identity with SEQ ID NO:4 or 16. The substitution of the amino acid residue at the position corresponding to position 181 of the amino acid sequence of SEQ ID NO: 2 with L is a common substitution when the parent lipase is a polypeptide having an amino acid sequence having at least the above-mentioned specified identity with SEQ ID NO: 4, 14 or 16 and having lipase activity.

[0062] In addition to the mutations at the above-mentioned predetermined positions, the lipase variant of the present invention may have a mutation (e.g., deletion, substitution, addition, insertion) at any other position relative to the parent lipase, so long as it does not interfere with its heterologous expression. The mutation may be naturally occurring or artificially introduced.

[0063] <2. Polynucleotide encoding the lipase variant of the present invention> The lipase variant of the present invention can be produced by using various mutagenesis techniques known in the art, for example, by mutating a polynucleotide encoding an amino acid residue to be substituted in a parent lipase gene (reference lipase gene) encoding the reference amino acid sequence to a polynucleotide encoding the substituted amino acid residue, and then expressing the variant from the mutant gene.

[0064] The polynucleotide encoding the lipase variant of the present invention may be in the form of single- or double-stranded DNA, RNA, or an artificial nucleic acid, or may be cDNA, or chemically synthesized DNA containing no introns.

[0065] In the present invention, various mutation introduction techniques known in the art can be used as a means for mutating amino acid residues of a parent lipase. For example, in a polynucleotide encoding the amino acid sequence of a parent lipase (hereinafter also referred to as a parent gene), a nucleotide sequence encoding an amino acid residue to be mutated is mutated to a nucleotide sequence encoding the mutated amino acid residue, thereby obtaining a polynucleotide encoding a lipase variant of the present invention.

[0066] The introduction of a desired mutation into a parent gene can basically be carried out using various site-directed mutagenesis methods well known to those skilled in the art. Site-directed mutagenesis can be carried out by any method, such as inverse PCR or annealing. Commercially available site-directed mutagenesis kits (e.g., Stratagene's QuickChange II Site-Directed Mutagenesis Kit, QuickChange Multi Site-Directed Mutagenesis Kit, etc.) can also be used.

[0067] Site-specific mutagenesis of a parent gene can be most commonly performed using a mutagenesis primer containing the nucleotide mutation to be introduced. The mutagenesis primer can be designed to anneal to a region containing a nucleotide sequence encoding the amino acid residue to be mutated in the parent gene, and to contain a nucleotide sequence having a nucleotide sequence (codon) encoding the mutated amino acid residue instead of the nucleotide sequence (codon) encoding the amino acid residue to be mutated. Those skilled in the art can appropriately recognize and select the nucleotide sequences (codons) encoding the amino acid residues before and after the mutation based on ordinary textbooks, etc. Alternatively, site-specific mutagenesis can be performed by a method in which DNA fragments obtained by amplifying the upstream and downstream sides of the mutation site using two complementary primers containing the nucleotide mutation to be introduced separately are linked together by SOE (splicing by overlap extension)-PCR (Gene, 1989, 77(1): p61-68).

[0068] The template DNA containing the parent gene can be prepared by extracting genomic DNA from the above-mentioned microorganisms that produce lipase, or by extracting RNA and synthesizing cDNA by reverse transcription. Alternatively, a corresponding nucleotide sequence may be chemically synthesized based on the amino acid sequence of the parent lipase and used as the template DNA. DNA sequences containing a base sequence encoding lipases consisting of the amino acid sequences shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 and 34 are shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15 and 33, respectively.

[0069] The mutation primer can be prepared by a well-known oligonucleotide synthesis method such as the phosphoramidite method (Nucleic Acids Research, 1989, 17:7059-7071). Such primer synthesis can also be performed using, for example, a commercially available oligonucleotide synthesizer (such as that manufactured by ABI). A primer set including the mutation primer is used to perform site-specific mutagenesis as described above using a parent gene as a template DNA, thereby obtaining a polynucleotide encoding the lipase mutant of the present invention having the desired mutation.

[0070] The polynucleotide encoding the lipase variant of the present invention may comprise single-stranded or double-stranded DNA, cDNA, RNA or other artificial nucleic acid. The DNA, cDNA and RNA may be chemically synthesized. The polynucleotide may comprise a nucleotide sequence of an untranslated region (UTR) in addition to an open reading frame (ORF). The polynucleotide may be codon-optimized according to the species of the transformant used to produce the mutant polypeptide of the present invention. Information on codons used by various organisms is available from the Codon Usage Database ([www.kazusa.or.jp / codon / ]).

[0071] <3. Vector or DNA fragment> The obtained polynucleotide encoding the lipase variant of the present invention can be incorporated into a vector. The type of vector containing the polynucleotide is not particularly limited and may be any vector, such as a plasmid, a phage, a phagemid, a cosmid, a virus, a YAC vector, or a shuttle vector. The vector is preferably, but not limited to, a vector that can be amplified in bacteria, preferably in Bacillus bacteria (e.g., Bacillus subtilis or a mutant thereof), and more preferably an expression vector that can induce expression of an introduced gene in Bacillus bacteria. Among them, a shuttle vector that is a vector that can be replicated in both Bacillus bacteria and other organisms can be suitably used for recombinantly producing the lipase variant of the present invention. Preferred examples of the vector include, but are not limited to, shuttle vectors such as pHA3040SP64, pHSP64R or pASP64 (Patent No. 3492935), pHY300PLK (an expression vector capable of transforming both Escherichia coli and Bacillus subtilis; Jpn J Genet, 1985, 60:235-243), and pAC3 (Nucleic Acids Res, 1988, 16:8732); and plasmid vectors that can be used for transformation of bacteria of the genus Bacillus, such as pUB110 (J Bacteriol, 1978, 134:318-329) and pTA10607 (Plasmid, 1987, 18:8-15). Plasmid vectors derived from E. coli (e.g., pET22b(+), pBR322, pBR325, pUC57, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.) can also be used.

[0072] The vector may contain a DNA replication origin region or a DNA region containing a replication origin. Alternatively, the vector may have a control sequence, such as a promoter region for initiating transcription of the gene, a terminator region, or a secretion signal region for secreting the expressed protein outside the cell, operably linked upstream of a polynucleotide encoding the lipase variant of the present invention (i.e., the lipase variant gene).

[0073] The types of the control sequences such as the promoter region, terminator region, and secretion signal region are not particularly limited, and promoters and secretion signal sequences that are commonly used can be appropriately selected and used depending on the host to be introduced. For example, suitable examples of the control sequences that can be incorporated into the vector include the promoter and secretion signal sequence of the cellulase gene of Bacillus sp. KSM-S237 strain.

[0074] Alternatively, the vector of the present invention may further incorporate a marker gene (e.g., a resistance gene to a drug such as ampicillin, neomycin, kanamycin, or chloramphenicol) for selecting a host into which the vector has been appropriately introduced. Alternatively, when an auxotrophic strain is used as a host, a gene encoding an enzyme for synthesizing the required nutrient may be incorporated as a marker gene into the vector. Furthermore, when a selective medium requiring a specific metabolism for growth is used, a gene related to the metabolism may be incorporated as a marker gene into the vector. An example of such a metabolism-related gene is the acetamidase gene for utilizing acetamide as a nitrogen source.

[0075] The polynucleotide encoding the lipase variant of the present invention can be ligated to a control sequence and a marker gene by a method known in the art, such as splicing by overlap extension (SOE)-PCR (Gene, 1989, 77:61-68). The procedure for introducing the ligated fragment into a vector is well known in the art.

[0076] 4. Transformed cells The transformed cell of the present invention can be obtained by introducing a vector containing a polynucleotide encoding the lipase variant of the present invention into a host, or by introducing a DNA fragment containing a polynucleotide encoding the lipase variant of the present invention into the genome of the host.

[0077] Examples of host cells include microorganisms such as bacteria and filamentous fungi. Examples of bacteria include bacteria belonging to the genera Escherichia coli, Staphylococcus, Enterococcus, Listeria, and Bacillus, among which Escherichia coli and Bacillus are preferred, Bacillus are more preferred, and Bacillus subtilis (e.g., Bacillus subtilis Marburg No. 168 (Bacillus subtilis 168 strain) or a mutant thereof) are even more preferred. Examples of Bacillus subtilis mutants include the KA8AX, a nine-fold protease deletion strain described in J. Biosci. Bioeng., 2007, 104(2):135-143, and the D8PA strain, an eight-fold protease deletion strain with improved protein folding efficiency described in Biotechnol. Lett., 2011, 33(9):1847-1852. Examples of filamentous fungi include the genera Trichoderma, Aspergillus, and Rhizopus.

[0078] The vector can be introduced into the host by a method commonly used in the art, such as the protoplast method, electroporation, etc. A strain into which the vector has been appropriately introduced can be selected based on the expression of a marker gene, nutritional requirements, etc., to obtain a desired transformant into which the vector has been introduced.

[0079] Alternatively, a fragment in which a polynucleotide encoding the lipase variant of the present invention, a control sequence, and a marker gene are linked can be directly introduced into the genome of a host. For example, a DNA fragment in which sequences complementary to the host genome are added to both ends of the linked fragment is constructed by SOE-PCR or the like, and this is introduced into a host to cause homologous recombination between the host genome and the DNA fragment, thereby introducing the polynucleotide encoding the lipase variant of the present invention into the genome of the host.

[0080] When the thus obtained transformant into which a polynucleotide encoding the lipase variant of the present invention or a vector containing the same has been introduced is cultured in an appropriate medium, the gene encoding the protein on the vector is expressed to produce the lipase variant of the present invention. The medium used for culturing the transformant can be appropriately selected by those skilled in the art depending on the type of microorganism used as the transformant.

[0081] Alternatively, the lipase variant of the present invention may be expressed from a polynucleotide encoding the lipase variant of the present invention or a transcription product thereof using a cell-free translation system. The "cell-free translation system" refers to an in vitro transcription / translation system or an in vitro translation system that is prepared by adding reagents such as amino acids required for protein translation to a suspension obtained by mechanically disrupting host cells.

[0082] The lipase variant of the present invention produced in the above culture or cell-free translation system can be isolated or purified by a general method used for protein purification, such as centrifugation, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc., either alone or in appropriate combination. The protein recovered from the culture may be further purified by known means.

[0083] <5. Method for improving heterologous expression of lipase> The thus obtained lipase variants of the present invention have improved heterologous expression properties compared to the parent lipase. "Improved heterologous expression" means that the expression level of the lipase is higher than that of the parent lipase when expressed in a species different from that of the parent lipase. Here, the heterologous species is not particularly limited as long as it is a species different from that of the parent lipase, but is preferably a microorganism widely used in protein expression systems, more preferably Escherichia coli or Bacillus bacteria, even more preferably Bacillus bacteria, and even more preferably Bacillus subtilis. The expression level of the lipase variant of the present invention may be measured by a method for measuring the expression level of a protein known to those skilled in the art, or may be measured using the lipase activity of the lipase variant as an index. The heterologous expression level of the lipase variant of the present invention (expression level of the lipase variant in a heterologous species) may be preferably 130% or more, more preferably 150% or more, compared to that of the parent lipase.

[0084] Therefore, in another aspect, the present invention provides a method for improving heterologous expression of a lipase, which comprises substituting an amino acid residue at a predetermined position in a parent lipase, as numbered based on SEQ ID NO: 2, with a predetermined amino acid residue. The details of the method are the same as those of the above-mentioned method for producing a lipase variant of the present invention.

[0085] <6. Uses of lipase mutants> Lipases catalyze various reactions such as ester synthesis, transesterification, and acidolysis in addition to ester hydrolysis. By utilizing this reactivity, lipases are used in various industrial applications such as cleaning, optical resolution, biofuel production, oil and fat processing, wastewater treatment, pulp production, and leather production. The lipase variants of the present invention have improved heterologous expression properties compared to the parent lipase, making it possible to produce them in large quantities industrially advantageously by heterologous expression, and can be suitably used for such industrial applications. In addition, the lipase variants of the present invention have a variety of sequences and various different characteristics, so they can be used to search for lipases suitable for substrates and applications. Thus, the lipase variants of the present invention can be used as detergents, optical resolution agents, catalysts for ester synthesis, catalysts for oil and fat processing, wastewater treatment agents, pulp treatment agents, leather treatment agents, and the like, and are preferably detergents. Examples of detergents include laundry detergents, dishwashing detergents, bleaching agents, detergents for cleaning hard surfaces, drain cleaners, denture cleaners, and germicidal cleaners for medical instruments.

[0086] The agent may be the lipase variant of the present invention alone or an enzyme composition containing the lipase variant. Such an enzyme composition may be a solid composition such as a powder or a liquid composition.

[0087] The enzyme composition containing the lipase variant of the present invention may contain, in addition to the lipase variant, any additives such as an inert carrier, a pH adjuster, a dispersant, a buffer, a preservative, etc. The enzyme composition may contain one type of lipase variant or a combination of two or more types of lipase variants.

[0088] The content of the lipase variant of the present invention in the enzyme composition of the present invention is not particularly limited as long as the lipase variant exhibits activity, but is, for example, preferably 0.1 mg or more, more preferably 1 mg or more, more preferably 5 mg or more, and preferably 50,000 mg or less, more preferably 5,000 mg or less, more preferably 500 mg or less, per 1 kg of the enzyme composition. Also, the content is preferably 0.1 to 50,000 mg, more preferably 1 to 5,000 mg, and more preferably 5 to 500 mg.

[0089] In relation to the above-described embodiment, the present invention further discloses the following aspects. <1> Any lipase variant selected from the following (a) to (v): (a) a lipase variant consisting of an amino acid sequence having at least 82% identity to the amino acid sequence of SEQ ID NO:2, wherein the lipase variant has D at the position corresponding to position 118 of the amino acid sequence of SEQ ID NO:2; (b) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:4, and having a Y at the position corresponding to position 31 of the amino acid sequence of SEQ ID NO:2; (c) a lipase variant consisting of an amino acid sequence having at least 91% identity to the amino acid sequence of SEQ ID NO:4, and having a G at the position corresponding to position 32 of the amino acid sequence of SEQ ID NO:2; (d) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:4 and having a C at the position corresponding to position 90 of the amino acid sequence of SEQ ID NO:2; (e) a lipase variant consisting of an amino acid sequence having at least 94% identity to the amino acid sequence of SEQ ID NO:4, and having an L at the position corresponding to position 181 of the amino acid sequence of SEQ ID NO:2; (f) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:6, and having A, T, H or G at the position corresponding to position 222 of the amino acid sequence of SEQ ID NO:2; (g) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8, and having a K at the position corresponding to position 39 of the amino acid sequence of SEQ ID NO:2; (h) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8, and having a T at the position corresponding to position 125 of the amino acid sequence of SEQ ID NO:2; (i) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8, and having a P at the position corresponding to position 126 of the amino acid sequence of SEQ ID NO:2; (j) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8, and having G at the position corresponding to position 293 of the amino acid sequence of SEQ ID NO:2; (k) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 10, and having a Y at the position corresponding to position 29 of the amino acid sequence of SEQ ID NO: 2; (l) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 10, and having a Y at the position corresponding to position 31 of the amino acid sequence of SEQ ID NO: 2; (m) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 10, and having G at the position corresponding to position 32 of the amino acid sequence of SEQ ID NO: 2; (n) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 10, and having G at the position corresponding to position 186 of the amino acid sequence of SEQ ID NO: 2; (o) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 12, and having a Q at a position corresponding to position 206 of the amino acid sequence of SEQ ID NO: 2; (p) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 14, and having a Y at the position corresponding to position 31 of the amino acid sequence of SEQ ID NO: 2; (q) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 14, and having G at the position corresponding to position 32 of the amino acid sequence of SEQ ID NO: 2; (r) a lipase variant consisting of an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 14, and having a K at the position corresponding to position 77 of the amino acid sequence of SEQ ID NO: 2; (s) a lipase variant consisting of an amino acid sequence having at least 83% identity to the amino acid sequence of SEQ ID NO: 14, and having an L at a position corresponding to position 181 of the amino acid sequence of SEQ ID NO: 2; (t) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 16 and having a C at the position corresponding to position 90 of the amino acid sequence of SEQ ID NO: 2; (u) a lipase variant consisting of an amino acid sequence having at least 81% identity to the amino acid sequence of SEQ ID NO: 16, and having an L at a position corresponding to position 181 of the amino acid sequence of SEQ ID NO: 2; (v) A lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 34, and having a Y at the position corresponding to position 29 of the amino acid sequence of SEQ ID NO: 2. <2> Any lipase variant selected from the following (aa) to (ae): (aa) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 4, and having at least two amino acid residues selected from Y at the position corresponding to position 31, G at the position corresponding to position 32, C at the position corresponding to position 90, and L at the position corresponding to position 181 of the amino acid sequence of SEQ ID NO: 2; (ab) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8, and having at least two amino acid residues selected from K at the position corresponding to position 39, T at the position corresponding to position 125, P at the position corresponding to position 126, and G at the position corresponding to position 293 of the amino acid sequence of SEQ ID NO:2; (ac) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 10, and having at least two amino acid residues selected from Y at the position corresponding to position 29, Y at the position corresponding to position 31, G at the position corresponding to position 32, and G at the position corresponding to position 186 of the amino acid sequence of SEQ ID NO: 2; (ad) a lipase variant consisting of an amino acid sequence having at least 83% identity to the amino acid sequence of SEQ ID NO: 14, and having at least two amino acid residues selected from Y at the position corresponding to position 31, G at the position corresponding to position 32, K at the position corresponding to position 77, and L at the position corresponding to position 181 of the amino acid sequence of SEQ ID NO: 2; (ae) A lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 16, and having a C at the position corresponding to position 90 and an L at the position corresponding to position 181 of the amino acid sequence of SEQ ID NO: 2. <3> The lipase variant is any one selected from the above (a), (c) to (g), (i) to (k), (n), (o), (r), and (t) to (v). <1> The lipase variant described in <4> Any lipase variant selected from the following (aa-1) to (ae): <2> Lipase variants according to (aa-1) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 4, and having a Y at the position corresponding to position 31 and a G at the position corresponding to position 32 of the amino acid sequence of SEQ ID NO: 2; (aa-2) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 4, and having C at the position corresponding to position 90 and L at the position corresponding to position 181 of the amino acid sequence of SEQ ID NO: 2; (aa-3) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 4, and having G at the position corresponding to position 32 and C at the position corresponding to position 90 of the amino acid sequence of SEQ ID NO: 2; (aa-4) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:4, and having Y at the position corresponding to position 31, G at the position corresponding to position 32, C at the position corresponding to position 90, and L at the position corresponding to position 181 of the amino acid sequence of SEQ ID NO:2; (ab-1) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8, and having a T at the position corresponding to position 125 and a P at the position corresponding to position 126 of the amino acid sequence of SEQ ID NO:2; (ab-2) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8, and having K at the position corresponding to position 39 and G at the position corresponding to position 293 of the amino acid sequence of SEQ ID NO:2; (ab-3) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8, and having a T at a position corresponding to position 125, a P at a position corresponding to position 126, and a G at a position corresponding to position 293 of the amino acid sequence of SEQ ID NO:2; (ab-4) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8, and having K at the position corresponding to position 39, T at the position corresponding to position 125, and P at the position corresponding to position 126 of the amino acid sequence of SEQ ID NO:2; (ab-5) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8, and having K at a position corresponding to position 39, P at a position corresponding to position 126, and G at a position corresponding to position 293 of the amino acid sequence of SEQ ID NO:2; (ab-6) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8, and having K at the position corresponding to position 39, T at the position corresponding to position 125, P at the position corresponding to position 126, and G at the position corresponding to position 293 of the amino acid sequence of SEQ ID NO:2; (ac-1) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 10, and having a Y at a position corresponding to position 29 and a G at a position corresponding to position 186 of the amino acid sequence of SEQ ID NO: 2; (ac-2) a lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 10, or having a Y at a position corresponding to position 29, a Y at a position corresponding to position 31, a G at a position corresponding to position 32, and a G at a position corresponding to position 186 of the amino acid sequence of SEQ ID NO: 2; (ad-1) a lipase variant consisting of an amino acid sequence having at least 83% identity with the amino acid sequence of SEQ ID NO: 14, and having a Y at the position corresponding to position 31 and a G at the position corresponding to position 32 of the amino acid sequence of SEQ ID NO: 2; (ad-2) a lipase variant consisting of an amino acid sequence having at least 83% identity to the amino acid sequence of SEQ ID NO: 14, and having K at a position corresponding to position 77 and L at a position corresponding to position 181 of the amino acid sequence of SEQ ID NO: 2; (ad-3) a lipase variant consisting of an amino acid sequence having at least 83% identity to the amino acid sequence of SEQ ID NO: 14, and having Y at the position corresponding to position 31, G at the position corresponding to position 32, and L at the position corresponding to position 181 of the amino acid sequence of SEQ ID NO: 2; (ad-4) a lipase variant consisting of an amino acid sequence having at least 83% identity to the amino acid sequence of SEQ ID NO: 14, and having Y at the position corresponding to position 31, G at the position corresponding to position 32, K at the position corresponding to position 77, and L at the position corresponding to position 181 of the amino acid sequence of SEQ ID NO: 2; (ae) A lipase variant consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 16, and having a cysteine ​​at a position corresponding to position 90 and a leucine at a position corresponding to position 181 of the amino acid sequence of SEQ ID NO: 2. <5> <1> ~ <4> 10. An enzyme composition comprising the lipase variant according to any one of claims 1 to 9. <6> a detergent, an optical resolution agent, an ester synthesis catalyst, an oil and fat processing catalyst, a wastewater treatment agent, a pulp treatment agent, or a leather treatment agent, preferably a detergent; <5> The enzyme composition described.

[0090] <7> <1> ~ <4> A polynucleotide encoding the lipase variant according to any one of claims 1 to 5. <8> <7> A vector or DNA fragment comprising the polynucleotide described in . <9> <7> A transformed cell containing the vector or DNA fragment described in . <10> It is a microorganism, <9> A transformed cell according to claim 1. <11> Escherichia coli or Bacillus bacteria, preferably Bacillus bacteria, more preferably Bacillus subtilis; <10> A transformed cell according to claim 1. <12> <9> ~ <11> A method for producing a lipase variant, comprising a step of culturing the transformed cell described in any one of the above.

[0091] <13> A method for producing a lipase variant, comprising any one of the steps selected from the following (i) to (xxii): (i) substituting an amino acid residue at a position corresponding to position 118 of the amino acid sequence of SEQ ID NO: 2 with D in a polypeptide having lipase activity and consisting of an amino acid sequence having at least 82% identity to the amino acid sequence of SEQ ID NO: 2; (ii) substituting an amino acid residue at a position corresponding to position 31 of the amino acid sequence of SEQ ID NO:2 with Y in a polypeptide having lipase activity and consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO:4; (iii) substituting, in a polypeptide having lipase activity and consisting of an amino acid sequence having at least 91% identity with the amino acid sequence of SEQ ID NO:4, an amino acid residue at a position corresponding to position 32 of the amino acid sequence of SEQ ID NO:2 with G; (iv) substituting, in a polypeptide having lipase activity and consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO:4, an amino acid residue at a position corresponding to position 90 of the amino acid sequence of SEQ ID NO:2 with C; (v) substituting an amino acid residue at a position corresponding to position 181 of the amino acid sequence of SEQ ID NO:2 with L in a polypeptide having lipase activity and consisting of an amino acid sequence having at least 94% identity with the amino acid sequence of SEQ ID NO:4; (vi) substituting an amino acid residue at a position corresponding to position 222 of the amino acid sequence of SEQ ID NO:2 with A, T, H, or G in a polypeptide having lipase activity and consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO:6; (vii) substituting an amino acid residue at a position corresponding to position 39 of the amino acid sequence of SEQ ID NO:2 with K in a polypeptide having lipase activity, the polypeptide having an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8; (viii) substituting, with T, an amino acid residue at a position corresponding to position 125 of the amino acid sequence of SEQ ID NO:2 in a polypeptide having lipase activity and consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO:8; (ix) substituting an amino acid residue at a position corresponding to position 126 of the amino acid sequence of SEQ ID NO:2 with P in a polypeptide having lipase activity, the polypeptide having an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8; (x) substituting, with G, an amino acid residue at a position corresponding to position 293 of the amino acid sequence of SEQ ID NO:2 in a polypeptide having lipase activity and consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8; (xi) substituting an amino acid residue at a position corresponding to position 29 of the amino acid sequence of SEQ ID NO:2 with Y in a polypeptide having lipase activity, the polypeptide having an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:10; (xii) substituting an amino acid residue at a position corresponding to position 31 of the amino acid sequence of SEQ ID NO:2 with Y in a polypeptide having lipase activity, the polypeptide having an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:10; (xiii) substituting, in a polypeptide having lipase activity, an amino acid residue at a position corresponding to position 32 of the amino acid sequence of SEQ ID NO:2 with G, the polypeptide having an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:10; (xiv) substituting, with G, an amino acid residue at a position corresponding to position 186 of the amino acid sequence of SEQ ID NO:2 in a polypeptide having lipase activity and consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:10; (xv) substituting an amino acid residue at a position corresponding to position 206 of the amino acid sequence of SEQ ID NO:2 with Q in a polypeptide having lipase activity, the polypeptide having an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:12; (xvi) substituting an amino acid residue at a position corresponding to position 31 of the amino acid sequence of SEQ ID NO: 2 with Y in a polypeptide having lipase activity, the polypeptide having an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 14; (xvii) substituting, with G, an amino acid residue at a position corresponding to position 32 of the amino acid sequence of SEQ ID NO:2 in a polypeptide having lipase activity and consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:14; (xviii) substituting an amino acid residue at a position corresponding to position 77 of the amino acid sequence of SEQ ID NO: 2 with K in a polypeptide having lipase activity, the polypeptide having an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 14; (xix) substituting an amino acid residue at a position corresponding to position 181 of the amino acid sequence of SEQ ID NO:2 with L in a polypeptide having lipase activity, the polypeptide having an amino acid sequence having at least 83% identity to the amino acid sequence of SEQ ID NO:14; (xx) substituting an amino acid residue at a position corresponding to position 90 of the amino acid sequence of SEQ ID NO:2 with C in a polypeptide having lipase activity, the polypeptide having an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:16; (xxi) substituting an amino acid residue at a position corresponding to position 181 of the amino acid sequence of SEQ ID NO: 2 with L in a polypeptide having lipase activity, the polypeptide having an amino acid sequence having at least 81% identity to the amino acid sequence of SEQ ID NO: 16; (xxii) replacing the amino acid residue at the position corresponding to position 29 of the amino acid sequence of SEQ ID NO: 2 with Y in a polypeptide having lipase activity and consisting of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 34. <14> A method for producing a lipase variant, comprising any one of the steps selected from the following (xxiii) to (xxvii): (xxiii) substituting amino acid residues at at least two positions selected from positions 31, 32, 90 and 181 of the amino acid sequence of SEQ ID NO:2 with Y at the position corresponding to position 31, G at the position corresponding to position 32, C at the position corresponding to position 90 and L at the position corresponding to position 181 in a polypeptide having an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:4 and having lipase activity; (xxiv) substituting amino acid residues at at least two positions selected from positions 39, 125, 126, and 293 of the amino acid sequence of SEQ ID NO:2 in a polypeptide having lipase activity, the polypeptide having an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8, with K at the position corresponding to position 39, with T at the position corresponding to position 125, with P at the position corresponding to position 126, and with G at the position corresponding to position 293; (xxv) substituting amino acid residues at at least two positions selected from positions 29, 31, 32, and 186 of the amino acid sequence of SEQ ID NO:2 with Y at the position corresponding to position 29, Y at the position corresponding to position 31, G at the position corresponding to position 32, and G at the position corresponding to position 186 in a polypeptide having an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:10 and having lipase activity; (xxvi) substituting amino acid residues at at least two positions selected from positions 31, 32, 77, and 181 of the amino acid sequence of SEQ ID NO:2 with Y at the position corresponding to position 31, G at the position corresponding to position 32, K at the position corresponding to position 77, and L at the position corresponding to position 181 in a polypeptide having a lipase activity, the polypeptide having an amino acid sequence having at least 83% identity to the amino acid sequence of SEQ ID NO:14; (xxvii) substituting an amino acid residue at a position corresponding to position 90 in the amino acid sequence of SEQ ID NO:2 with C and substituting an amino acid residue at a position corresponding to position 181 in the amino acid sequence of SEQ ID NO:2 with L in a polypeptide having lipase activity, the polypeptide having an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:16. <15> A method for improving heterologous expression of lipase, comprising any one of the steps selected from (i) to (xxii) above. <16> A method for improving heterologous expression of lipase, comprising any one of the steps selected from the above (xxiii) to (xxvii). <17> The step is any one of steps selected from the above steps (i), (iii) to (vii), (ix) to (xi), (xiv), (xv), (xviii) and (xx) to (xxii). <13> or <15> The method according to <18> The step is any one of steps selected from the following steps (xxiii-1) to (xxvii): <14> or <16> Method to do this: (xxiii-1) substituting an amino acid residue at a position corresponding to position 31 of the amino acid sequence of SEQ ID NO:2 with Y and substituting an amino acid residue at a position corresponding to position 32 of the amino acid sequence of SEQ ID NO:2 with G in a polypeptide having a lipase activity, the polypeptide having an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:4; (xxiii-2) substituting an amino acid residue at a position corresponding to position 90 in the amino acid sequence of SEQ ID NO:2 with C and substituting an amino acid residue at a position corresponding to position 181 in the amino acid sequence of SEQ ID NO:2 with L in a polypeptide having a lipase activity, the polypeptide having an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:4; (xxiii-3) substituting an amino acid residue at a position corresponding to position 32 in the amino acid sequence of SEQ ID NO:2 with G and substituting an amino acid residue at a position corresponding to position 90 in the amino acid sequence of SEQ ID NO:2 with C in a polypeptide having a lipase activity, the polypeptide having an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:4; (xxiii-4) substituting an amino acid residue at a position corresponding to position 31 of the amino acid sequence of SEQ ID NO:2 with Y, an amino acid residue at a position corresponding to position 32 with G, an amino acid residue at a position corresponding to position 90 with C, and an amino acid residue at a position corresponding to position 181 with L in a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO:4 and having lipase activity; (xxiv-1) substituting an amino acid residue at a position corresponding to position 125 of the amino acid sequence of SEQ ID NO:2 with T and substituting an amino acid residue at a position corresponding to position 126 of the amino acid sequence of SEQ ID NO:2 with P in a polypeptide having a lipase activity, the polypeptide having an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8; (xxiv-2) substituting an amino acid residue at a position corresponding to position 39 of the amino acid sequence of SEQ ID NO:2 with K and substituting an amino acid residue at a position corresponding to position 293 of the amino acid sequence of SEQ ID NO:2 with G in a polypeptide having a lipase activity, the polypeptide having an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:8; (xxiv-3) substituting an amino acid residue at a position corresponding to position 125 of the amino acid sequence of SEQ ID NO:2 with T, an amino acid residue at a position corresponding to position 126 of the amino acid sequence of SEQ ID NO:2 with P, and an amino acid residue at a position corresponding to position 293 of the amino acid sequence of SEQ ID NO:2 with G, in a polypeptide having a lipase activity and consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO:8; (xxiv-4) substituting an amino acid residue at a position corresponding to position 39 of the amino acid sequence of SEQ ID NO:2 with K, substituting an amino acid residue at a position corresponding to position 125 of the amino acid sequence of SEQ ID NO:2 with T, and substituting an amino acid residue at a position corresponding to position 126 of the amino acid sequence of SEQ ID NO:2 with P, in a polypeptide having a lipase activity and consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO:8; (xxiv-5) substituting an amino acid residue at a position corresponding to position 39 of the amino acid sequence of SEQ ID NO:2 with K, substituting an amino acid residue at a position corresponding to position 126 of the amino acid sequence of SEQ ID NO:2 with P, and substituting an amino acid residue at a position corresponding to position 293 of the amino acid sequence of SEQ ID NO:2 with G, in a polypeptide having a lipase activity and consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO:8; (xxiv-6) substituting an amino acid residue at a position corresponding to position 39 of the amino acid sequence of SEQ ID NO:2 with K, an amino acid residue at a position corresponding to position 125 with T, an amino acid residue at a position corresponding to position 126 with P, and an amino acid residue at a position corresponding to position 293 with G in a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO:8 and having lipase activity; (xxv-1) substituting an amino acid residue at a position corresponding to position 29 of the amino acid sequence of SEQ ID NO:2 with Y and substituting an amino acid residue at a position corresponding to position 186 of the amino acid sequence of SEQ ID NO:2 with G in a polypeptide having a lipase activity, the polypeptide having an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:10; (xxv-2) substituting an amino acid residue at a position corresponding to position 29 of the amino acid sequence of SEQ ID NO:2 with Y, an amino acid residue at a position corresponding to position 31 of the amino acid sequence of SEQ ID NO:2 with Y, an amino acid residue at a position corresponding to position 32 of the amino acid sequence of SEQ ID NO:2 with G, and an amino acid residue at a position corresponding to position 186 of the amino acid sequence of SEQ ID NO:2 with G in a polypeptide having lipase activity, the polypeptide having an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:10; (xxvi-1) substituting an amino acid residue at a position corresponding to position 31 of the amino acid sequence of SEQ ID NO:2 with Y and substituting an amino acid residue at a position corresponding to position 32 of the amino acid sequence of SEQ ID NO:2 with G in a polypeptide having a lipase activity, the polypeptide having an amino acid sequence having at least 83% identity to the amino acid sequence of SEQ ID NO:14; (xxvi-2) substituting an amino acid residue at a position corresponding to position 77 of the amino acid sequence of SEQ ID NO:2 with K and substituting an amino acid residue at a position corresponding to position 181 of the amino acid sequence of SEQ ID NO:2 with L, in a polypeptide having a lipase activity and consisting of an amino acid sequence having at least 83% identity to the amino acid sequence of SEQ ID NO:14; (xxvi-3) substituting an amino acid residue at a position corresponding to position 31 of the amino acid sequence of SEQ ID NO:2 with Y, an amino acid residue at a position corresponding to position 32 with G, and an amino acid residue at a position corresponding to position 181 with L in a polypeptide having a lipase activity, the polypeptide having an amino acid sequence having at least 83% identity with the amino acid sequence of SEQ ID NO:14; (xxvi-4) substituting an amino acid residue at a position corresponding to position 31 of the amino acid sequence of SEQ ID NO:2 with Y, an amino acid residue at a position corresponding to position 32 with G, an amino acid residue at a position corresponding to position 77 with K, and an amino acid residue at a position corresponding to position 181 with L in a polypeptide having an amino acid sequence having at least 83% identity with the amino acid sequence of SEQ ID NO:14 and having lipase activity; (xxvii) substituting an amino acid residue at a position corresponding to position 90 in the amino acid sequence of SEQ ID NO:2 with C and substituting an amino acid residue at a position corresponding to position 181 in the amino acid sequence of SEQ ID NO:2 with L in a polypeptide having lipase activity, the polypeptide having an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:16. <19> The lipase variants have improved heterologous expression compared to the parent lipase. <13> ~ <18> 2. The method according to claim 1 . EXAMPLES

[0092] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples.

[0093] (1) Construction of lipase expression plasmid The lipase expression plasmid was constructed using as a template a plasmid for expressing VHH of SEQ ID NO: 26, which contains a promoter derived from the Bacillus subtilis spoVG gene described in WO2021 / 153129. Each lipase gene was replaced by an In-Fusion reaction onto the full-length ORF containing the VHH gene of the above plasmid. The artificially synthesized lipase genes SspLip, PfLip, EtLip, YeLip, AnLip, YfLip, PspLip, PspLip2, SaLip, BbLip, EspLip, AspLip, YeLip2, YmLip, EspLip2, MiLip, and PaLip (polynucleotides of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, respectively, and SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, Plasmids pHY-SspLip, pHY-PfLip, pHY-EtLip, pHY-YeLip, pHY-AnLip, pHY-YfLip, pHY-PspLip, pHY-PspLip2, pHY-SaLip, pHY-BbLip, pHY-EspLip, pHY-AspLip, pHY-YeLip2, pHY-YmLip, pHY-EspLip2, pHY-MiLip, and pHY-PaLip were constructed from the lipases (encoding the amino acid sequences of 24, 26, 28, 30, 32, and 34, respectively). Mutations were introduced into each lipase by site-specific introduction using PCR with complementary primer pairs (see Nucleic Acids Research, 2004, 32(14):e115). The mutations introduced into each lipase and the positions of the mutations (positions based on each parent enzyme and positions based on SEQ ID NO: 2) are shown in Table 2. In the following examples, the positions of the mutations in each lipase are shown based on the positions in the parent enzyme.

[0094] [Table 2]

[0095] (2) Expression of wild-type lipase Sixteen diverse sequences belonging to the Proteus / Yersinia clade (SspLip, PfLip, EtLip, YeLip, AnLip, YfLip, PspLip, PspLip2, SaLip, BbLip, EspLip, AspLip, YeLip2, YmLip, EspLip2, MiLip (SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, and 32, respectively)) were selected from public databases and their heterologous expression was evaluated. The wild-type lipase expression plasmid or empty vector pHY300PLK (TaKaRa) obtained in (1) was introduced into Bacillus subtilis Dpr9 strain (see Microbial cell factories 20.1 (2021): 1-13.) by the protoplast method, and cultured in 2x L-maltose medium (2% tryptone, 1% yeast extract, 1% NaCl, 7.5% maltose, 7.5 ppm manganese sulfate pentahydrate, 0.04% calcium chloride dihydrate, 10 ppm tetracycline; % is (w / v)%) at 30 ° C for 3 days, and the culture supernatant was collected by centrifugation. The culture supernatant was mixed with an equal volume of 2x Laemmli Sample buffer (Bio-Rad) containing 100 mM DTT and incubated at 100 ° C for 3 minutes. Each sample was incubated at Any kD TM Mini-PROTEAN® TGX Stain-Free TM After application to Protein Gel (Bio-Rad), electrophoresis was performed at a constant voltage of 200 V. The gel after electrophoresis was photographed using a Chemi Doc MP Imaging system (Bio-Rad). In comparison with the empty vector, those for which a clear band was observed near the molecular weight of lipase are indicated with a "◯", those for which a faint band was observed with a "△", and those for which no band was observed with a "-" are shown in Table 3. Of the 16 sequences examined, only three sequences were able to clearly confirm the lipase band, indicating that there is an issue with Proteus / Yersinia clade lipases in that there is little diversity in sequences that can be heterologously expressed in large quantities.

[0096] [Table 3]

[0097] (3) Expression evaluation of lipase mutants Heterologous expression of lipase mutants was performed using the same method as in (2). Lipase PS Amano SD (Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in 20 mM Tris-HCl (pH 7.0), and the concentration was measured using DC protein assay (Bio-Rad) with BSA as a standard. The culture supernatant was subjected to SDS-PAGE using the same method as in (2) together with lipase PS Amano SD of known concentration. The lipase concentration in the culture supernatant was quantified from the band intensity using lipase PS Amano SD as a standard (Table 4). Heterologous expression of various lipases of the Proteus / Yersinia clade was significantly improved by site-specific modification.

[0098] [Table 4]

[0099] (4) Calculation of sequence identity Sequence identities between mutants with significantly improved heterologous expression (SspLip E118D, PfLip F30Y E31G A89C V180L, EtLip I222G, YeLip V38K V124T E125P S292G, AnLip C24Y F26Y A27G R181G, YfLip G206Q, PspLip F30Y D31G R76K M179L, PspLip2 A89C V180L) were calculated using Genetyx (Table 5). These mutants significantly expand the heterologously expressible sequence diversity of lipases of the Proteus / Yersinia clade.

[0100] [Table 5]

[0101] (5) Expression of PaLip mutants Heterologous expression of PaLip (SEQ ID NO: 34), a lipase closely related to lipases of the Proteus / Yersinia clade, was carried out in the same manner as in (2). 2 μL of culture supernatant diluted 100-fold with 20 mM Tris-HCl (pH 7.0) and 100 μL of substrate solution were mixed in each well of a 96-well assay plate, and the absorbance change at 405 nm (OD / min) was measured at 30 °C. 2 mM 4-nitrophenyl octanoate in 20 mM Tris-HCl (pH 7.0) was used as the substrate solution. No esterase activity was detected in the culture supernatant of wild-type PaLip, suggesting that active lipase was not heterologously expressed, but esterase activity derived from lipase was detected in the culture supernatant of the PaLip M28Y mutant (Figure 1).

Claims

1. Any lipase variant selected from the following (a) to (v): (a) A lipase variant consisting of an amino acid sequence having at least 82% identity with the amino acid sequence of SEQ ID NO: 2 and having aspartic acid at the position corresponding to position 118 of the amino acid sequence of SEQ ID NO: 2; (b) A lipase variant consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 4 and having tyrosine at the position corresponding to position 31 of the amino acid sequence of SEQ ID NO: 2; (c) A lipase variant consisting of an amino acid sequence having at least 91% identity with the amino acid sequence of SEQ ID NO: 4 and having glycine at the position corresponding to position 32 of the amino acid sequence of SEQ ID NO: 2; (d) A lipase variant consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 4 and having cysteine at the position corresponding to position 90 of the amino acid sequence of SEQ ID NO: 2; (e) A lipase variant consisting of an amino acid sequence having at least 94% identity with the amino acid sequence of SEQ ID NO: 4 and having leucine at the position corresponding to position 181 of the amino acid sequence of SEQ ID NO: 2; (f) A lipase variant consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 6 and having alanine, threonine, histidine or glycine at the position corresponding to position 222 of the amino acid sequence of SEQ ID NO: 2; (g) A lipase variant consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 8 and having lysine at the position corresponding to position 39 of the amino acid sequence of SEQ ID NO: 2; (h) A lipase variant consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 8 and having threonine at the position corresponding to position 125 of the amino acid sequence of SEQ ID NO: 2; (i) A lipase variant consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 8 and having proline at the position corresponding to position 126 of the amino acid sequence of SEQ ID NO: 2; (j) A lipase variant consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 10 and having glycine at the position corresponding to position 29 of the amino acid sequence of SEQ ID NO: 2; (k) A lipase variant consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 10 and having tyrosine at the position corresponding to position 29 of the amino acid sequence of SEQ ID NO: 2; (l) a lipase mutant consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 10 and having tyrosine at a position corresponding to position 31 of the amino acid sequence of SEQ ID NO: 2; (m) a lipase mutant consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 10 and having glycine at a position corresponding to position 32 of the amino acid sequence of SEQ ID NO: 2; (n) a lipase mutant consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 10 and having glycine at a position corresponding to position 186 of the amino acid sequence of SEQ ID NO: 2; (o) a lipase mutant consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 12 and having glutamine at a position corresponding to position 206 of the amino acid sequence of SEQ ID NO: 2; (p) a lipase mutant consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 14 and having tyrosine at a position corresponding to position 31 of the amino acid sequence of SEQ ID NO: 2; (q) a lipase mutant consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 14 and having glycine at a position corresponding to position 32 of the amino acid sequence of SEQ ID NO: 2; (r) a lipase mutant consisting of an amino acid sequence having at least 97% identity with the amino acid sequence of SEQ ID NO: 14 and having lysine at a position corresponding to position 77 of the amino acid sequence of SEQ ID NO: 2; (s) a lipase mutant consisting of an amino acid sequence having at least 83% identity with the amino acid sequence of SEQ ID NO: 14 and having leucine at a position corresponding to position 181 of the amino acid sequence of SEQ ID NO: 2; (t) a lipase mutant consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 16 and having cysteine at a position corresponding to position 90 of the amino acid sequence of SEQ ID NO: 2; (u) a lipase mutant consisting of an amino acid sequence having at least 81% identity with the amino acid sequence of SEQ ID NO: 16 and having leucine at a position corresponding to position 181 of the amino acid sequence of SEQ ID NO: 2; (v) a lipase mutant consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 34 and having tyrosine at a position corresponding to position 29 of the amino acid sequence of SEQ ID NO:

2.

2. Any lipase mutant selected from the following (aa) to (ae): A lipase variant comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 4, and having at least two amino acid residues selected from tyrosine at the position corresponding to position 31 of the amino acid sequence of SEQ ID NO: 2, glycine at the position corresponding to position 32, cysteine at the position corresponding to position 90, and leucine at the position corresponding to position 181; A lipase variant comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 8, and having at least two amino acid residues selected from lysine at the position corresponding to position 39 of the amino acid sequence of SEQ ID NO: 2, threonine at the position corresponding to position 125, proline at the position corresponding to position 126, and glycine at the position corresponding to position 293; A lipase variant comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 10, and having at least two amino acid residues selected from tyrosine at the position corresponding to position 29 of the amino acid sequence of SEQ ID NO: 2, tyrosine at the position corresponding to position 31, glycine at the position corresponding to position 32, and glutamine at the position corresponding to position 186; A lipase variant comprising an amino acid sequence having at least 83% identity with the amino acid sequence of SEQ ID NO: 14, and having at least two amino acid residues selected from tyrosine at the position corresponding to position 31 of the amino acid sequence of SEQ ID NO: 2, glycine at the position corresponding to position 32, lysine at the position corresponding to position 77, and leucine at the position corresponding to position 181; A lipase variant comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 16, and having cysteine at the position corresponding to position 90 and leucine at the position corresponding to position 181 of the amino acid sequence of SEQ ID NO:

2.

3. The lipase variant according to claim 1, which is any one of the lipase variants selected from (a), (c) to (g), (i) to (k), (n), (o), (r), and (t) to (v).

4. The lipase variant according to claim 2, which is any one of the lipase variants selected from the following (aa-1) to (ae): A lipase mutant comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 4 and having tyrosine at the position corresponding to position 31 and glycine at the position corresponding to position 32 of the amino acid sequence of SEQ ID NO: 2; A lipase mutant comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 4 and having cysteine at the position corresponding to position 90 and leucine at the position corresponding to position 181 of the amino acid sequence of SEQ ID NO: 2; A lipase mutant comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 4 and having glycine at the position corresponding to position 32 and cysteine at the position corresponding to position 90 of the amino acid sequence of SEQ ID NO: 2; A lipase mutant comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 4 and having tyrosine at the position corresponding to position 31, glycine at the position corresponding to position 32, cysteine at the position corresponding to position 90, and L at the position corresponding to position 181 of the amino acid sequence of SEQ ID NO: 2; A lipase mutant comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 8 and having threonine at the position corresponding to position 125 and proline at the position corresponding to position 126 of the amino acid sequence of SEQ ID NO: 2; A lipase mutant comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 8 and having lysine at the position corresponding to position 39 and glycine at the position corresponding to position 293 of the amino acid sequence of SEQ ID NO: 2; A lipase mutant comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 8 and having threonine at the position corresponding to position 125, proline at the position corresponding to position 126, and glycine at the position corresponding to position 293 of the amino acid sequence of SEQ ID NO: 2; A lipase mutant comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 8 and having lysine at the position corresponding to position 39, threonine at the position corresponding to position 125, and proline at the position corresponding to position 126 of the amino acid sequence of SEQ ID NO: 2; A lipase variant comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 8, and having lysine at the position corresponding to position 39 of the amino acid sequence of SEQ ID NO: 2, proline at the position corresponding to position 126, and glycine at the position corresponding to position 293; A lipase variant comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 8, and having lysine at the position corresponding to position 39 of the amino acid sequence of SEQ ID NO: 2, threonine at the position corresponding to position 125, proline at the position corresponding to position 126, and glycine at the position corresponding to position 293; A lipase variant comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 10, and having tyrosine at the position corresponding to position 29 of the amino acid sequence of SEQ ID NO: 2 and glycine at the position corresponding to position 186; A lipase variant comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 10, or having tyrosine at the position corresponding to position 29 of the amino acid sequence of SEQ ID NO: 2, tyrosine at the position corresponding to position 31, glycine at the position corresponding to position 32, and glycine at the position corresponding to position 186; A lipase variant comprising an amino acid sequence having at least 83% identity with the amino acid sequence of SEQ ID NO: 14, and having tyrosine at the position corresponding to position 31 of the amino acid sequence of SEQ ID NO: 2 and glycine at the position corresponding to position 32; A lipase variant comprising an amino acid sequence having at least 83% identity with the amino acid sequence of SEQ ID NO: 14, and having lysine at the position corresponding to position 77 of the amino acid sequence of SEQ ID NO: 2 and leucine at the position corresponding to position 181; A lipase variant comprising an amino acid sequence having at least 83% identity with the amino acid sequence of SEQ ID NO: 14, and having tyrosine at the position corresponding to position 31 of the amino acid sequence of SEQ ID NO: 2, glycine at the position corresponding to position 32, and leucine at the position corresponding to position 181; An amino acid sequence having at least 83% identity with the amino acid sequence of SEQ ID NO: 14, and having tyrosine at the position corresponding to position 31 of the amino acid sequence of SEQ ID NO: 2, glycine at the position corresponding to position 32, lysine at the position corresponding to position 77, and leucine at the position corresponding to position 181; a lipase mutant An amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 16, and having cysteine at the position corresponding to position 90 of the amino acid sequence of SEQ ID NO: 2 and leucine at the position corresponding to position 181; a lipase mutant **Claim 5** An enzyme composition containing the lipase mutant according to any one of Claims 1 to 4 **Claim 6** A polynucleotide encoding the lipase mutant according to any one of Claims 1 to 4 **Claim 7** A vector or DNA fragment containing the polynucleotide according to Claim 6 **Claim 8** A transformed cell containing the vector or DNA fragment according to Claim 7 **Claim 9** The transformed cell according to Claim 8, which is a microorganism **Claim 10** The transformed cell according to Claim 9, which is Escherichia coli or a bacterium belonging to the genus Bacillus **Claim 11** A method for producing a lipase mutant, comprising the step of culturing the transformed cell according to Claim 8 **Claim 12** A method for producing a lipase mutant, comprising any one of the following steps (i) to (xxii): (i) In a polypeptide having an amino acid sequence having at least 82% identity with the amino acid sequence of SEQ ID NO: 2 and having lipase activity, substituting the amino acid residue at the position corresponding to position 118 of the amino acid sequence of SEQ ID NO: 2 with aspartic acid; (ii) In a polypeptide having an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 4 and having lipase activity, substituting the amino acid residue at the position corresponding to position 31 of the amino acid sequence of SEQ ID NO: 2 with tyrosine; (iii) In a polypeptide having an amino acid sequence having at least 91% identity with the amino acid sequence of SEQ ID NO: 4 and having lipase activity, substituting the amino acid residue at the position corresponding to position 32 of the amino acid sequence of SEQ ID NO: 2 with glycine; (iv) a step of substituting the amino acid residue at the position corresponding to position 90 of the amino acid sequence of SEQ ID NO: 2 with cysteine in a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 4 and having lipase activity; (v) a step of substituting the amino acid residue at the position corresponding to position 181 of the amino acid sequence of SEQ ID NO: 2 with leucine in a polypeptide consisting of an amino acid sequence having at least 94% identity with the amino acid sequence of SEQ ID NO: 4 and having lipase activity; (vi) a step of substituting the amino acid residue at the position corresponding to position 222 of the amino acid sequence of SEQ ID NO: 2 with alanine, threonine, histidine or glycine in a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 6 and having lipase activity; (vii) a step of substituting the amino acid residue at the position corresponding to position 39 of the amino acid sequence of SEQ ID NO: 2 with lysine in a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 8 and having lipase activity; (viii) a step of substituting the amino acid residue at the position corresponding to position 125 of the amino acid sequence of SEQ ID NO: 2 with threonine in a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 8 and having lipase activity; (ix) a step of substituting the amino acid residue at the position corresponding to position 126 of the amino acid sequence of SEQ ID NO: 2 with proline in a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 8 and having lipase activity; (x) a step of substituting the amino acid residue at the position corresponding to position 293 of the amino acid sequence of SEQ ID NO: 2 with glycine in a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 8 and having lipase activity; (xi) a step of substituting the amino acid residue at the position corresponding to position 29 of the amino acid sequence of SEQ ID NO: 2 with tyrosine in a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 10 and having lipase activity; (xii) a step of substituting the amino acid residue at the position corresponding to position 31 of the amino acid sequence of SEQ ID NO: 2 with tyrosine in a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 10 and having lipase activity; (xiii) A step of substituting the amino acid residue at the position corresponding to position 32 of the amino acid sequence of SEQ ID NO: 2 with glycine in a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 10 and having lipase activity; (xiv) A step of substituting the amino acid residue at the position corresponding to position 186 of the amino acid sequence of SEQ ID NO: 2 with glycine in a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 10 and having lipase activity; (xv) A step of substituting the amino acid residue at the position corresponding to position 206 of the amino acid sequence of SEQ ID NO: 2 with glutamine in a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 12 and having lipase activity; (xvi) A step of substituting the amino acid residue at the position corresponding to position 31 of the amino acid sequence of SEQ ID NO: 2 with tyrosine in a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 14 and having lipase activity; (xvii) A step of substituting the amino acid residue at the position corresponding to position 32 of the amino acid sequence of SEQ ID NO: 2 with glycine in a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 14 and having lipase activity; (xviii) A step of substituting the amino acid residue at the position corresponding to position 77 of the amino acid sequence of SEQ ID NO: 2 with lysine in a polypeptide consisting of an amino acid sequence having at least 97% identity with the amino acid sequence of SEQ ID NO: 14 and having lipase activity; (xix) A step of substituting the amino acid residue at the position corresponding to position 181 of the amino acid sequence of SEQ ID NO: 2 with leucine in a polypeptide consisting of an amino acid sequence having at least 83% identity with the amino acid sequence of SEQ ID NO: 14 and having lipase activity; (xx) A step of substituting the amino acid residue at the position corresponding to position 90 of the amino acid sequence of SEQ ID NO: 2 with cysteine in a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 16 and having lipase activity; (xxi) A step of substituting the amino acid residue at the position corresponding to position 181 of the amino acid sequence of SEQ ID NO: 2 with leucine in a polypeptide consisting of an amino acid sequence having at least 81% identity with the amino acid sequence of SEQ ID NO: 16 and having lipase activity; (xxii) A polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 34 and having lipase activity, wherein the amino acid residue at the position corresponding to position 29 of the amino acid sequence of SEQ ID NO: 2 is substituted with tyrosine. **Claim 13** A method for producing a lipase variant, comprising any one of the following steps (xxiii) to (xxvii): (xxiii) A polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 4 and having lipase activity, wherein at least two amino acid residues at positions selected from the positions corresponding to positions 31, 32, 90 and 181 of the amino acid sequence of SEQ ID NO: 2 are substituted with tyrosine at the position corresponding to position 31, glycine at the position corresponding to position 32, cysteine at the position corresponding to position 90, and leucine at the position corresponding to position 181; (xxiv) A polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 8 and having lipase activity, wherein at least two amino acid residues at positions selected from the positions corresponding to positions 39, 125, 126 and 293 of the amino acid sequence of SEQ ID NO: 2 are substituted with lysine at the position corresponding to position 39, threonine at the position corresponding to position 125, proline at the position corresponding to position 126, and glycine at the position corresponding to position 293; (xxv) A polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 10 and having lipase activity, wherein at least two amino acid residues at positions selected from the positions corresponding to positions 29, 31, 32 and 186 of the amino acid sequence of SEQ ID NO: 2 are substituted with tyrosine at the position corresponding to position 29, tyrosine at the position corresponding to position 31, glycine at the position corresponding to position 32, and glycine at the position corresponding to position 186; (xxvi) A polypeptide consisting of an amino acid sequence having at least 83% identity with the amino acid sequence of SEQ ID NO: 14 and having lipase activity, wherein at least two amino acid residues at positions selected from the positions corresponding to positions 31, 32, 77 and 181 of the amino acid sequence of SEQ ID NO: 2 are substituted with tyrosine at the position corresponding to position 31, glycine at the position corresponding to position 32, lysine at the position corresponding to position 77, and leucine at the position corresponding to position 181; (xxvii) A step of substituting the amino acid residue at the position corresponding to position 90 of the amino acid sequence of SEQ ID NO: 2 with cysteine and substituting the amino acid residue at the position corresponding to position 181 with leucine in a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 16 and having lipase activity. **Claim 14** A method for improving the heterologous expression of lipase, comprising any one of the following steps (i) to (xxii): (i) A step of substituting the amino acid residue at the position corresponding to position 118 of the amino acid sequence of SEQ ID NO: 2 with aspartic acid in a polypeptide consisting of an amino acid sequence having at least 82% identity with the amino acid sequence of SEQ ID NO: 2 and having lipase activity; (ii) A step of substituting the amino acid residue at the position corresponding to position 31 of the amino acid sequence of SEQ ID NO: 2 with tyrosine in a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 4 and having lipase activity; (iii) A step of substituting the amino acid residue at the position corresponding to position 32 of the amino acid sequence of SEQ ID NO: 2 with glycine in a polypeptide consisting of an amino acid sequence having at least 91% identity with the amino acid sequence of SEQ ID NO: 4 and having lipase activity; (iv) A step of substituting the amino acid residue at the position corresponding to position 90 of the amino acid sequence of SEQ ID NO: 2 with cysteine in a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 4 and having lipase activity; (v) A step of substituting the amino acid residue at the position corresponding to position 181 of the amino acid sequence of SEQ ID NO: 2 with leucine in a polypeptide consisting of an amino acid sequence having at least 94% identity with the amino acid sequence of SEQ ID NO: 4 and having lipase activity; (vi) A step of substituting the amino acid residue at the position corresponding to position 222 of the amino acid sequence of SEQ ID NO: 2 with alanine, threonine, histidine or glycine in a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 6 and having lipase activity; (vii) A step of substituting the amino acid residue at the position corresponding to position 39 of the amino acid sequence of SEQ ID NO: 2 with lysine in a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 8 and having lipase activity; (viii) A step of substituting the amino acid residue at the position corresponding to position 125 of the amino acid sequence of SEQ ID NO: 2 with threonine in a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 8 and having lipase activity; (ix) A step of substituting the amino acid residue at the position corresponding to position 126 of the amino acid sequence of SEQ ID NO: 2 with proline in a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 8 and having lipase activity; (x) A step of substituting the amino acid residue at the position corresponding to position 293 of the amino acid sequence of SEQ ID NO: 2 with glycine in a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 8 and having lipase activity; (xi) A step of substituting the amino acid residue at the position corresponding to position 29 of the amino acid sequence of SEQ ID NO: 2 with tyrosine in a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 10 and having lipase activity; (xii) A step of substituting the amino acid residue at the position corresponding to position 31 of the amino acid sequence of SEQ ID NO: 2 with tyrosine in a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 10 and having lipase activity; (xiii) A step of substituting the amino acid residue at the position corresponding to position 32 of the amino acid sequence of SEQ ID NO: 2 with glycine in a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 10 and having lipase activity; (xiv) A step of substituting the amino acid residue at the position corresponding to position 186 of the amino acid sequence of SEQ ID NO: 2 with glycine in a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 10 and having lipase activity; (xv) A step of substituting the amino acid residue at the position corresponding to position 206 of the amino acid sequence of SEQ ID NO: 2 with glutamine in a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 12 and having lipase activity; (xvi) A step of substituting the amino acid residue at the position corresponding to position 31 of the amino acid sequence of SEQ ID NO: 2 with tyrosine in a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 14 and having lipase activity; (xvii) A step of substituting the amino acid residue at the position corresponding to position 32 of the amino acid sequence of SEQ ID NO: 2 with glycine in a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 14 and having lipase activity; (xviii) A step of substituting the amino acid residue at the position corresponding to position 77 of the amino acid sequence of SEQ ID NO: 2 with lysine in a polypeptide consisting of an amino acid sequence having at least 97% identity with the amino acid sequence of SEQ ID NO: 14 and having lipase activity; (xix) A step of substituting the amino acid residue at the position corresponding to position 181 of the amino acid sequence of SEQ ID NO: 2 with leucine in a polypeptide consisting of an amino acid sequence having at least 83% identity with the amino acid sequence of SEQ ID NO: 14 and having lipase activity; (xx) A step of substituting the amino acid residue at the position corresponding to position 90 of the amino acid sequence of SEQ ID NO: 2 with cysteine in a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 16 and having lipase activity; (xxi) A step of substituting the amino acid residue at the position corresponding to position 181 of the amino acid sequence of SEQ ID NO: 2 with leucine in a polypeptide consisting of an amino acid sequence having at least 81% identity with the amino acid sequence of SEQ ID NO: 16 and having lipase activity; (xxii) A step of substituting the amino acid residue at the position corresponding to position 29 of the amino acid sequence of SEQ ID NO: 2 with tyrosine in a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 34 and having lipase activity. (Claim 15) (A method for improving the heterologous expression of lipase, comprising any one of the following steps (xxiii) to (xxvii): (xxiii) In a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 4 and having lipase activity, substituting at least two amino acid residues selected from the positions corresponding to positions 31, 32, 90, and 181 of the amino acid sequence of SEQ ID NO: 2 with tyrosine at the position corresponding to position 31, glycine at the position corresponding to position 32, cysteine at the position corresponding to position 90, and leucine at the position corresponding to position 181; (xxiv) a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 8 and having lipase activity, wherein at least two amino acid residues at positions selected from the positions corresponding to positions 39, 125, 126 and 293 of the amino acid sequence of SEQ ID NO: 2 are lysine at the position corresponding to position 39, threonine at the position corresponding to position 125, proline at the position corresponding to position 126, and glycine at the position corresponding to position 293; (xxv) a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 10 and having lipase activity, wherein at least two amino acid residues at positions selected from the positions corresponding to positions 29, 31, 32 and 186 of the amino acid sequence of SEQ ID NO: 2 are tyrosine at the position corresponding to position 29, tyrosine at the position corresponding to position 31, glycine at the position corresponding to position 32, and glycine at the position corresponding to position 186; (xxvi) a polypeptide consisting of an amino acid sequence having at least 83% identity with the amino acid sequence of SEQ ID NO: 14 and having lipase activity, wherein at least two amino acid residues at positions selected from the positions corresponding to positions 31, 32, 77 and 181 of the amino acid sequence of SEQ ID NO: 2 are tyrosine at the position corresponding to position 31, glycine at the position corresponding to position 32, lysine at the position corresponding to position 77, and leucine at the position corresponding to position 181; (xxvii) a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 16 and having lipase activity, wherein the amino acid residue at the position corresponding to position 90 of the amino acid sequence of SEQ ID NO: 2 is substituted with cysteine and the amino acid residue at the position corresponding to position 181 is substituted with leucine.

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