Polypeptide having esterification activity for L-menthol and / or hydrolysis activity for L-menthol ester

Abstract

The present invention provides a technology capable of further improving substrate specificity for L-menthol and / or its esters in the production of L-menthol and / or its esters. A120G, Q88A, Q88G, Q88D, Q88M, Q88L mutants of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, and any different site other than the amino acid residue into which the above substitution is introduced in the mutant, a polypeptide having esterification activity for L-menthol and / or hydrolysis activity for L-menthol esters, and having improved substrate specificity for L-menthol and / or L-menthol esters compared to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, can further improve the optical purity of the product in the production of L-menthol and / or its esters.

Description

Technical Field

[0001] The present invention relates to a polypeptide having an esterification activity for L-menthol and / or an activity for hydrolyzing L-menthol esters. More specifically, the present invention relates to a polypeptide having an esterification activity for L-menthol and / or an activity for hydrolyzing L-menthol esters and having improved substrate specificity for L-menthol and / or L-menthol esters, a DNA encoding the polypeptide, a recombinant vector, a transformant, an enzyme composition, an enzyme preparation, a method for producing the polypeptide, and a method for producing L-menthol esters and L-menthol using the polypeptide. Background Art

[0002] L-menthol is an important substance commonly used in the fields of fragrance, food, medicine, etc. due to its characteristics such as refreshing flavor and refreshing skin feeling. In addition, menthol esters are not only used as raw materials for L-menthol, but also L-menthol esters themselves are used in the fields of fragrance, food, medicine, etc.

[0003] These components are manufactured industrially by artificial synthesis. On the other hand, the synthesized L-menthol and its esters are mixed with the D-type, which is its optical isomer. The quality of L-menthol and its esters is greatly affected by the mixing of the D-type. Therefore, it is necessary to obtain L-menthol and its esters with high optical purity.

[0004] As methods for optically selectively obtaining L-menthol and its esters, chemical methods and enzymatic methods can be cited. As chemical methods, methods such as reacting an optically active acid or base with DL-menthol to selectively crystallize the L-form can be cited. In addition, as enzymatic methods, methods such as allowing lipase to act on DL-menthol esters in an aqueous solvent to specifically hydrolyze the L-form and methods such as allowing lipase to act on DL-menthol in an organic solvent to specifically esterify the L-form can be cited.

[0005] Among them, various studies are being conducted on enzymatic methods from the perspective of the high specificity of enzymes. For example, non-patent document 1 states that racemic lauric acid menthyl ester is hydrolyzed in an aqueous medium using lipase from Candida rugosa to preferentially produce L-menthol (ee: 70%). Such enantioselectivity can also be observed when racemic menthol is esterified with lauric acid. For example, racemic menthol is enantioselectively esterified with lauric acid in a non-aqueous medium using lipase from Candida rugosa to preferentially produce L-menthyl laurate (ee: 95%).

[0006] Furthermore, Non-Patent Document 2 describes that racemic menthol is esterified with acetic anhydride, propionic anhydride and butyric anhydride using a lipase derived from Candida rugosa in a specific enantiomer-selective manner, particularly with butyric anhydride in n-hexane, to preferentially produce L-menthyl butyrate (ee: 86%).

[0007] Furthermore, Non-Patent Document 3 describes that racemic menthol is esterified with propionic anhydride using lipase derived from Candida rugosa to produce L-menthyl propionate having a very high optical purity (ee: 95%).

[0008] Prior art literature

[0009] Non-patent literature

[0010] Non-patent document 1: Tetrahedron Letters, Vol. 27, No. 1, pp 29-32, 1986

[0011] Non-patent document 2: Enzyme and Microbial Technology Volume 18, Issue 7, 1996, pp536-539

[0012] Non-patent document 3: Applied Microbiology and Biotechnology. 1995, Volume 43, Issue 4, pp 639-643 Summary of the invention

[0013] Technical problem to be solved by the invention

[0014] However, there is still room for improvement in the enantioselectivity obtained in the production of L-menthol and / or its esters.

[0015] Therefore, an object of the present invention is to provide a technique capable of further improving substrate specificity for the L-form in the production of L-menthol and / or its esters.

[0016] Technical solutions for solving technical problems

[0017] As a result of the inventor's intensive research, the inventor focused on the high enantiomeric selectivity of the lipase from Burkholderia cepacia, and further comprehensively examined the substrate specificity for L-menthol and / or its esters in the production of L-menthol and / or its esters by introducing mutations into more than 840 lipase mutants obtained by introducing mutations into various parts of the lipase. As a result, it was found that a polypeptide consisting of an amino acid sequence in which the amino acid residue at position 120 of the lipase is replaced by a glycine residue and a polypeptide consisting of an amino acid sequence in which the amino acid residue at position 88 is replaced by an alanine residue, a glycine residue, an aspartic acid residue, a methionine residue or a leucine residue can improve the substrate specificity for L-menthol. The present invention is completed based on this knowledge. That is, the present invention provides an invention of the following disclosed mode.

[0018] Item 1. A polypeptide represented by any one of the following (1) to (3):

[0019] (1) a polypeptide consisting of an amino acid sequence in which the amino acid residue at position 120 in the amino acid sequence of SEQ ID NO: 1 is substituted with a glycine residue,

[0020] (2) an amino acid sequence in which the amino acid residue at position 120 in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with a glycine residue, and one or more amino acid residues other than the amino acid residue into which the substitution is introduced are substituted, added, inserted or deleted, and a polypeptide having an esterification activity for L-menthol and / or a hydrolysis activity for L-menthol ester and having improved substrate specificity for L-menthol and / or L-menthol ester compared to a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1;

[0021] (3) An amino acid sequence in which the amino acid residue at position 120 in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with a glycine residue, wherein the sequence identity with respect to the amino acid sequence set forth in SEQ ID NO: 1 excluding the amino acid residue into which the above substitution has been introduced is 80% or more, and a polypeptide having an esterification activity for L-menthol and / or a hydrolysis activity for L-menthol ester and having improved substrate specificity for L-menthol and / or L-menthol ester compared to a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1.

[0022] Item 2. A polypeptide represented by any one of the following (4) to (6):

[0023] (4) a polypeptide consisting of an amino acid sequence in which the amino acid residue at position 88 in the amino acid sequence of SEQ ID NO: 1 is substituted with an alanine residue, a glycine residue, an aspartic acid residue, a methionine residue or a leucine residue,

[0024] (5) an amino acid sequence in which the amino acid residue at position 88 in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with an alanine residue, a glycine residue, an aspartic acid residue, a methionine residue or a leucine residue, and one or more amino acid residues other than the amino acid residue into which the substitution is introduced are substituted, added, inserted or deleted, and a polypeptide having an esterification activity for L-menthol and / or an activity for hydrolyzing L-menthol esters and having improved substrate specificity for L-menthol and / or L-menthol esters compared to a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1;

[0025] (6) An amino acid sequence in which the amino acid residue at position 88 in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with an alanine residue, a glycine residue, an aspartic acid residue, a methionine residue or a leucine residue, wherein the sequence identity with respect to the amino acid sequence set forth in SEQ ID NO: 1 excluding the amino acid residue into which the above substitution is introduced is 80% or more, and a polypeptide having an esterification activity for L-menthol and / or a hydrolysis activity for L-menthol ester and having improved substrate specificity for L-menthol and / or L-menthol ester compared to a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1.

[0026] Item 3. A DNA encoding the polypeptide described in Item 1 or 2.

[0027] Item 4. A recombinant vector comprising the DNA described in Item 3.

[0028] Item 5. A transformant obtained by transforming a host with the recombinant vector described in Item 4.

[0029] Item 6. A method for producing the polypeptide according to claim 1 or 2, comprising the step of culturing the transformant according to Item 5.

[0030] Item 7. An enzyme composition comprising the polypeptide described in Item 1 or 2.

[0031] Item 8. An enzyme preparation comprising the polypeptide described in Item 1 or 2 or the enzyme composition described in Item 7.

[0032] Item 9. A method for producing an L-menthol ester, comprising the step of allowing the polypeptide according to Item 1 or 2, the enzyme composition according to Item 7, or the enzyme preparation according to Item 8 to act on a mixture containing L-menthol and D-menthol to esterify L-menthol.

[0033] Item 10. A method for producing L-menthol, comprising the step of allowing the polypeptide according to Item 1 or 2, the enzyme composition according to Item 7, or the enzyme preparation according to Item 8 to act on a mixture containing L-menthol ester and D-menthol ester to hydrolyze the L-menthol ester.

[0034] Effects of the Invention

[0035] According to the present invention, a technique capable of further improving substrate specificity for the L-form in the production of L-menthol and / or its ester can be provided. DETAILED DESCRIPTION

[0036] The present invention is described in detail below. It should be noted that, outside the sequence table, the 20 kinds of amino acid residues in the amino acid sequence are sometimes represented by single-letter symbols. That is, glycine (Gly) is G, alanine (Ala) is A, valine (Val) is V, leucine (Leu) is L, isoleucine (Ile) is I, phenylalanine (Phe) is F, tyrosine (Tyr) is Y, tryptophan (Trp) is W, serine (Ser) is S, threonine (Thr) is T, cysteine ​​(Cys) is C, methionine (Met) is M, aspartic acid (Asp) is D, glutamic acid (Glu) is E, asparagine (Asn) is N, glutamine (Gln) is Q, lysine (Lys) is K, arginine (Arg) is R, histidine (His) is H, and proline (Pro) is P.

[0037] In the present specification, the amino acid sequence shown is the N-terminus on the left and the C-terminus on the right.

[0038] In the present specification, "A120G" and the like are expressions indicating amino acid substitutions. For example, "A120G" indicates that the amino acid A at position 120 from the N-terminal side in a specific amino acid sequence is substituted with the amino acid G.

[0039] In this specification, "non-polar amino acids" include alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan. "Non-charged amino acids" include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. "Acidic amino acids" include aspartic acid and glutamic acid. "Basic amino acids" include lysine, arginine, and histidine.

[0040] In this specification, "replacement" includes not only the case where an amino acid residue replacement is artificially introduced, but also the case where an amino acid residue replacement is introduced by a natural method, i.e., the original amino acid residue is different. In this specification, the replacement of amino acid residues can be artificial replacement or natural replacement, preferably artificial replacement.

[0041] 1. Peptides

[0042] The polypeptide of the present invention is a polypeptide represented by any one of the following (1) to (3), or a polypeptide represented by any one of the following (4) to (6).

[0043] (1) a polypeptide consisting of an amino acid sequence in which the amino acid residue (alanine residue) at position 120 in the amino acid sequence shown in SEQ ID NO: 1 is substituted with a glycine residue,

[0044] (2) an amino acid sequence in which the amino acid residue at position 120 in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with a glycine residue, wherein one or more amino acid residues other than the amino acid residue into which the substitution is introduced are substituted, added, inserted or deleted, and a polypeptide having an esterification activity for L-menthol and / or a hydrolysis activity for L-menthol ester and having improved substrate specificity for L-menthol and / or L-menthol ester compared to a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1;

[0045] (3) An amino acid sequence in which the amino acid residue at position 120 in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with a glycine residue, wherein the sequence identity with respect to the amino acid sequence set forth in SEQ ID NO: 1 excluding the amino acid residue into which the above substitution has been introduced is 80% or more, and a polypeptide having an esterification activity for L-menthol and / or a hydrolysis activity for L-menthol ester and having improved substrate specificity for L-menthol and / or L-menthol ester compared to a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1.

[0046] (4) a polypeptide consisting of an amino acid sequence in which the amino acid residue at position 88 (glutamine residue) in the amino acid sequence shown in SEQ ID NO: 1 is substituted with an alanine residue, a glycine residue, an aspartic acid residue, a methionine residue or a leucine residue,

[0047] (5) an amino acid sequence in which the amino acid residue at position 88 in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with an alanine residue, a glycine residue, an aspartic acid residue, a methionine residue or a leucine residue, and one or more amino acid residues other than the amino acid residue into which the substitution is introduced are substituted, added, inserted or deleted, and a polypeptide having an esterification activity for L-menthol and / or a hydrolysis activity for L-menthol ester and having improved substrate specificity for L-menthol and / or L-menthol ester compared to a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1;

[0048] (6) An amino acid sequence in which the amino acid residue at position 88 in the amino acid sequence set forth in SEQ ID NO: 1 is substituted with an alanine residue, a glycine residue, an aspartic acid residue, a methionine residue or a leucine residue, wherein the sequence identity with respect to the amino acid sequence set forth in SEQ ID NO: 1 excluding the amino acid residue into which the above substitution is introduced is 80% or more, and a polypeptide having an esterification activity for L-menthol and / or a hydrolysis activity for L-menthol ester and having improved substrate specificity for L-menthol and / or L-menthol ester compared to a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1.

[0049] The polypeptides described in (1) to (6) above have esterification activity for L-menthol and / or hydrolysis activity for L-menthol esters, and have improved substrate specificity for L-menthol and / or L-menthol esters. Preferably, the polypeptides described in (1) to (6) above have improved substrate specificity for L-menthol and L-menthol esters.

[0050] The polypeptide of SEQ ID NO: 1 is a wild-type lipase (mature form) derived from Burkholderia cepacia.

[0051] The polypeptides described in (1) to (6) above include not only polypeptides obtained by artificial substitution but also polypeptides originally having such amino acid sequences.

[0052] The polypeptide of (1) and the polypeptide of (4) also include a polypeptide comprising both substitution at position 120 and substitution at position 88.

[0053] Hereinafter, the polypeptides of (2) and (3) and (5) and (6) above, other than the amino acid residue at position 120 or the amino acid residue at position 88 of sequence number 1, are sometimes described as "any difference site". In the present specification, the term "any difference site" refers to a site that is allowed to be different as long as it does not significantly affect the properties of the polypeptide. In addition, in the present specification, a substance that has a substrate specificity for L-menthol and / or L-menthol esters that is equal to or higher than that of the polypeptide of (1) or (4) is referred to as a variant of the polypeptide of (1) or (4). In addition, the variant of the polypeptide preferably has a difference in amino acid sequence at any difference site compared to the polypeptide of (1) or (4), but has substantially the same properties as that of the polypeptide of (1) or (4). It should be noted that "substantially the same" means having substrate specificity for L-menthol and / or L-menthol esters. The polypeptides of (2) and (3) are variants of the polypeptide of (1), and the polypeptides of (5) and (6) are variants of the polypeptide of (4).

[0054] The difference in amino acids in the polypeptides of (2) and (5) above may include only one difference among substitution, addition, insertion and deletion (e.g. substitution), or may include two or more differences (e.g. substitution and insertion). In the polypeptides of (2) and (5) above, the number of amino acid differences at any difference site may be one or more, and may include, for example, 1 to 50, preferably 1 to 20, 1 to 10, 1 to 8, 1 to 7, 1 to 6, 1 to 5, or 1 to 4, more preferably 1 to 3, and particularly preferably 1 or 2 or 1.

[0055] In addition, in the polypeptides of (2) and (5) above, the sequence identity with respect to each amino acid sequence shown in sequence number 1, excluding the site where the aforementioned amino acid is substituted, may be 80% or more, preferably 85% or more or 90% or more, further preferably 95% or more, 96% or more, 97% or more, or 98% or more, and particularly preferably 99% or more.

[0056] Here, in the polypeptides of (3) and (6), the sequence identity with respect to each amino acid sequence shown in SEQ ID NO: 1, excluding the site where the above amino acid is substituted, is the sequence identity calculated by extracting only the above arbitrary difference site from each amino acid sequence shown in SEQ ID NO: 1 and comparing only the arbitrary difference site. In addition, "sequence identity" means the value of the identity of the amino acid sequence obtained by using BLASTPACKAGE [sgi32 bit edition, Version 2.0.12; available from National Center for Biotechnology Information (NCBI)] bl2seq program (Tatiana A. Tatsusova, Thomas L. Madden, FEMS Microbiol. Lett., Vol. 174, p247-250, 1999). The parameters can be set to Gap insertion Cost value: 11, Gap extension Cost value: 1.

[0057] It is believed that the amino acids at positions 87 (serine), 264 (aspartic acid), and 286 (histidine) in the amino acid sequence shown in SEQ ID NO: 1 in the polypeptides of (2) and (3) and (5) and (6) above contribute to the esterification activity for L-menthol and / or the hydrolysis activity for L-menthol ester, and therefore it is desirable not to introduce substitutions or deletions at these positions.

[0058] When amino acid substitution is introduced into the polypeptides of (2) and (3) and (5) and (6), examples of the amino acid substitution include conservative substitution. That is, examples of the amino acid substitution introduced into the polypeptides of (2) and (3) and (5) and (6) relative to the amino acid sequence shown in SEQ ID NO: 1 include: when the non-polar amino acid of the amino acid before substitution is substituted with another non-polar amino acid, when the non-charged amino acid of the amino acid before substitution is substituted with another non-charged amino acid, when the acidic amino acid of the amino acid before substitution is substituted with another acidic amino acid, and when the basic amino acid of the amino acid before substitution is substituted with another basic amino acid.

[0059] In the polypeptides of (2) and (3) above and (5) and (6) above, the term “a polypeptide having an esterification activity for L-menthol and / or a hydrolysis activity for L-menthol ester, and having improved substrate specificity for L-menthol and / or L-menthol ester compared to the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1” means (i) having an esterification activity for L-menthol and / or a hydrolysis activity for L-menthol ester, and (ii-a) with respect to substrate specificity for L-menthol, the optical purity of L-menthol acetate measured under the conditions of the following Test Example 2 is 1.007 times or more, preferably 1.009 times or more, and more preferably 1.010 times or more, of that of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1. (ii-b) with respect to substrate specificity for L-menthol esters, the ratio of the conversion rate to L-menthol to the conversion rate to D-menthol (L / D conversion rate) measured under the conditions of the following Test Example 1 is 1.05 times or more, preferably 1.12 times or more, more preferably 1.19 times or more, further preferably 1.26 times or more, and even more preferably 1.32 times or more of the L / D conversion rate of the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1.

[0060] 2. DNA

[0061] The DNA encoding the polypeptide of the present invention (hereinafter, sometimes referred to as "DNA of the present invention") can be obtained, for example, by introducing the aforementioned amino acid mutation into the DNA encoding the amino acid sequence of the wild-type lipase (SEQ ID NO: 1). In addition, the DNA of the present invention can also be artificially synthesized by a total gene synthesis method.

[0062] DNA encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 is known, for example, the base sequence shown in SEQ ID NO: 2, and can be isolated from genomic DNA of Burkholderia cepacia strain M-12-33 by a conventional method using PCR.

[0063] Methods for introducing specific mutations into specific sites of base sequences are well known, and for example, site-specific mutation introduction methods of DNA can be used. As methods for converting bases in DNA, for example, commercially available kits (QuickChange Lightning Site-Directed Mutagenesis kit: manufactured by Stratagene, KOD-Plus-Mutagenesis kit: manufactured by Toyobo, etc.) can be cited.

[0064] The DNA after the mutation is introduced into the base sequence can be confirmed by using a DNA sequencer. Once the base sequence is determined, the DNA encoding the above polypeptide can be obtained by chemical synthesis, PCR using a cloned probe as a template, or hybridization using a DNA fragment having the base sequence as a probe.

[0065] In addition, a mutant DNA encoding the aforementioned peptide and having the same function as before the mutation can also be synthesized by site-directed mutagenesis, etc. It should be noted that when introducing mutations into the DNA encoding the aforementioned peptide, it can be carried out by known methods such as the Kunkel method, the Gapped duplex method, and the megaprimer PCR method.

[0066] The DNA of the present invention preferably has codon usage frequency optimized to be suitable for the host, and more preferably has codon usage frequency optimized to be suitable for Escherichia coli.

[0067] As an index representing the frequency of codon utilization, the total of the host's optimal codon utilization frequency of each codon can be selected. The optimal codon can be defined as the codon with the highest utilization frequency among the codons corresponding to the same amino acid. The frequency of codon utilization is not particularly limited as long as it is optimized to be suitable for the host. For example, as an example of the optimal codon for Escherichia coli, the following examples can be cited. F: phenylalanine (ttt), L: leucine (ctg), I: isoleucine (att), M: methionine (atg), V: valine (gtg), Y: tyrosine (tat), stop codon (taa), H: histidine (cat), Q: glutamine (cag), N: asparagine (aat), K: lysine (aaa), D: aspartic acid (gat), E: glutamic acid (gaa), S: serine (agc), P: proline (ccg), T: threonine (acc), A: alanine (gcg), C: cysteine ​​(tgc), W: tryptophan (tgg), R: arginine (cgc), G: glycine (ggc).

[0068] Examples of the DNA of the present invention include DNAs comprising the base sequences shown in sequence numbers 3 and 13 to 17. The DNA consisting of the base sequence shown in sequence number 3 encodes a polypeptide in which the amino acid residue at position 120 of the polypeptide shown in sequence number 1 described in (1) above is substituted with a glycine residue. The DNA consisting of the base sequences shown in sequence numbers 13, 14, 15, 16, and 17 encodes a polypeptide in which the amino acid residue at position 88 of the amino acid sequence of the polypeptide shown in sequence number 1 described in (4) above is substituted with an alanine residue, a glycine residue, an aspartic acid residue, a methionine residue, and a leucine residue, respectively.

[0069] Other examples of the DNA of the present invention include DNAs encoding a polypeptide having improved substrate specificity for L-menthol and / or L-menthol esters compared to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, and which hybridize under stringent conditions with a DNA comprising a complementary base sequence to a DNA consisting of the base sequences shown in SEQ ID NOs: 3 and 13 to 17, respectively.

[0070] Here, "stringent conditions" refers to the conditions of incubation at 50°C to 65°C for 4 hours to overnight in 6×SSC (1×SSC is 0.15M NaCl, 0.015M sodium citrate, pH 7.0) containing 0.5% SDS, 5×Denhartz's [Denhartz's, 0.1% bovine serum albumin (BSA), 0.1% polyvinyl pyrrolidone, 0.1% FICOLL400] and 100μg / ml fish sperm DNA.

[0071] Hybridization under stringent conditions is specifically performed by the following method. That is, a nylon membrane with a DNA library or cDNA library fixed thereon is prepared, and the nylon membrane is blocked at 65°C in a prehybridization solution containing 6×SSC, 0.5% SDS, 5×Denhartz's, and 100 μg / ml fish sperm DNA. 32 Each probe labeled with P was incubated overnight at 65° C. The nylon membrane was washed in 6×SSC at room temperature for 10 minutes, in 2×SSC containing 0.1% SDS at room temperature for 10 minutes, and in 0.2×SSC containing 0.1% SDS at 45° C. for 30 minutes, and then autoradiography was performed to detect DNA that was specifically hybridized with the probe.

[0072] Another example of the DNA of the present invention includes a DNA encoding a polypeptide having improved substrate specificity for L-menthol and / or L-menthol esters compared to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, and having 80% or more homology with the DNA consisting of the base sequences shown in SEQ ID NOs: 3 and 13 to 17, respectively. The homology is preferably 85% or more or 90% or more, more preferably 95% or more, 96% or more, or 97% or more, and particularly preferably 98% or more or 99% or more.

[0073] Here, the "homology" of DNA can be calculated using an algorithm that compares the reference sequence with the query sequence, a public or commercially available software. Specifically, BLAST, FASTA or GENETYX (Software Development Co., Ltd.) etc. can be used, and these can be used with the setting of default parameters.

[0074] 3. Recombinant vector

[0075] A recombinant vector comprising a DNA encoding the peptide of the present invention (hereinafter, sometimes referred to as "the recombinant vector of the present invention") can be obtained by inserting the DNA of the present invention into an expression vector.

[0076] The recombinant vector of the present invention includes a promoter and other control factors that are operably linked to the DNA of the present invention. Representative examples of control factors include promoters, and transcription factors such as enhancers, CCAAT boxes, TATA boxes, and SPI sites may be included as needed. In addition, operably linked means that various control factors such as promoters and boxes that regulate the DNA of the present invention are linked to the DNA of the present invention in an operably state in a host cell.

[0077] As an expression vector, an expression vector constructed for gene recombination purposes by a phage, plasmid, or virus that can autonomously proliferate in a host is preferred. Such expression vectors are well known, and for example, as commercially available expression vectors, pQE vectors (QIAGEN Co., Ltd.), pDR540, pRIT2T (GE Healthcare Bioscience Co., Ltd.), pET vectors (Merck Co., Ltd.), etc. can be listed. A suitable combination of an expression vector and a host cell can be selected and used. For example, when Escherichia coli is used as a host cell, a combination of a pET vector and a DH5α Escherichia coli strain, a combination of a pET vector and a BL21 (DE3) Escherichia coli strain, or a combination of a pDR540 vector and a JM109 Escherichia coli strain can be preferably listed.

[0078] 4. Transformants

[0079] By transforming a host with the recombinant vector of the present invention, a transformant (hereinafter sometimes referred to as "the transformant of the present invention") can be obtained.

[0080] As the host used in the preparation of the transformant, there is no particular limitation as long as the recombinant vector is stable and can proliferate autonomously and can express the properties of the exogenous gene, and examples thereof include bacteria belonging to the genus Escherichia, such as Escherichia coli, bacteria belonging to the genus Bacillus, such as Bacillus subtilis, and bacteria belonging to the genus Pseudomonas, such as Pseudomonas putida; yeast, etc. are preferred examples, and animal cells, insect cells, plants, etc. may also be used. Among these, Escherichia coli is particularly preferred.

[0081] The transformant of the present invention can be obtained by introducing the recombinant vector of the present invention into the host, and the conditions for introducing the recombinant vector into the host can be appropriately set according to the type of the host, etc. When the host is bacteria, for example, methods using competent cells obtained by calcium ion treatment and electroporation methods can be mentioned. When the host is yeast, for example, electroporation methods (electroporation method), protoplast method and lithium acetate method can be mentioned. When the host is animal cells, for example, electroporation methods, calcium phosphate methods and liposome methods can be mentioned. When the host is insect cells, for example, calcium phosphate methods, liposome methods and electroporation methods can be mentioned. When the host is plant cells, for example, electroporation methods, Agrobacterium methods, gene gun methods, and PEG methods can be mentioned.

[0082] Whether the recombinant vector of the present invention is integrated into the host can be confirmed by PCR, DNA hybridization, Northern hybridization, and the like.

[0083] When confirming whether the recombinant vector of the present invention is integrated into the host by the PCR method, for example, the recombinant vector can be isolated and purified from the transformant.

[0084] Regarding the separation / purification of the recombinant vector, for example, in the case of host bacteria, it can be carried out based on the lysate obtained by lysing the bacteria. As a lysis method, for example, lysozymes such as lysozyme can be used for treatment, and proteases and other enzymes and surfactants such as sodium dodecyl sulfate (SDS) can be used in combination as needed.

[0085] Furthermore, physical disruption methods such as freeze-thaw and French press treatment may be combined. DNA can be isolated and purified from the lysate by, for example, a suitable combination of deproteinization treatments such as phenol treatment and protease treatment, nuclease treatment, alcohol precipitation treatment, and commercially available kits.

[0086] DNA cleavage can be performed by conventional methods, for example, using restriction enzyme treatment. As restriction enzymes, for example, type II restriction enzymes that act on specific nucleic acid sequences are used. DNA and expression vectors are connected using, for example, DNA ligase.

[0087] Then, using the separated / purified DNA as a template, designing primers specific to the DNA of the present invention and performing PCR. The amplified product obtained by PCR is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., and stained with ethidium bromide and SYBR Green solution, and then the amplified product is detected as a band, thereby confirming that transformation has occurred.

[0088] Alternatively, the amplification product may be detected by performing PCR using primers previously labeled with a fluorescent dye, etc. Furthermore, a method may be used in which the amplification product is bound to a solid phase such as a microplate and confirmed by fluorescence or enzyme reaction, etc.

[0089] 5. Method for producing polypeptide

[0090] The polypeptide of the present invention can be obtained by a production method comprising the step of culturing the transformant of the present invention.

[0091] The culture conditions of the transformant may be appropriately set in consideration of the nutritional and physiological properties of the host, and liquid culture is preferred. In addition, aeration and stirring culture is preferred for industrial production.

[0092] As the nutrient source of the medium, a nutrient source necessary for the growth of the transformant can be used. As the carbon source, any assimilable carbon compound may be used, and examples thereof include glucose, sucrose, lactose, maltose, molasses, and pyruvic acid.

[0093] The nitrogen source may be any assimilable nitrogen compound, and examples thereof include peptone, meat extract, yeast extract, casein hydrolyzate, and soybean meal alkali extract.

[0094] In addition to the carbon source and the nitrogen source, for example, phosphates, carbonates, sulfates, salts such as magnesium, calcium, potassium, iron, manganese and zinc, specific amino acids and specific vitamins can be used as needed.

[0095] The culture temperature can be appropriately set within the range in which the transformant of the present invention can grow and produce the polypeptide of the present invention, and is preferably about 15 to 37° C. The time when the polypeptide of the present invention reaches the maximum yield can be predicted and the culture can be completed at an appropriate time, which is usually about 12 to 48 hours.

[0096] The transformant of the present invention is cultured, the culture supernatant or the bacterial cells are recovered by a method such as centrifuging the culture solution, the bacterial cells are treated mechanically by ultrasonication and French press or by lysozymes such as lysozyme, and if necessary, they are solubilized using enzymes such as proteases and surfactants such as sodium dodecyl sulfate (SDS), thereby obtaining a water-soluble fraction containing the polypeptide of the present invention.

[0097] In addition, the expressed polypeptide of the present invention can be secreted into the culture medium by selecting an appropriate expression vector and host.

[0098] The water-soluble fraction containing the polypeptide of the present invention obtained as described above may be directly subjected to purification treatment, or the polypeptide of the present invention in the water-soluble fraction may be concentrated and then subjected to purification treatment.

[0099] The concentration can be performed, for example, by reduced pressure concentration, membrane concentration, salting-out treatment, or a fractional precipitation method using a hydrophilic organic solvent (eg, methanol, ethanol, and acetone).

[0100] The polypeptide of the present invention can be purified by, for example, appropriately combining methods such as gel filtration, adsorption chromatography, ion exchange chromatography, and affinity chromatography.

[0101] The above purification treatment is already known and can be performed with reference to appropriate literature, magazines, textbooks, etc. The polypeptide of the present invention purified in this way can be pulverized by freeze drying, vacuum drying, spray drying, etc. as needed and distributed on the market.

[0102] 6. Enzyme Composition

[0103] The polypeptide of the present invention can be provided in the form of a composition in which other components coexist, for example. Examples of the form of the enzyme composition include: a culture solution obtained during the production process of the polypeptide of the present invention and containing the polypeptide, a water-soluble fraction obtained from the culture solution and containing the polypeptide, or a composition in which the degree of purification of the polypeptide is increased to an arbitrary level by using the water-soluble fraction; an enzyme preparation described in "7. Enzyme preparation" below; a reaction mixture containing unreacted polypeptide obtained by using the polypeptide of the present invention for the production of L-menthol and / or its esters, etc.

[0104] Examples of other components contained in the enzyme composition include arbitrary components added, produced or mixed in the process of preparing the enzyme composition, such as: foreign protein components and / or components other than protein from the culture medium used for producing the polypeptide of the present invention; additives or bases shown in "7. Enzyme preparation" described later; unreacted raw materials and products contained in the reaction mixture obtained in the production of L-menthol and / or its esters, etc.

[0105] The enzyme composition may further comprise other enzymes. Examples of other enzymes include amylases (α-amylase, β-amylase, glucoamylase), glucosidases (α-glucosidases, β-glucosidases), galactosidases (α-galactosidases, β-galactosidases), proteases (acid proteases, neutral proteases, alkaline proteases), peptidases (leucine peptidases, aminopeptidases), lipases, esterases, cellulases, phosphatases (acid phosphatases, alkaline phosphatases), nucleases, deaminases, oxidases, dehydrogenases, glutaminases, pectinases, catalases, glucanases, transglutaminases, protein deaminases, pullulanase, and the like.

[0106] The content of the polypeptide of the present invention in the enzyme composition is not particularly limited, but preferably 10% by mass or more of the total protein in the enzyme composition can be cited, and more preferably 30% by mass or more of the total protein in the enzyme composition can be cited. The form of the enzyme composition is not particularly limited, and examples thereof include liquid, powder, granules, etc. The enzyme composition can be prepared by a generally known method or the method described in "8-3. Method for producing L-menthol ester" described later.

[0107] 7. Enzyme preparations

[0108] The polypeptide of the present invention or the enzyme composition comprising the polypeptide of the present invention can be provided in the form of an enzyme preparation, for example. The enzyme preparation is an enzyme composition prepared for the polypeptide of the present invention for use in the "8. Uses" described later, and comprises the polypeptide of the present invention as an active ingredient. In the enzyme preparation, in addition to the polypeptide of the present invention, additives or bases such as excipients, buffers, suspending agents, stabilizers, preservatives, preservatives, physiological saline, solvents, etc. can also be contained. As excipients, starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, white sugar, glycerol, etc. can be used. As buffers, phosphates, citrates, acetates, etc. can be used. As stabilizers, propylene glycol, ascorbic acid, etc. can be used. As preservatives, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methyl parahydroxybenzoate, etc. can be used. As preservatives, ethanol, benzalkonium chloride, parahydroxybenzoic acid, chlorobutanol, etc. can be used. Furthermore, the enzyme preparation may contain other ingredients (e.g., any ingredients added, produced, or mixed in during the production of the polypeptide as an active ingredient, etc.) to the extent that the effects of the present invention are not affected. The content of the polypeptide in the enzyme preparation may be appropriately set within the range in which the polypeptide can exert its effects.

[0109] 8. Purpose

[0110] The polypeptide of the present invention can be used for applications requiring esterification of L-menthol and hydrolysis of L-menthol esters. Applications requiring esterification of L-menthol include the production of L-menthol esters, and applications requiring hydrolysis of L-menthol esters include the production of L-menthol. More specific examples of these applications include: production of flavor compounds; production of additives for food and beverages, cosmetics, pharmaceuticals, or quasi-drugs; production of active ingredients for cosmetics, pharmaceuticals, or quasi-drugs; production of intermediates for active ingredients for cosmetics, pharmaceuticals, or quasi-drugs, etc.

[0111] 8-1. Substrate

[0112] L-menthol and / or L-menthol esters that are substrates for the polypeptide of the present invention are known as flavoring compounds; additives for foods, beverages, cosmetics, pharmaceuticals, or quasi-drugs; active ingredients for cosmetics, pharmaceuticals, or quasi-drugs; and intermediates for active ingredients for cosmetics, pharmaceuticals, or quasi-drugs.

[0113] L-Menthol is (1R, 2S, 5R)-5-methyl-2-(1-ethylethyl)cyclohexanol. The L-menthol ester is not particularly limited as long as it is an ester of L-menthol and a carboxylic acid, and specific examples thereof include compounds represented by the following formula (1).

[0114] [Chemical formula 1]

[0115]

[0116] In formula (1), R 1 It represents a linear or branched alkyl group having 1 to 20 carbon atoms, preferably 1 to 6 carbon atoms, a cycloalkyl group having 3 to 8 carbon atoms, an aryl group having 6 to 14 carbon atoms, an aralkyl group having 7 to 15 carbon atoms, an alkoxy group having 1 to 20 carbon atoms, or an alkylamino group having 1 to 20 carbon atoms. The above alkyl group, the above cycloalkyl group, the above aryl group, the above aralkyl group, the alkyl group of the above alkoxy group, and the alkyl group of the above alkylamino group may be unsubstituted or substituted, and examples of the substituents when substituted include a hydroxyl group, a formyl group, an alkoxy group having 1 to 6 carbon atoms, a carboxyl group, a mercapto group, a sulfo group, an amino group, an alkylamino group having 1 to 6 carbon atoms, a nitro group, and a halogen group.

[0117] Preferred examples of L-menthol esters include L-menthyl acetate, L-menthyl benzoate, L-menthyl isovalerate, L-menthyl lactate, L-menthyl succinate, L-menthyl propionate, and L-menthyl butyrate, and preferably L-menthyl acetate is used.

[0118] 8-2. How to use peptides

[0119] The use of the polypeptide of the present invention is not particularly limited, and examples thereof include free polypeptide and immobilized polypeptide. An immobilized polypeptide is a method of using the polypeptide of the present invention by fixing it to a carrier (e.g., ion exchange resin, porous resin, ceramic, calcium carbonate, etc.) according to a conventional method.

[0120] Furthermore, as the polypeptide of the present invention, one species may be used alone or a plurality of species may be used in combination.

[0121] 8-3. Method for producing L-menthol ester

[0122] The method for producing L-menthol ester of the present invention comprises the step of allowing the polypeptide of the present invention, the enzyme composition of the present invention, or the enzyme preparation of the present invention (hereinafter referred to as "polypeptide of the present invention, etc.") to act on a mixture containing L-menthol and D-menthol to esterify L-menthol.

[0123] In the method for producing L-menthol ester of the present invention, the polypeptide of the present invention and the like are preferably used in the form of an immobilized polypeptide in which the polypeptide is immobilized on a carrier.

[0124] It should be noted that the polypeptides of the present invention not only improve the substrate specificity for L-menthol, but also can achieve an excellent ester conversion rate of L-menthol compared to their transesterification activity values. Therefore, the polypeptides of the present invention used in the method for producing L-menthol esters can achieve a high ester conversion rate of L-menthol even if their transesterification activity values ​​are lower than the transesterification activity value of the wild-type lipase of sequence number 1. From such a viewpoint, the transesterification activity value of the polypeptides of the present invention can be, for example, 0.3 to 0.8 times, preferably 0.45 to 0.7 times, and more preferably 0.55 to 0.6 times relative to the transesterification activity value of the wild-type lipase of sequence number 1 of the same mass (ratio of transesterification activity values).

[0125] (Method for deriving the ratio of transesterification activity values)

[0126] The wild-type lipase of sequence number 1 or the improved lipase of the present invention was allowed to act on phenylethanol (20 parts by weight) and vinyl acetate (80 parts by weight) as substrates at 30°C for 20 minutes to carry out an ester exchange reaction. The obtained phenylethyl acetate was quantified by HPLC analysis. The amount of phenylethyl acetate obtained by the improved lipase of the present invention (ester activity value of the improved lipase of the present invention) was taken as the ratio of the ester exchange activity value when the amount of phenylethyl acetate obtained by the wild-type lipase (ester exchange activity value of the wild-type lipase) was set to 1.

[0127] The polypeptide or the like of the present invention can be used in an amount of, for example, 0.01 to 200 mg, preferably 0.03 to 20 mg, and more preferably 0.05 to 1 mg, based on 1 g of L-menthol.

[0128] Examples of the acylating agent include: 2 Carboxylic acid represented by COOH and its ester, as the ester, there can be mentioned the general formula R 2 COOR 3 The carboxylic acid alkyl ester shown in the general formula R 2 Vinyl carboxylate represented by COOCH=CH2, etc.

[0129] In the above general formula, R 2 represents a linear or branched alkyl group having 1 to 20 carbon atoms, preferably 1 to 6 carbon atoms, a cycloalkyl group having 3 to 8 carbon atoms, an aryl group having 6 to 14 carbon atoms, an aralkyl group having 7 to 15 carbon atoms, an alkoxy group having 1 to 20 carbon atoms, or an alkylamino group having 1 to 20 carbon atoms, and R 3represents a linear or branched alkyl group having 1 to 20 carbon atoms, preferably 1 to 6 carbon atoms. The above alkyl group, the above cycloalkyl group, the above aryl group, the above aralkyl group, the above alkyl group of the alkoxy group, and the above alkyl group of the alkylamino group may be unsubstituted or substituted, and examples of the substituents when substituted include hydroxyl, formyl, alkoxy group having 1 to 6 carbon atoms, carboxyl, mercapto, sulfo, amino, alkylamino group having 1 to 6 carbon atoms, nitro, and halogen groups. These acylating agents may be used alone or in combination of two or more.

[0130] In the method for producing L-menthol ester of the present invention, the polypeptide of the present invention and an acylating agent may be allowed to act on a mixture containing L-menthol and D-menthol. Preferred examples of the acylating agent include esters of carboxylic acids, more preferably vinyl carboxylate, further preferably vinyl acetate, vinyl propionate, vinyl butyrate, vinyl caproate, vinyl n-octanoate, vinyl caprate, vinyl laurate, vinyl myristate, vinyl palmitate, vinyl stearate, vinyl pivalate, vinyl octanoate, vinyl benzoate, vinyl isovalerate, acetic anhydride, vinyl butyrate, vinyl chloroacetate, propionic anhydride, etc., and more preferably vinyl acetate.

[0131] The acylating agent can be used in an amount of, for example, 0.5 to 3 mol, preferably 0.8 to 2 mol, more preferably 1 to 1.5 mol, and further preferably 1.1 to 1.3 mol, based on 1 mol of L-menthol.

[0132] In the method for producing L-menthol ester of the present invention, the solvent used in the mixture containing L-menthol and D-menthol is a non-aqueous solvent. The non-aqueous solvent is an organic solvent that does not substantially contain water. Examples of such organic solvents include hydrocarbon solvents such as pentane, hexane, heptane, and octane; ether solvents such as diethyl ether, methyl tert-butyl ether, dibutyl ether, and tetrahydrofuran; aromatic solvents such as toluene, xylene, and benzene; and halogen solvents such as dichloromethane and chloroform. Among these organic solvents, hydrocarbon solvents are preferred, and heptane is more preferred. These organic solvents may be used alone or in combination. It should be noted that substantially free of water means that in addition to the case of no water at all, a trace amount of water to the extent that does not affect the esterification reaction is also allowed. As a specific amount of trace water, for example, a trace amount of water obtained by mixing with moisture in the solvent storage environment, etc., can be cited as a specific example, 1000 ppm or less, preferably 500 ppm or less.

[0133] The amount of these solvents used may be an amount capable of completely dissolving the mixture containing L-menthol and D-menthol, and for example, 0.3 to 3 ml, preferably 0.5 to 2 ml, more preferably 0.8 to 1.5 ml, relative to 1 g of the total weight of L-menthol and D-menthol.

[0134] The specific operation in the method for producing L-menthol ester of the present invention is not particularly limited as long as a transesterification system can be constructed in which a mixture of L-menthol and D-menthol, an acylating agent, and the polypeptide of the present invention coexist. For example, L-menthol and D-menthol (DL-menthol (racemic form)) can be dissolved in the above-mentioned solvent, an acylating agent can be added thereto and mixed, and then the polypeptide of the present invention can be mixed. The transesterification system can specifically transesterify L-menthol among L-menthol and D-menthol.

[0135] The polypeptide of the present invention has excellent substrate specificity for L-menthol, so the method for producing L-menthol ester of the present invention can obtain L-menthol ester with high optical purity even if the transesterification rate of L-menthol is high (that is, a large amount of D-menthol is present in the transesterification system). From this viewpoint, in the method for producing L-menthol ester of the present invention, the timing of terminating the reaction in the transesterification system can be set to the timing when the transesterification rate of L-menthol reaches 80% or more, 85% or more, 90% or more, 93% or more, or 95% or more. In addition, the upper limit of the range of the transesterification rate of L-menthol is not particularly limited, and from the viewpoint of obtaining high optical purity, it can be set to, for example, the timing when it reaches 99% or less, preferably 97% or less, and more preferably 96% or less.

[0136] The obtained L-menthol ester can be purified by fractional extraction, fractional distillation, column chromatography or the like.

[0137] The L-menthol ester thus obtained can be used as a flavoring compound; an additive for food and beverages, cosmetics, pharmaceuticals, or quasi-drugs; an active ingredient for cosmetics, pharmaceuticals, or quasi-drugs; an intermediate for the active ingredient of cosmetics, pharmaceuticals, or quasi-drugs, etc., and can also be used as a raw material for L-menthol. When the L-menthol ester obtained using the L-menthol ester of the present invention is used as a raw material for L-menthol, L-menthol can be produced by chemical hydrolysis using an acid or an alkali.

[0138] 8-4. Method for producing L-menthol

[0139] The method for producing L-menthol of the present invention comprises the step of allowing the polypeptide of the present invention, the enzyme composition of the present invention, or the enzyme preparation of the present invention (the polypeptide of the present invention, etc.) to act on a mixture containing L-menthol ester and D-menthol ester to hydrolyze the L-menthol ester.

[0140] In the method for producing L-menthol of the present invention, the polypeptide of the present invention is preferably used in the form of a free polypeptide.

[0141] It should be noted that the polypeptides of the present invention not only improve the substrate specificity for L-menthol esters, but also achieve excellent L-menthol conversion rates compared to their lipase activity values. Therefore, the polypeptides of the present invention used in the method for producing L-menthol, etc., even if their lipase activity values ​​are lower than the lipase activity values ​​of the wild-type lipase of sequence number 1, also exert efficient L-menthol conversion rates. From such a viewpoint, the lipase activity values ​​of the polypeptides of the present invention, etc., can be, for example, 0.1 to 0.95 times, preferably 0.2 to 0.9 times, more preferably 0.3 to 0.8 times, further preferably 0.4 to 0.7 times, and even more preferably 0.5 to 0.6 times relative to the lipase activity value of the wild-type lipase of sequence number 1 of equal mass (ratio of lipase activity values).

[0142] (Method for deriving the ratio of lipase activity values)

[0143] The lipase activity value can be measured by the following steps using a lipase kit S (DS PharmaBiomedical Co., Ltd.). 1 mL of the colorimetric solution attached to the kit, 20 μL of the esterase inhibitory solution attached to the kit, 1 mL of the buffer attached to the kit, 100 μL of the substrate solution attached to the kit, and 8 mL of water are mixed to prepare an activity measurement solution. 10 μL of an enzyme solution prepared by diluting the wild-type lipase of sequence number 1 or the polypeptide of the present invention with 20 mM potassium phosphate buffer (pH 7.0) to an appropriate concentration is added to 100 μL of the activity measurement solution, and the absorbance at 412 nm after reaction at 37° C. for 15 minutes is measured. In the blank (control group), 20 mM potassium phosphate buffer (pH 7.0) is used instead of the enzyme solution. The value obtained by multiplying the absorbance difference between the wild-type lipase or the polypeptide of the present invention and the blank by a coefficient of 1.3 and the dilution factor is calculated as the lipase activity value (U / mL), and the ratio of the value obtained by the improved lipase of the present invention when the value obtained by the wild-type lipase is set to 1 is calculated.

[0144] The polypeptide of the present invention can be used in an amount of, for example, 0.1 to 1000 mg, preferably 1 to 100 mg, per 1 g of L-menthol ester. The polypeptide of the present invention can be used in an amount of, for example, 500 to 50,000 U, preferably 1,000 to 30,000 U, in terms of lipase activity per 1 g of L-menthol ester.

[0145] In the method for producing L-menthol of the present invention, the solvent used in the mixture containing L-menthol ester and D-menthol ester contains at least water. Furthermore, the solvent may be mixed with an organic solvent in addition to water, and examples of such organic solvents include hydrocarbon solvents such as pentane, hexane, heptane, and octane; ether solvents such as ethyl ether, methyl tert-butyl ether, butyl ether, and tetrahydrofuran; aromatic solvents such as toluene, xylene, and benzene; halogen solvents such as dichloromethane and chloroform; alcohol solvents such as methanol, ethanol, propanol, and isopropanol; and ketone solvents such as acetone, methyl ethyl ketone, and methyl isobutyl ketone. These organic solvents may be used alone or in combination.

[0146] Moreover, as a reaction temperature, 10-50 degreeC is mentioned, for example, Preferably it is 20-45 degreeC, More preferably, it is 30-40 degreeC, More preferably, it is 33-38 degreeC.

[0147] The specific operation in the method for producing L-menthol of the present invention is not particularly limited as long as a hydrolysis system can be constructed in which a mixture containing L-menthol ester and D-menthol ester and water coexists with the polypeptide of the present invention, for example, L-menthol ester and D-menthol ester (DL-menthol ester (racemic form)) can be mixed in the above-mentioned solvent, and then the polypeptide of the present invention can be mixed. The hydrolysis system can specifically hydrolyze L-menthol ester among L-menthol ester and D-menthol ester.

[0148] The polypeptide of the present invention has excellent substrate specificity for L-menthol esters, and therefore the method for producing L-menthol of the present invention can obtain L-menthol with high optical purity even if the exchange rate for L-menthol is high.

[0149] The obtained L-menthol can be purified by removing unreacted substances by fractional extraction, fractional distillation, column chromatography or the like.

[0150] Example

[0151] The present invention is specifically described below with reference to the following examples, but the present invention is not limited to the following examples.

[0152] [Test Example 1: Substrate Specificity for L-menthol Ester in Hydrolysis - 1]

[0153] [1-1. When D-menthyl acetate and L-menthyl acetate are used as substrates]

[0154] In the present test example, lipase derived from Burkholderia cepacia (PS lipase) or various mutants thereof (free enzyme) were reacted with D-menthyl acetate (purity 98% or more, manufactured by Tokyo Chemical Industry Co., Ltd., oily form, d=0.9250 to 0.9280) and L-menthyl acetate (purity 98% or more, manufactured by Tokyo Chemical Industry Co., Ltd., oily form, d=0.9250 to 0.9280) as substrates, respectively, and the obtained reaction products were analyzed by gas chromatography to investigate the substrate specificity for L-menthol esters in the hydrolysis.

[0155] As mutants of lipase derived from Burkholderia cepacia, mutants described in Table 1 were prepared. Specifically, an enzyme extract containing each mutant was prepared by the following method.

[0156] (Construction of plasmid for E. coli expression)

[0157] When constructing the E. coli expression system, the genes of Burkholderia cepacia M12-33 (LipA; lipase LipA gene (E. coli codon optimized): sequence number 4, LipX; chaperone gene (LipX) wild type: sequence number 5) were fully synthesized and codon-optimized in a manner suitable for E. coli expression.

[0158] The fully synthesized structural gene (LipA; sequence number 4) was used as a template for PCR amplification (PrimeSTAR GXL DNA polymerase (TaKaRa)). Primers (forward primer: 5'-TTTTCCATGGCTCGTTCTATGCGTTCTCG-3': sequence number 6, reverse primer: 5'-AAAAAAGCTTAAACACCCGCCAGTTTCAGACGG-3': sequence number 7)) were used to add adapter sequences (Nco I, Hind III) and then purified (NucleoSpin Gel and PCR Clean-up (MACHEREY-NAGEL)) to obtain a gene fragment (BCL-LipA).

[0159] The gene fragment (BCL-LipA) and pETDuet-1 (Novagen) were treated with restriction enzymes (Nco I (TaKaRa), HindIII (TaKaRa)) and then ligated (DNA Ligation Kit <Mighty Mix> (TaKaRa)), and transformed into Escherichia coli DH5α (TaKaRa) to obtain Escherichia coli BCL-LipA. When extracting the plasmid from Escherichia coli BCL-LipA, it was inoculated in LBBroth Base (invitrogen) + Amp: 100 μg / mL: 5 mL and shake-cultured (37°C, 16 h, 140 rpm), and then used NucleoSpin Plasmid EasyPure (MACHEREY-NAGEL) to obtain the plasmid (pETBCL-LipA).

[0160] The same operation was performed for the chaperone gene (LipX). That is, a fully synthetic chaperone gene (LipX; chaperone gene LipX (codon optimized in E. coli: sequence number 8) was used as a template for PCR amplification (using primers (forward primer: 5'-TTTTCATATGACCGCACGTGAAGGTCGCGC-3': sequence number 9, reverse primer: 5'-AAAACTCGAGTTACTGTGCAGAACCCGCACCG-3': sequence number 10) and adding a linker sequence (Nde I, Xho I) and then purified (NucleoSpin Geland PCR Clean-up (MACHEREY-NAGEL)) to obtain a gene fragment (BCL-LipX).

[0161] The gene fragment (BCL-LipX) and pETBCL-LipA were treated with restriction enzymes (NdeI (TaKaRa), Xho I (TaKaRa)) and then ligated and transformed into E. coli DH5α to obtain E. coli BCL-LipAX. When extracting the plasmid from E. coli BCL-LipAX, it was inoculated in LB Broth Base + Amp: 100 μg / mL: 5 mL and shake-cultured (37°C, 16 h, 140 rpm), and then NucleoSpin Plasmid EasyPure (MACHEREY-NAGEL) was used to obtain the E. coli expression plasmid (pETBCL-LipAX).

[0162] (Construction of E. coli expression system)

[0163] The obtained E. coli expression plasmid (pETBCL-LipAX) was transformed into E. coli BL21 (DE3) (Nippongene) to obtain the E. coli expression strain: E. coli BL21 (BCL-LipAX).

[0164] (Preparation of random mutant strains)

[0165] Primers were designed to prepare a saturation mutation library for the mutation introduction site.

[0166] A120X Primer:

[0167] (Forward primer: 5'-NNKGATTTCGTTCAGGGCGTTCTGGC-3': SEQ ID NO: 11, Reverse primer: 5'-GAATTCAGAACCGCGATGCGGAGTG-3': SEQ ID NO: 12)

[0168] When introducing mutations into the mutation point, the plasmid (pETBCL-LipAX) was used as a template by PCR amplification using Primer (PrimeSTAR GXL DNA polymerase (TaKaRa)). After PCR amplification, the template plasmid was treated with Dpn I (TaKaRa) (37°C, 16h), and T4 polymerase (Toyobo) and Ligation High (Toyobo) were used for ligation reaction (16°C, o / n), and then transformed into Escherichia coli BL21 (DE3) to obtain a random mutant strain (Escherichia coli BL21 (BCL-A120X)) into which random mutations were introduced into the mutation point.

[0169] (Using Terriffic Broth (Amp: 100 μg / mL) to prepare a mutation library)

[0170] In order to prepare a mutant library for the mutation point, the mutant strain selected above was inoculated into 1 mL of Terriffic Broth (invitrogen) (Amp: 100 μg / mL), and then cultured with shaking in a shaking culture machine (Taitec) (33°C, 48h). The induction of enzyme expression was carried out by adding IPTG to the culture solution at a final concentration of 0.1mM at 24h of culture. After culture, the bacteria were recovered by centrifugation (3300g×15min, 4°C), and then lysed (25°C, 1000rpm) using B-PER (ThermoFisher), and then centrifuged (3300g×15min, 4°C), and the supernatant was recovered to obtain an enzyme extract containing improved lipase.

[0171] The obtained enzyme extract containing the improved lipase 200 μL (lipase activity was used in the range of 10 to 200 U. The solvent was water. In addition, the protein concentration was about 20 mg / mL.) and the substrate 12 μL were added to a reaction vessel (96-well plate) and mixed, and hydrolyzed using a well plate shaker at 35°C, 1000 rpm, and 72 hours. It should be noted that the wild-type lipase and the substrate 12 μL were added to a reaction vessel (96-well plate) at a protein concentration of about 20 mg / mL and mixed, and hydrolyzed using a well plate shaker at 35°C, 1,000 rpm, and 72 hours.

[0172] (Method for measuring the ratio of lipase activity values)

[0173] The lipase activity value is measured using a lipase kit S (DSPharma Biomedical Co., Ltd.) according to the following steps. 1 mL of the colorimetric solution attached to the kit, 20 μL of the esterase inhibitory solution attached to the kit, 1 mL of the buffer attached to the kit, 100 μL of the substrate solution attached to the kit, and 8 mL of water are mixed to prepare an activity measurement solution. 10 μL of an enzyme solution in which the wild-type lipase of sequence number 1 or the polypeptide of the present invention is diluted to an appropriate concentration with 20 mM potassium phosphate buffer (pH 7.0) is added to 100 μL of the activity measurement solution, and the absorbance 412 nm after 15 minutes of reaction is measured at 37 ° C. In the blank, 20 mM potassium phosphate buffer (pH 7.0) is used instead of the enzyme solution. The value obtained by multiplying the absorbance difference between the wild-type lipase or the polypeptide of the present invention and the blank by the coefficient 1.3 and the dilution multiple is calculated as the lipase activity value (U / mL), and the ratio of the value obtained by the improved lipase of the present invention when the value obtained by the wild-type lipase is set to 1 is calculated.

[0174] The reaction mixture was transferred to a 1.5 mL Eppendorf tube, 12 μL of 6M hydrochloric acid solution and 200 μL of heptane were added, mixed by a vortex mixer and centrifuged (15000 rpm, 10 min, 25° C.), 150 μL of the heptane layer (upper layer) was recovered into a vial for instrumental analysis, thereby extracting the reaction product. 150 μL of heptane was further added to the obtained heptane layer for dilution, thereby preparing 300 μL of analysis sample.

[0175] 300 μL of the analysis sample was subjected to gas chromatography under the following conditions, and the amounts of the remaining raw materials and the reaction product were analyzed based on the chromatographic peak areas.

[0176] (Gas chromatography analysis conditions)

[0177] Chromatographic column: CP-Chiral-DEX CB (0.25mmID×25m, J&W)

[0178] Injection volume: 1μL

[0179] Inlet temperature: 200℃

[0180] Injection method: split 1:100

[0181] Carrier gas: He

[0182] Flow rate: 1.3mL / min

[0183] Column oven: 130°C, 8 minutes

[0184] Detector: FID, 300°C (H2: 40 mL / min, O2: 400 mL / min)

[0185] The conversion rate of L-menthol when L-menthyl acetate was used as a substrate was calculated based on the following formula. The results are shown in Table 1.

[0186] [Mathematical formula 1]

[0187]

[0188] When D-menthyl acetate was used as a substrate, the D-menthol conversion rate (%) was calculated in the same manner as the above formula, and the L / D conversion rate ratio (substrate specificity for the L form) was calculated based on the following formula. The results are shown in Table 1.

[0189] [Mathematical formula 2]

[0190]

[0191] [Table 1]

[0192]

[0193] As shown in Table 1, among the 120th mutants of PS lipase (wild type; Comparative Example 1), only the A120G mutant (Example 1) showed a substrate specificity for the L-type significantly higher than that of the wild type. It was also found that the A120G mutant also had an excellent conversion rate for L-menthol.

[0194] [1-2. Case of using DL-menthyl acetate as a substrate]

[0195] Hydrolysis was carried out in the same manner as in the above item 1-1 except that the wild-type lipase (Comparative Example 1) or the improved lipase of the present invention (Example 1) was used as the enzyme and the substrate was changed to DL-menthyl acetate (purity 98% or more, manufactured by Tokyo Chemical Industry Co., Ltd., oily, d=0.9250 to 0.9280).

[0196] In addition, the lipase activity values ​​of equal masses of the wild-type lipase (Comparative Example 1) and the improved lipase of the present invention (Example 1) were measured, and the ratio thereof was derived.

[0197] [Table 2]

[0198] PS lipase Lipase activity ratio Comparative Example 1 wild type 1 Example 1 A120G 0.54

[0199] The optical purity of L-menthol when DL-menthyl acetate was used as a substrate was calculated based on the following formula.

[0200] [Mathematical formula 3]

[0201]

[0202] [Table 3]

[0203] PS lipase L-type conversion rate (%) Optical purity (%ee) Comparative Example 1 wild type 12.6 96.4 Example 1 A120G 48.3 97.5

[0204] As shown in Table 3, the improved lipase (Example 1) of the present invention has a high L-type conversion rate compared to the wild-type lipase (Comparative Example 1), but an improvement in optical purity can be observed. In addition, as shown in Table 2, the improved lipase (Example 1) of the present invention has a significantly reduced lipase activity compared to the wild-type lipase (Comparative Example 1), but an improvement in L-type conversion rate as shown in Table 3 is observed.

[0205] [Test Example 2: Substrate Specificity for L-Menthol in Transesterification Reaction]

[0206] In this test example, a mutant (immobilized enzyme) in which the A at position 120 of the lipase from Burkholderia cepacia (PS lipase) was changed to G was reacted with DL-menthol (purity 98% or more, manufactured by Tokyo Chemical Industry Co., Ltd., racemic form, solid) as a substrate, and the obtained reaction product was analyzed by gas chromatography to investigate the substrate specificity for L-menthol in the transesterification reaction.

[0207] A modified lipase from Burkholderia cepacia (PS lipase) in which the A at position 120 was changed to G was fixed on a carrier (silica particles) according to a conventional method to obtain an immobilized enzyme (Example 2). In addition, a wild-type lipase was also immobilized in the same manner to obtain an immobilized enzyme (Comparative Example 20). It should be noted that the wild-type lipase or modified lipase accounted for about 0.2 to 2% by weight of the immobilized enzyme obtained.

[0208] The ratio of the transesterification activity values ​​of the immobilized wild-type lipase (Comparative Example 20) and the immobilized improved lipase (Example 2) of equal mass was measured by the following method.

[0209] (Method for measuring the transesterification activity ratio)

[0210] Phenylethanol (20 parts by weight) and vinyl acetate (80 parts by weight) were used as substrates, and each immobilized enzyme was allowed to act at 30°C for 20 minutes to perform an ester exchange reaction. The obtained phenylethyl acetate was quantified by HPLC analysis. The amount of phenylethyl acetate obtained by the improved lipase of the present invention (ester exchange activity value of the improved lipase of the present invention) was taken as the ratio of the ester exchange activity value when the amount of phenylethyl acetate obtained by the wild-type lipase (ester exchange activity value of the wild-type lipase) was set to 1.

[0211] [Table 4]

[0212] PS lipase Transesterification activity ratio Comparative Example 20 Wild type (fixed) 1 Example 2 A120G(Fixed) 0.58

[0213] Dissolve 50 g of DL-menthol in 52.5 ml of heptane, and then add 15.4 g of vinyl acetate as an acylating agent and mix (before enzyme reaction). Add 2 g of immobilized enzyme, stir at 25°C to start the reaction. After reacting for 18 hours, filter with filter paper to remove the immobilized enzyme to obtain a reaction filtrate. Add 20 μl of internal standard solution to 100 μl of the reaction filtrate, and dilute 25 μl of it with 1 ml of heptane to prepare an analytical sample.

[0214] The analysis sample was subjected to gas chromatography under the following conditions to analyze the reaction product.

[0215] (Gas chromatography analysis conditions)

[0216] Chromatographic column: CP-Chiral-DEX CB (0.25mmID×25m, J&W)

[0217] Injection volume: 1μL

[0218] Inlet temperature: 25℃

[0219] Injection method: split 1:100

[0220] Carrier gas:

[0221] Flow rate: 1.3mL / min

[0222] Column oven: 110°C, 25 minutes

[0223] Detector: FID, 300℃

[0224] The conversion rate of L-menthyl acetate in the reaction product (L-form conversion rate) was calculated based on the following formula.

[0225] [Formula 4]

[0226]

[0227] The optical purity of the reaction product was calculated based on the following formula.

[0228] [Formula 5]

[0229]

[0230] [Table 5]

[0231] PS lipase L-type conversion rate (%) Optical purity ee (%) Comparative Example 20 Wild type (fixed) 95.6 98.3 Example 2 A120G(Fixed) 95 99.5

[0232] As shown in Table 5, the L-type conversion rate of the improved lipase of the present invention (Example 2) is equivalent to that of the wild-type lipase (Comparative Example 20), but an improvement in optical purity can be observed. In addition, as shown in Table 4, the ester activity value of the improved lipase of the present invention (Example 2) is greatly reduced compared with the wild-type lipase (Comparative Example 20), but as shown in Table 5, a decrease in the L-type conversion rate is hardly observed.

[0233] [Test Example 3: Substrate Specificity for L-menthol Ester in Hydrolysis-2]

[0234] The same operation as in Test Example 1 was performed except that the polypeptides listed in Table 6 were prepared as mutants of the Burkholderia cepacia lipase using appropriately designed primers, and the substrate specificity of the mutants for L-menthol esters in hydrolysis was examined. The results are shown in Table 6.

[0235] [Table 6]

[0236]

[0237] As shown in Table 6, among the 88th mutants of PS lipase (wild type; Comparative Example 21), only the Q88A mutant (Example 3), Q88G mutant (Example 4), Q88D mutant (Example 5), Q88M mutant (Example 6), and Q88L mutant (Example 7) showed substrate specificity for the L-type higher than that of the wild type. In addition, it was found that the conversion rate of L-menthol of the Q88A mutant, Q88G mutant, Q88D mutant, Q88M mutant, and Q88L mutant was also extremely excellent.

[0238] Sequence Listing Free Text

[0239] Sequence No. 3 is a DNA encoding a lipase A120G mutant from Burkholderia cepacia.

[0240] Sequence number 6 is the forward primer of LipA.

[0241] Sequence number 7 is the reverse primer of LipA.

[0242] Sequence number 9 is the forward primer of LipX.

[0243] Sequence number 10 is the reverse primer of LipX.

[0244] Sequence number 11 is the forward primer of A120X.

[0245] Sequence number 12 is the reverse primer of A120X.

[0246] Sequence No. 13 is a DNA encoding the Q88A mutant of lipase from Burkholderia cepacia.

[0247] Sequence No. 14 is a DNA encoding the Q88G mutant of lipase from Burkholderia cepacia.

[0248] Sequence No. 15 is a DNA encoding the Q88D mutant of lipase from Burkholderia cepacia.

[0249] Sequence No. 16 is a DNA encoding the Q88M mutant of lipase from Burkholderia cepacia.

[0250] Sequence No. 17 is a DNA encoding the Q88L mutant of lipase from Burkholderia cepacia. Sequence Listing <110> AMANO ENZYME INC. <120> Polypeptide having esterification activity for L-menthol and / or hydrolysis activity for L-menthol ester <130> FP221856JP <150> JP2020-044455 <151> 2020-03-13 <160> 17 <170> PatentIn version 3.5 <210> 1 <211> 320 <212> PRT <213> Burkholderia cepacia <400> 1 Ala Asp Asn Tyr Ala Ala Thr Arg Tyr Pro Ile Ile Leu Val His Gly 1 5 10 15 Leu Thr Gly Thr Asp Lys Tyr Ala Gly Val Leu Glu Tyr Trp Tyr Gly 20 25 30 Ile Gln Glu Asp Leu Gln Gln Arg Gly Ala Thr Val Tyr Val Ala Asn 35 40 45 Leu Ser Gly Phe Gln Ser Asp Asp Gly Pro Asn Gly Arg Gly Glu Gln 50 55 60 Leu Leu Ala Tyr Val Lys Thr Val Leu Ala Ala Thr Gly Ala Thr Lys 65 70 75 80 Val Asn Leu Val Gly His Ser Gln Gly Gly Leu Thr Ser Arg Tyr Val 85 90 95 Ala Ala Val Ala Pro Asp Leu Val Ala Ser Val Thr Thr Ile Gly Thr 100 105 110 Pro His Arg Gly Ser Glu Phe Ala Asp Phe Val Gln Gly Val Leu Ala 115 120 125 Tyr Asp Pro Thr Gly Leu Ser Ser Thr Val Ile Ala Ala Phe Val Asn 130 135 140 Val Phe Gly Ile Leu Thr Ser Ser Ser Asn Asn Thr Asn Gln Asp Ala 145 150 155 160 Leu Ala Ala Leu Lys Thr Leu Thr Thr Ala Gln Ala Ala Thr Tyr Asn 165 170 175 Gln Asn Tyr Pro Ser Ala Gly Leu Gly Ala Pro Gly Ser Cys Gln Thr 180 185 190 Gly Ala Pro Thr Glu Thr Val Gly Gly Asn Thr His Leu Leu Tyr Ser 195 200 205 Trp Ala Gly Thr Ala Ile Gln Pro Thr Ile Ser Val Phe Gly Val Thr 210 215 220 Gly Ala Thr Asp Thr Ser Thr Ile Pro Leu Val Asp Pro Ala Asn Ala 225 230 235 240 Leu Asp Pro Ser Thr Leu Ala Leu Phe Gly Thr Gly Thr Val Met Val 245 250 255 Asn Arg Gly Ser Gly Gln Asn Asp Gly Val Val Ser Lys Cys Ser Ala 260 265 270 Leu Tyr Gly Gln Val Leu Ser Thr Ser Tyr Lys Trp Asn His Leu Asp 275 280 285 Glu Ile Asn Gln Leu Leu Gly Val Arg Gly Ala Asn Ala Glu Asp Pro 290 295 300 Val Ala Val Ile Arg Thr His Ala Asn Arg Leu Lys Leu Ala Gly Val 305 310 315 320 <210> 2 <211> 1095 <212> DNA <213> Burkholderia cepacia <400> 2 atggccagat cgatgcgttc cagggtggtg gcaggggcag tggcatgcgc gatgagcgtc 60 gcgccgttcg cggggatgac cgcggcgatg acgctcgcga cgacgcgcgc ggcaatggcg 120 gcgagcgcgc ccgccgacaa ctacgcggcg acgcgttatc cgatcattct cgtgcacggg 180 ctcacgggca ccgacaaata cgcaggtgtg ctcgagtact ggtacgggat ccaggaggac 240 ctgcagcagc gtggcgcgac cgtctatgtc gctaacctgt cgggcttcca gagcgacgac 300 ggcccgaacg ggcgcggcga acagttgctg gcctacgtga agacggtgct cgccgcgacg 360 ggggcgacca aggtcaacct cgtcggccac agccagggcg ggctgacgtc gcgctatgtc 420 gcggccgtcg cgcccgatct ggtcgcgtcg gtgacgacga tcggcacgcc gcatcgcggc 480 tccgagttcg ccgacttcgt gcagggcgtg ctcgcgtacg atccgaccgg gctgtcgtcg 540 acggtgatcg ccgcgttcgt caatgtgttc ggaatcctca cgagcagcag caacaacacg 600 aaccaggacg cgctcgcggc gctgaagacg ctgacgaccg cgcaggccgc cacgtacaac 660 cagaactacc ctagcgcggg cctcggcgcg ccgggcagtt gccagaccgg cgcgccgacg 720 gaaaccgtcg gcggcaacac gcatctgctg tattcgtggg ccggcacggc gatccagccg 780 acgatctccg tgttcggcgt cacgggtgcg acggatacga gcaccattcc gctcgtcgat 840 ccggcgaacg cgctcgaccc gtcgacgctc gcgctgttcg gcaccggcac ggtgatggtc 900 aaccgcggtt cgggccagaa cgacggggtc gtgtcgaagt gcagcgcgct gtacggccag 960 gtgctgagca cgagctacaa gtggaaccat ctcgacgaga tcaaccagtt gctcggcgtg 1020 cgcggcgcga atgcggaaga tccggtcgcg gtgatccgca cgcatgcgaa ccggctgaag 1080 ctcgcgggcg tgtga 1095 <210> 3 <211> 1095 <212> DNA <213> Artificial Sequence <220> <223> DNA encoding mutant A120G lipase from Burkholderia cepacia <400> 3 atggccagat cgatgcgttc cagggtggtg gcaggggcag tggcatgcgc gatgagcgtc 60 gcgccgttcg cggggatgac cgcggcgatg acgctcgcga cgacgcgcgc ggcaatggcg 120 gcgagcgcgc ccgccgacaa ctacgcggcg acgcgttatc cgatcattct cgtgcacggg 180 ctcacgggca ccgacaaata cgcaggtgtg ctcgagtact ggtacgggat ccaggaggac 240 ctgcagcagc gtggcgcgac cgtctatgtc gctaacctgt cgggcttcca gagcgacgac 300 ggcccgaacg ggcgcggcga acagttgctg gcctacgtga agacggtgct cgccgcgacg 360 ggggcgacca aggtcaacct cgtcggccac agccagggcg ggctgacgtc gcgctatgtc 420 gcggccgtcg cgcccgatct ggtcgcgtcg gtgacgacga tcggcacgcc gcatcgcggc 480 tccgagttcg gcgacttcgt gcagggcgtg ctcgcgtacg atccgaccgg gctgtcgtcg 540 acggtgatcg ccgcgttcgt caatgtgttc ggaatcctca cgagcagcag caacaacacg 600 aaccaggacg cgctcgcggc gctgaagacg ctgacgaccg cgcaggccgc cacgtacaac 660 cagaactacc ctagcgcggg cctcggcgcg ccgggcagtt gccagaccgg cgcgccgacg 720 gaaaccgtcg gcggcaacac gcatctgctg tattcgtggg ccggcacggc gatccagccg 780 acgatctccg tgttcggcgt cacgggtgcg acggatacga gcaccattcc gctcgtcgat 840 ccggcgaacg cgctcgaccc gtcgacgctc gcgctgttcg gcaccggcac ggtgatggtc 900 aaccgcggtt cgggccagaa cgacggggtc gtgtcgaagt gcagcgcgct gtacggccag 960 gtgctgagca cgagctacaa gtggaaccat ctcgacgaga tcaaccagtt gctcggcgtg 1020 cgcggcgcga atgcggaaga tccggtcgcg gtgatccgca cgcatgcgaa ccggctgaag 1080 ctcgcgggcg tgtga 1095 <210> 4 <211> 1095 <212> DNA <213> Burkholderia cepacia <400> 4 atggctcgtt ctatgcgttc tcgtgttgtg gcaggtgctg tggcatgcgc aatgtctgtg 60 gcgccgtttg caggcatgac cgcagcgatg accctggcga ccacccgtgc ggcgatggct 120 gcgtctgcac cggctgacaa ctacgcagct actcgttacc cgatcatcct ggtgcatggt 180 ctgactggca ccgataagta tgctggtgtt ctggagtact ggtacggtat tcaggaagac 240 ctgcagcagc gtggcgcgac tgtttacgtt gcgaacctgt ctggtttcca gtccgacgac 300 ggcccgaacg gtcgcggcga acagctgctg gcgtatgtga aaactgtgct ggcggctacc 360 ggcgcaacca aagttaacct ggttggccac tcccagggtg gcctgacttc tcgctacgtg 420 gcagcggtgg ctccggacct ggtggcgagc gttactacca ttggcactcc gcatcgcggt 480 tctgaattcg cggatttcgt tcagggcgtt ctggcgtatg acccgactgg cctgtcttct 540 accgtgatcg cggcatttgt taacgttttt ggtatcctga cctcttccag caacaacact 600 aaccaggacg ctctggctgc actgaaaacc ctgaccaccg cgcaggctgc tacctacaac 660 cagaactatc cgtctgcggg tctgggcgct ccgggttctt gccagaccgg tgcgccgacc 720 gagactgtgg gtggcaacac tcacctgctg tactcttggg cgggtactgc gatccagccg 780 accatctctg ttttcggtgt tactggtgcg actgatacct ctactatccc gctggtggat 840 ccggcaaacg cactggaccc gtccactctg gcgctgtttg gtaccggcac cgttatggtg 900 aaccgtggta gcggtcagaa cgatggtgtg gtgtctaagt gctctgcgct gtacggccag 960 gttctgtcta cctcttacaa atggaaccac ctggacgaga tcaaccagct gctgggtgtt 1020 cgtggtgcta acgcggaaga tccggtggcg gtgattcgca ctcacgcaaa ccgtctgaaa 1080 ctggcgggtg tttaa 1095 <210> 5 <211> 1034 <212> DNA <213> Burkholderia cepacia <400> 5 atgacggcac gtgaagggcg cgcgccgctg gcgcggcgcg ctgtggtcta cggttcgtgg 60 ggctggcggc gatcgccggc gtcgcgatgt ggagcggtgc gggatggcat cgcgggacgg 120 gcacggccgg cgagttgccg gacgcggcag cggcaggcgg ggcggctgcc gcaccgccgc 180 aggccgctct gccggcgagc acgggcctgc cgtcgtcgct ggccggctcc agtgcgccgc 240 ggctgccgct cgatgccggc ggccatcttg cgaagtcgcg cgcggtgcgc gatttcttcg 300 actactgcct gaccgcgcag agtgacctga gcgcggccgc gctcgatgcg ttcgtcgtac 360 gccagatcgc cgcgcagctc gacggcacgg tcgcgcaggc cgaggcgctc gacgtctggc 420 accggtaccg cgcgtatctc gacgcgctcg cgaagttgcg cgatgccggc gcggtcgaca 480 agtccgacct gggtgcgctg cagctcgcgc tcgaccagcg cgcgtcgatc gcgtaccgca 540 cgctcggcga ctggagccag ccgtttttcg gcgcggagca gtggcggcag cgctacgatc 600 tcgcgcgact gaagatcgcg caggatcgta cgctgacgga tgcgcagaag gccgagcggc 660 tcgcggcgct tgagcagcag atgccagccg acgaacgcgc ggcgcagcag cgggtcgacc 720 agcagcgggc cgcgatcgac cggatcgcgc aactgcagaa gagcggcgcg acgcccgatg 780 cgatgcgcgc gcaactgacg cagacgctcg gcccggaagc cgccgcgcgc gtcgcgcaga 840 tgcagcagga cgacgcatcg tggcagagcc gctacgcgga ctatgcgacg cagcgtgcgc 900 agatcgagtc ggccggcctg tcgccgcagg atcgcgacgc ccagatcgcc gcattgcggc 960 agcgcacgtt cacgaaaccc ggcgaagcgg tgcgggcggc atcgctcgat cgcggcgcgg 1020 gcagcgcgca gtga 1034 <210> 6 <211> 29 <212> DNA <213> Artificial sequence <220> <223> Forward primer of LipA <400> 6 ttttccatgg ctcgttctat gcgttctcg 29 <210> 7 <211> 33 <212> DNA <213> Artificial sequence <220> <223> Reverse primer of LipA <400> 7 aaaaaagctt aaacacccgc cagtttcaga cgg 33 <210> 8 <211> 1035 <212> DNA <213> Burkholderia cepacia <400> 8 atgaccgcac gtgaaggtcg cgctccgctg gctcgtcgcg cagtggttta cggtgttgtt 60 ggtctggctg ctatcgcagg tgttgcgatg tggtccggtg ctggttggca tcgtggtacc 120 ggcactgcgg gcgaactgcc ggatgcagcg gctgcaggtg gtgctgcggc agctccgccg 180 caggcggctc tgccggcgtc tactggtctg ccgagcagcc tggcgggctc ttctgctccg 240 cgcctgccgc tggacgcggg tggtcacctg gctaaaagcc gtgctgttcg cgacttcttc 300 gactactgcc tgaccgcgca gagcgacctg agcgcagcag ctctggacgc ttttgttgtt 360 cgtcagattg cggctcagct ggatggcact gttgcgcagg ctgaagcact ggacgtgtgg 420 caccgttacc gtgcttacct ggatgcactg gcaaaactgc gtgatgcggg tgcagtggac 480 aaatctgatc tgggcgcact gcagctggcg ctggatcagc gtgcgtctat cgcgtaccgt 540 accctgggtg attggtctca gccgttcttc ggtgcggaac agtggcgtca gcgttacgac 600 ctggcgcgtc tgaaaatcgc gcaggatcgt accctgaccg acgcgcagaa agcggaacgt 660 ctggcggcac tggaacagca gatgccggct gatgagcgtg cagctcagca gcgtgtggac 720 cagcagcgcg cagctatcga tcgtatcgct cagctgcaga aatctggtgc gaccccggac 780 gcgatgcgtg cacagctgac tcagaccctg ggtccggagg cggcagctcg cgttgcacag 840 atgcagcagg acgatgcttc ctggcagtct cgctacgcgg actacgcgac ccagcgtgcg 900 cagattgaga gcgcgggtct gtctccgcag gaccgtgacg ctcagattgc tgcgctgcgt 960 cagcgtacct tcaccaaacc gggtgaagcg gttcgtgcgg cgtctctgga tcgcggtgcg 1020 ggttctgcac agtaa 1035 <210> 9 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Forward primer of LipX <400> 9 ttttcatatg accgcacgtg aaggtcgcgc 30 <210> 10 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Reverse primer of LipX <400> 10 aaaactcgag ttactgtgca gaacccgcac cg 32 <210> 11 <211> 26 <212> DNA <213> Artificial sequence <220> <223> A120X forward primer <220> <221> misc_feature <222> (1)..(2) <223> n is a, c, g, or t <220> <221> misc_feature <222> (3) <223> k is g or t <400> 11 nnkgatttcg ttcagggcgt tctggc 26 <210> 12 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Reverse primer for A120X <400> 12 gaattcagaa ccgcgatgcg gagtg 25 <210> 13 <211> 1095 <212> DNA <213> Artificial sequence <220> <223> DNA encoding mutant Q88A of lipase from Burkholderia cepacia <400> 13 atggccagat cgatgcgttc cagggtggtg gcaggggcag tggcatgcgc gatgagcgtc 60 gcgccgttcg cggggatgac cgcggcgatg acgctcgcga cgacgcgcgc ggcaatggcg 120 gcgagcgcgc ccgccgacaa ctacgcggcg acgcgttatc cgatcattct cgtgcacggg 180 ctcacgggca ccgacaaata cgcaggtgtg ctcgagtact ggtacgggat ccaggaggac 240 ctgcagcagc gtggcgcgac cgtctatgtc gctaacctgt cgggcttcca gagcgacgac 300 ggcccgaacg ggcgcggcga acagttgctg gcctacgtga agacggtgct cgccgcgacg 360 ggggcgacca aggtcaacct cgtcggccac agcgcgggcg ggctgacgtc gcgctatgtc 420 gcggccgtcg cgcccgatct ggtcgcgtcg gtgacgacga tcggcacgcc gcatcgcggc 480 tccgagttcg ccgacttcgt gcagggcgtg ctcgcgtacg atccgaccgg gctgtcgtcg 540 acggtgatcg ccgcgttcgt caatgtgttc ggaatcctca cgagcagcag caacaacacg 600 aaccaggacg cgctcgcggc gctgaagacg ctgacgaccg cgcaggccgc cacgtacaac 660 cagaactacc ctagcgcggg cctcggcgcg ccgggcagtt gccagaccgg cgcgccgacg 720 gaaaccgtcg gcggcaacac gcatctgctg tattcgtggg ccggcacggc gatccagccg 780 acgatctccg tgttcggcgt cacgggtgcg acggatacga gcaccattcc gctcgtcgat 840 ccggcgaacg cgctcgaccc gtcgacgctc gcgctgttcg gcaccggcac ggtgatggtc 900 aaccgcggtt cgggccagaa cgacggggtc gtgtcgaagt gcagcgcgct gtacggccag 960 gtgctgagca cgagctacaa gtggaaccat ctcgacgaga tcaaccagtt gctcggcgtg 1020 cgcggcgcga atgcggaaga tccggtcgcg gtgatccgca cgcatgcgaa ccggctgaag 1080 ctcgcgggcg tgtga 1095 <210> 14 <211> 1095 <212> DNA <213> Artificial Sequence <220> <223> DNA encoding mutant Q88G lipase from Burkholderia cepacia <400> 14 atggccagat cgatgcgttc cagggtggtg gcaggggcag tggcatgcgc gatgagcgtc 60 gcgccgttcg cggggatgac cgcggcgatg acgctcgcga cgacgcgcgc ggcaatggcg 120 gcgagcgcgc ccgccgacaa ctacgcggcg acgcgttatc cgatcattct cgtgcacggg 180 ctcacgggca ccgacaaata cgcaggtgtg ctcgagtact ggtacgggat ccaggaggac 240 ctgcagcagc gtggcgcgac cgtctatgtc gctaacctgt cgggcttcca gagcgacgac 300 ggcccgaacg ggcgcggcga acagttgctg gcctacgtga agacggtgct cgccgcgacg 360 ggggcgacca aggtcaacct cgtcggccac agcgggggcg ggctgacgtc gcgctatgtc 420 gcggccgtcg cgcccgatct ggtcgcgtcg gtgacgacga tcggcacgcc gcatcgcggc 480 tccgagttcg ccgacttcgt gcagggcgtg ctcgcgtacg atccgaccgg gctgtcgtcg 540 acggtgatcg ccgcgttcgt caatgtgttc ggaatcctca cgagcagcag caacaacacg 600 aaccaggacg cgctcgcggc gctgaagacg ctgacgaccg cgcaggccgc cacgtacaac 660 cagaactacc ctagcgcggg cctcggcgcg ccgggcagtt gccagaccgg cgcgccgacg 720 gaaaccgtcg gcggcaacac gcatctgctg tattcgtggg ccggcacggc gatccagccg 780 acgatctccg tgttcggcgt cacgggtgcg acggatacga gcaccattcc gctcgtcgat 840 ccggcgaacg cgctcgaccc gtcgacgctc gcgctgttcg gcaccggcac ggtgatggtc 900 aaccgcggtt cgggccagaa cgacggggtc gtgtcgaagt gcagcgcgct gtacggccag 960 gtgctgagca cgagctacaa gtggaaccat ctcgacgaga tcaaccagtt gctcggcgtg 1020 cgcggcgcga atgcggaaga tccggtcgcg gtgatccgca cgcatgcgaa ccggctgaag 1080 ctcgcgggcg tgtga 1095 <210> 15 <211> 1095 <212> DNA <213> Artificial Sequence <220> <223> DNA encoding mutant Q88D lipase from Burkholderia cepacia <400> 15 atggccagat cgatgcgttc cagggtggtg gcaggggcag tggcatgcgc gatgagcgtc 60 gcgccgttcg cggggatgac cgcggcgatg acgctcgcga cgacgcgcgc ggcaatggcg 120 gcgagcgcgc ccgccgacaa ctacgcggcg acgcgttatc cgatcattct cgtgcacggg 180 ctcacgggca ccgacaaata cgcaggtgtg ctcgagtact ggtacgggat ccaggaggac 240 ctgcagcagc gtggcgcgac cgtctatgtc gctaacctgt cgggcttcca gagcgacgac 300 ggcccgaacg ggcgcggcga acagttgctg gcctacgtga agacggtgct cgccgcgacg 360 ggggcgacca aggtcaacct cgtcggccac agcgacggcg ggctgacgtc gcgctatgtc 420 gcggccgtcg cgcccgatct ggtcgcgtcg gtgacgacga tcggcacgcc gcatcgcggc 480 tccgagttcg ccgacttcgt gcagggcgtg ctcgcgtacg atccgaccgg gctgtcgtcg 540 acggtgatcg ccgcgttcgt caatgtgttc ggaatcctca cgagcagcag caacaacacg 600 aaccaggacg cgctcgcggc gctgaagacg ctgacgaccg cgcaggccgc cacgtacaac 660 cagaactacc ctagcgcggg cctcggcgcg ccgggcagtt gccagaccgg cgcgccgacg 720 gaaaccgtcg gcggcaacac gcatctgctg tattcgtggg ccggcacggc gatccagccg 780 acgatctccg tgttcggcgt cacgggtgcg acggatacga gcaccattcc gctcgtcgat 840 ccggcgaacg cgctcgaccc gtcgacgctc gcgctgttcg gcaccggcac ggtgatggtc 900 aaccgcggtt cgggccagaa cgacggggtc gtgtcgaagt gcagcgcgct gtacggccag 960 gtgctgagca cgagctacaa gtggaaccat ctcgacgaga tcaaccagtt gctcggcgtg 1020 cgcggcgcga atgcggaaga tccggtcgcg gtgatccgca cgcatgcgaa ccggctgaag 1080 ctcgcgggcg tgtga 1095 <210> 16 <211> 1095 <212> DNA <213> Artificial Sequence <220> DNA encoding mutant Q88M of lipase from Burkholderia cepacia <400> 16 atggccagat cgatgcgttc cagggtggtg gcaggggcag tggcatgcgc gatgagcgtc 60 gcgccgttcg cggggatgac cgcggcgatg acgctcgcga cgacgcgcgc ggcaatggcg 120 gcgagcgcgc ccgccgacaa ctacgcggcg acgcgttatc cgatcattct cgtgcacggg 180 ctcacgggca ccgacaaata cgcaggtgtg ctcgagtact ggtacgggat ccaggaggac 240 ctgcagcagc gtggcgcgac cgtctatgtc gctaacctgt cgggcttcca gagcgacgac 300 ggcccgaacg ggcgcggcga acagttgctg gcctacgtga agacggtgct cgccgcgacg 360 ggggcgacca aggtcaacct cgtcggccac agcatgggcg ggctgacgtc gcgctatgtc 420 gcggccgtcg cgcccgatct ggtcgcgtcg gtgacgacga tcggcacgcc gcatcgcggc 480 tccgagttcg ccgacttcgt gcagggcgtg ctcgcgtacg atccgaccgg gctgtcgtcg 540 acggtgatcg ccgcgttcgt caatgtgttc ggaatcctca cgagcagcag caacaacacg 600 aaccaggacg cgctcgcggc gctgaagacg ctgacgaccg cgcaggccgc cacgtacaac 660 cagaactacc ctagcgcggg cctcggcgcg ccgggcagtt gccagaccgg cgcgccgacg 720 gaaaccgtcg gcggcaacac gcatctgctg tattcgtggg ccggcacggc gatccagccg 780 acgatctccg tgttcggcgt cacgggtgcg acggatacga gcaccattcc gctcgtcgat 840 ccggcgaacg cgctcgaccc gtcgacgctc gcgctgttcg gcaccggcac ggtgatggtc 900 aaccgcggtt cgggccagaa cgacggggtc gtgtcgaagt gcagcgcgct gtacggccag 960 gtgctgagca cgagctacaa gtggaaccat ctcgacgaga tcaaccagtt gctcggcgtg 1020 cgcggcgcga atgcggaaga tccggtcgcg gtgatccgca cgcatgcgaa ccggctgaag 1080 ctcgcgggcg tgtga 1095 <210> 17 <211> 1095 <212> DNA <213> Artificial Sequence <220> <223> DNA encoding mutant Q88L lipase from Burkholderia cepacia <400> 17 atggccagat cgatgcgttc cagggtggtg gcaggggcag tggcatgcgc gatgagcgtc 60 gcgccgttcg cggggatgac cgcggcgatg acgctcgcga cgacgcgcgc ggcaatggcg 120 gcgagcgcgc ccgccgacaa ctacgcggcg acgcgttatc cgatcattct cgtgcacggg 180 ctcacgggca ccgacaaata cgcaggtgtg ctcgagtact ggtacgggat ccaggaggac 240 ctgcagcagc gtggcgcgac cgtctatgtc gctaacctgt cgggcttcca gagcgacgac 300 ggcccgaacg ggcgcggcga acagttgctg gcctacgtga agacggtgct cgccgcgacg 360 ggggcgacca aggtcaacct cgtcggccac agcctgggcg ggctgacgtc gcgctatgtc 420 gcggccgtcg cgcccgatct ggtcgcgtcg gtgacgacga tcggcacgcc gcatcgcggc 480 tccgagttcg ccgacttcgt gcagggcgtg ctcgcgtacg atccgaccgg gctgtcgtcg 540 acggtgatcg ccgcgttcgt caatgtgttc ggaatcctca cgagcagcag caacaacacg 600 aaccaggacg cgctcgcggc gctgaagacg ctgacgaccg cgcaggccgc cacgtacaac 660 cagaactacc ctagcgcggg cctcggcgcg ccgggcagtt gccagaccgg cgcgccgacg 720 gaaaccgtcg gcggcaacac gcatctgctg tattcgtggg ccggcacggc gatccagccg 780 acgatctccg tgttcggcgt cacgggtgcg acggatacga gcaccattcc gctcgtcgat 840 ccggcgaacg cgctcgaccc gtcgacgctc gcgctgttcg gcaccggcac ggtgatggtc 900 aaccgcggtt cgggccagaa cgacggggtc gtgtcgaagt gcagcgcgct gtacggccag 960 gtgctgagca cgagctacaa gtggaaccat ctcgacgaga tcaaccagtt gctcggcgtg 1020 cgcggcgcga atgcggaaga tccggtcgcg gtgatccgca cgcatgcgaa ccggctgaag 1080 ctcgcgggcg tgtga 1095

Claims

1. A polypeptide consisting of an amino acid sequence in which the amino acid residue at position 120 in the amino acid sequence shown in SEQ ID NO: 1 is substituted with a glycine residue.

2. A polypeptide consisting of an amino acid sequence in which the amino acid residue at position 88 in the amino acid sequence shown in SEQ ID NO: 1 is substituted with an alanine residue, a glycine residue, an aspartic acid residue, a methionine residue or a leucine residue.

3. A DNA encoding the polypeptide according to claim 1 or 2. A recombinant vector comprising the DNA according to claim 3.

5. A transformant obtained by transforming a host with the recombinant vector according to claim 4. A method for producing the polypeptide according to claim 1 or 2, comprising the step of culturing the transformant according to claim 5.

7. An enzyme composition comprising the polypeptide according to claim 1 or 2.

8. An enzyme preparation comprising the polypeptide according to claim 1 or 2 or the enzyme composition according to claim 7.

9. A method for producing an L-menthol ester, comprising the step of allowing the polypeptide according to claim 1 or 2, the enzyme composition according to claim 7, or the enzyme preparation according to claim 8 to act on a mixture containing L-menthol and D-menthol to esterify L-menthol.

10. A method for producing L-menthol, comprising the step of allowing the polypeptide according to claim 1 or 2, the enzyme composition according to claim 7, or the enzyme preparation according to claim 8 to act on a mixture containing L-menthol ester and D-menthol ester to hydrolyze the L-menthol ester.

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