A polypeptide having esterification activity against L-menthol and / or hydrolysis activity against L-menthol ester

Abstract

Provided is a technique that can further improve substrate specificity to an L-form during L-menthol and / or L-menthol ester production. A polypeptide that: has A120G, Q88A, Q88G, Q88D, Q88M, Q88L variants of a polypeptide comprising the amino acid sequence represented by sequence no. 1, and in said variants, has a random different moiety other than an amino-acid residue into which a substitution has been introduced; has esterification activity for L-menthol and / or hydrolyzing activity for L-menthol ester; and improves substrate specificity to L-menthol and / or L-menthol ester compared to a polypeptide comprising the amino acid sequence represented by sequence no. 1. During L-menthol and / or L-menthol ester production, the polypeptide can further improve the optical purity of the product.

Description

Technical Field

[0001] The present invention relates to a polypeptide having esterification activity against L-menthol and / or hydrolysis activity against L-menthol ester. More specifically, the present invention relates to a polypeptide having esterification activity against L-menthol and / or hydrolysis activity against L-menthol ester, with improved substrate specificity against L-menthol and / or L-menthol ester, a DNA encoding the polypeptide, a recombinant vector, a transformant, an enzyme composition, an enzyme agent, a method for producing the polypeptide, and a method for producing L-menthol ester and a method for producing L-menthol using the polypeptide.

Background Art

[0002] L-menthol is an important substance widely used in the fields of fragrances, foods, pharmaceuticals, etc. due to its properties such as a refreshing flavor and a refreshing skin feeling. In addition to being a raw material for L-menthol, menthol esters are themselves used in the fields of fragrances, foods, pharmaceuticals, etc.

[0003] These components are industrially produced by artificial synthesis. On the other hand, the L-menthol and its esters obtained by synthesis are contaminated with their optical isomers, the D-form. Both L-menthol and its esters are greatly affected in their quality by the contamination of the D-form. Therefore, it is required 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 mentioned. As chemical methods, there are methods such as selectively crystallizing the L-form by reacting DL-menthol with an optically active acid or base. As enzymatic methods, there are methods such as specifically hydrolyzing the L-form by allowing lipase to act on DL-menthol ester in an aqueous solvent, and methods such as specifically esterifying the L-form by allowing lipase to act on DL-menthol in an organic solvent.

[0005] Among them, enzymatic methods have been variously studied due to the high specificity of enzymes. For example, Non-Patent Document 1 describes that racemic menthyl laurate is hydrolyzed in an aqueous medium by lipase derived from Candida rugosa to preferentially obtain L-menthol (ee: 70%). Such enantioselectivity is also observed when racemic menthol is esterified with lauric acid. For example, lipase derived from Candida rugosa causes racemic menthol to undergo enantioselective esterification with lauric acid in a non-aqueous medium, preferentially producing L-menthyl laurate (ee: 95%).

[0006] In addition, Non-Patent Document 2 describes that racemic menthol undergoes esterification with acetic anhydride, propionic anhydride, and butyric anhydride with specific enantioselectivity by lipase derived from Candida rugosa. In particular, esterification with butyric anhydride in n-hexane preferentially produced L-menthyl butyrate (ee: 86%).

[0007] Furthermore, Non-Patent Document 3 describes that racemic menthol produced L-menthyl propionate with very high optical purity (ee: 95%) by esterification with propionic anhydride.

Prior Art Documents

Non-Patent Documents

[0008] [Non-Patent Document 1] Tetrahedron Letters, Vo1.27, No-l, pp 29-32, 1986 [Non-Patent Document 2] Enzyme and Microbial Technology Volume 18, Issue 7, 1996, pp536-539 [Non-Patent Document 3] Applied Microbiology and Biotechnology. 1995, Volume 43, Issue 4, pp 639-643 [Summary of the Invention] [Problems to be Solved by the Invention]

[0009] However, there is still room for improvement in the enantioselectivity obtained in the production of L-menthol and / or its esters.

[0010] Therefore, an object of the present invention is to provide a technique capable of further improving the substrate specificity for the L-form in the production of L-menthol and / or its esters. [Means for Solving the Problems]

[0011] As a result of intensive studies, the present inventors focused on the high enantioselectivity of lipase derived from Burkholderia cepacia, and further comprehensively verified the substrate specificity for the L-form in the production of L-menthol and / or its esters for more than 840 lipase mutants in which mutations were introduced into various sites of the lipase. As a result, it was found that a polypeptide consisting of an amino acid sequence in which the 120th amino acid residue of the lipase was substituted with a glycine residue and a polypeptide consisting of an amino acid sequence in which the 88th amino acid residue was substituted with an alanine residue, a glycine residue, an aspartic acid residue, a methionine residue, or a leucine residue can improve the substrate specificity for the L-form. The present invention has been completed based on this finding. That is, the present invention provides an invention in the following aspects.

[0012] Item 1. A polypeptide shown in any one of the following (1) to (3): (1) A polypeptide consisting of an amino acid sequence in which the 120th amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 is substituted with a glycine residue, (2) In the amino acid sequence in which the 120th amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 is substituted with a glycine residue, one or several amino acid residues other than the amino acid residue into which the substitution is introduced are substituted, added, inserted or deleted, having esterification activity against L-menthol and / or hydrolysis activity against L-menthol ester, and having improved substrate specificity for L-menthol and / or L-menthol ester as compared with the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1. (3) A polypeptide having 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, having a sequence identity of 80% or more excluding the amino acid residue into which the substitution has been introduced with respect to the amino acid sequence shown in SEQ ID NO: 1, having esterification activity against L-menthol and / or hydrolysis activity against L-menthol ester, and having improved substrate specificity for L-menthol and / or L-menthol ester as compared with the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1. Item 2. A polypeptide shown in any one of the following (4) to (6): (4) 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. (5) A polypeptide in which one or several amino acid residues other than the amino acid residue into which the substitution has been introduced are substituted, added, inserted, or deleted in the 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, having esterification activity against L-menthol and / or hydrolysis activity against L-menthol ester, and having improved substrate specificity for L-menthol and / or L-menthol ester as compared with the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1. (6) A polypeptide having 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, having a sequence identity of 80% or more excluding the amino acid residue into which the substitution has been introduced with respect to the amino acid sequence shown in SEQ ID NO: 1, having esterification activity against L-menthol and / or hydrolysis activity against L-menthol ester, and having improved substrate specificity for L-menthol and / or L-menthol ester as compared with the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1. Item 3. DNA encoding the polypeptide according to Item 1 or 2. Item 4. A recombinant vector containing the DNA according to Item 3. Item 5. A transformant obtained by transforming a host with the recombinant vector according to Item 4. 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. Item 7. An enzyme composition containing the polypeptide according to Item 1 or 2. Item 8. An enzyme agent containing the polypeptide according to Item 1 or 2, or the enzyme composition according to Item 7. Item 9. The polypeptide according to Item 1 or 2, the enzyme composition according to Item 7, or the enzyme agent according to Item 8 is allowed to act on a mixture containing L-menthol and D-menthol to esterify L-menthol. A method for producing L-menthol ester, comprising the step of esterifying L-menthol. 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 agent according to Item 8 to act on a mixture containing L-menthol ester and D-menthol ester to hydrolyze L-menthol ester.

Advantages of the Invention

[0013] According to the present invention, there is provided a technique capable of further improving the substrate specificity for the L-form in the production of L-menthol and / or its ester.

Modes for Carrying Out the Invention

[0014] Hereinafter, the present invention will be described in detail. In addition to the sequence listing, the 20 types of amino acid residues in the amino acid sequence may be represented by one-letter abbreviations. That is, glycine (Gly) is represented by G, alanine (Ala) is represented by A, valine (Val) is represented by V, leucine (Leu) is represented by L, isoleucine (Ile) is represented by I, phenylalanine (Phe) is represented by F, tyrosine (Tyr) is represented by Y, tryptophan (Trp) is represented by W, serine (Ser) is represented by S, threonine (Thr) is represented by T, cysteine (Cys) is represented by C, methionine (Met) is represented by M, aspartic acid (Asp) is represented by D, glutamic acid (Glu) is represented by E, asparagine (Asn) is represented by N, glutamine (Gln) is represented by Q, lysine (Lys) is represented by K, arginine (Arg) is represented by R, histidine (His) is represented by H, and proline (Pro) is represented by P.

[0015] In this specification, for the amino acid sequence to be displayed, the left end is the N-terminus and the right end is the C-terminus.

[0016] Expressions such as "A120G" in this specification are notations for amino acid substitutions. For example, "A120G" means that the 120th amino acid A from the N-terminal side in a specific amino acid sequence is substituted with the amino acid G.

[0017] In this specification, "non-polar amino acids" include alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan. "uncharged 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.

[0018] In this specification, "substitution" includes not only the case where an artificial substitution of an amino acid residue is introduced, but also the case where a natural substitution of an amino acid residue is introduced, that is, the case where the amino acid residues were originally different. In this specification, the substitution of an amino acid residue may be an artificial substitution or a natural substitution, but an artificial substitution is preferred.

[0019] 1. Polypeptide The polypeptide of the present invention is a polypeptide shown in any one of the following (1) to (3), or a polypeptide shown in any one of the following (4) to (6).

[0020] (1) A polypeptide consisting of an amino acid sequence in which the amino acid residue at position 120 (alanine residue) in the amino acid sequence shown in SEQ ID NO: 1 is substituted with a glycine residue. (2) In the 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, one or several amino acid residues other than the amino acid residue into which the substitution has been introduced are substituted, added, inserted or deleted, having esterification activity against L-menthol and / or hydrolysis activity against L-menthol ester, and having improved substrate specificity against L-menthol and / or L-menthol ester as compared with the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1. (3) In the 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, the sequence identity excluding the amino acid residue into which the substitution has been introduced with respect to the amino acid sequence shown in SEQ ID NO: 1 is 80% or more, having esterification activity against L-menthol and / or hydrolysis activity against L-menthol ester, and having improved substrate specificity against L-menthol and / or L-menthol ester as compared with the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1.

[0021] (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. (5) In the amino acid sequence shown in SEQ ID NO: 1, the 88th amino acid residue is substituted with an alanine residue, a glycine residue, an aspartic acid residue, a methionine residue, or a leucine residue, and one or several amino acid residues other than the amino acid residue into which the substitution has been introduced are substituted, added, inserted, or deleted, and it has esterification activity against L-menthol and / or hydrolysis activity against L-menthol ester, and has improved substrate specificity for L-menthol and / or L-menthol ester as compared with the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1. (6) In the amino acid sequence shown in SEQ ID NO: 1, the 88th amino acid residue is substituted with an alanine residue, a glycine residue, an aspartic acid residue, a methionine residue, or a leucine residue, and the sequence identity excluding the amino acid residue into which the substitution has been introduced with respect to the amino acid sequence shown in SEQ ID NO: 1 is 80% or more, and it has esterification activity against L-menthol and / or hydrolysis activity against L-menthol ester, and has improved substrate specificity for L-menthol and / or L-menthol ester as compared with the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1.

[0022] The polypeptides shown in (1) to (6) above have esterification activity against L-menthol and / or hydrolysis activity against L-menthol ester, and have improved substrate specificity for L-menthol and / or L-menthol ester. Preferably, the polypeptides shown in (1) to (6) above have improved substrate specificity for L-menthol and L-menthol ester.

[0023] The polypeptide of SEQ ID NO: 1 is a wild-type lipase (mature form) derived from Burkholderia cepacia.

[0024] The polypeptides of (1) to (6) above include not only polypeptides obtained by artificial substitution but also polypeptides originally having such amino acid sequences.

[0025] The polypeptides of (1) and the polypeptides of (4) also include polypeptides containing both the substitution at position 120 and the substitution at position 88.

[0026] Hereinafter, in the polypeptides of (2) and (3), and in (5) and (6), positions other than the amino acid residue at position 120 or the amino acid residue at position 88 of SEQ ID NO: 1 may also be referred to as "optional difference sites". In this specification, the term "optional difference site" refers to a site where differences are allowed as long as they do not significantly affect the properties of the polypeptide. Also, in this specification, compared with the polypeptide of (1) or (4), although a difference in the amino acid sequence is observed at the optional difference site, those having a substrate specificity for L-menthol and / or L-menthol ester equal to or higher than that of the polypeptide of (1) or (4) are referred to as variants of the polypeptide of (1) or (4). Further, the variant of the polypeptide preferably has substantially the same properties as the polypeptide of (1) or (4), although a difference in the amino acid sequence is observed at the optional difference site compared with the polypeptide of (1) or (4). Note that "substantially the same" refers to those having a substrate specificity for L-menthol and / or L-menthol ester. 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).

[0027] The amino acid differences in the polypeptides of (2) and (5) may include only one type of difference (e.g., substitution) from among substitution, addition, insertion, and deletion, or may include two or more differences (e.g., substitution and insertion). In the polypeptides of (2) and (5), the number of amino acid differences at the optional difference site may be one or several, 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, particularly preferably 1 or 2 or 1.

[0028] In addition, in the polypeptides (2) and (5), the sequence identity excluding the sites where the amino acid substitutions are made with respect to each amino acid sequence shown in SEQ ID NO: 1 may be 80% or more, preferably 85% or more, 90% or more, more preferably 95% or more, 96% or more, 97% or more, or 98% or more, and particularly preferably 99% or more.

[0029] Here, in the polypeptides (3) and (6), the sequence identity excluding the sites where the amino acid substitutions are made with respect to each amino acid sequence shown in SEQ ID NO: 1 is the sequence identity calculated by extracting only the arbitrary different sites from each amino acid sequence shown in SEQ ID NO: 1 and comparing only the arbitrary different sites. Also, "sequence identity" indicates the value of the amino acid sequence identity obtained by the bl2seq program of BLAST PACKAGE [sgi32 bit edition, Version 2.0.12; available from National Center for Biotechnology Information (NCBI)] (Tatiana A. Tatsusova, Thomas L. Madden, FEMS Microbiol. Lett., Vol. 174, p247 - 250, 1999). The parameters may be set to Gap insertion Cost value: 11 and Gap extension Cost value: 1.

[0030] In the polypeptides (2) and (3) and the polypeptides (5) and (6), the amino acids at positions 87 (serine), 264 (aspartic acid), and 286 (histidine) in the amino acid sequence shown in SEQ ID NO: 1 are considered to contribute to the esterification activity with respect to L - menthol and / or the hydrolysis activity with respect to L - menthol ester. Therefore, it is desirable not to introduce substitutions or deletions at these sites.

[0031] When amino acid substitutions are introduced into the polypeptides of (2) and (3) and (5) and (6) above, conservative substitutions are included as modes of amino acid substitution. That is, in the polypeptides of (2) and (3) and (5) and (6) above, examples of amino acid substitutions introduced with respect to the amino acid sequence shown in SEQ ID NO: 1 include substitution with another nonpolar amino acid if the amino acid before substitution is a nonpolar amino acid, substitution with another uncharged amino acid if the amino acid before substitution is an uncharged amino acid, substitution with another acidic amino acid if the amino acid before substitution is an acidic amino acid, and substitution with another basic amino acid if the amino acid before substitution is a basic amino acid.

[0032] In the polypeptides of (2) and (3) and (5) and (6) above, the “polypeptide having esterification activity for L-menthol and / or hydrolysis activity for L-menthol ester and having improved substrate specificity for L-menthol and / or L-menthol ester as compared with the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1” means (i) having esterification activity for L-menthol and / or hydrolysis activity for L-menthol ester, and (ii-a) regarding the substrate specificity for L-menthol, the optical purity of L-menthol acetate measured under the conditions of Test Example 2 below is 1.007 times or more, preferably 1.009 times or more, more preferably 1.010 times or more, still more preferably 1.012 times or more of the optical purity by the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 (however, when the L-menthol ester conversion rate is made equivalent, specifically 99 to 101%, between the polypeptide of the present invention and the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1), and (ii-b) regarding the substrate specificity for L-menthol ester, 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 Test Example 1 below is 1.05 times or more, preferably 1.12 times or more, more preferably 1.19 times or more, still more preferably 1.26 times or more, even more preferably 1.32 times or more of the L / D conversion rate by the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1.

[0033] 2. DNA The DNA encoding the polypeptide of the present invention (hereinafter sometimes referred to as "the DNA of the present invention") can be obtained, for example, by introducing the amino acid mutation into the DNA encoding the amino acid sequence of wild-type lipase (SEQ ID NO: 1). Further, the DNA of the present invention can also be artificially synthesized by the total gene synthesis method.

[0034] The DNA encoding the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 is known, for example, as the base sequence shown in SEQ ID NO: 2, and can be isolated from the genomic DNA of the M-12-33 strain of Burkholderia cepacia by a conventional method using PCR.

[0035] Methods for introducing specific mutations into specific sites of the base sequence are known, and for example, site-directed mutagenesis methods of DNA can be used. Specific methods for converting bases in DNA include, for example, the use of commercially available kits (QuickChange Lightning Site-Directed Mutagenesis kit: manufactured by Stratagene, KOD-Plus-Mutagenesis kit: manufactured by Toyobo, etc.).

[0036] The DNA into which a mutation has been introduced into the base sequence can be confirmed for the base sequence using a DNA sequencer. Once the base sequence is determined, thereafter, the DNA encoding the polypeptide can be obtained by chemical synthesis, PCR using the cloned probe as a template, or hybridization using the DNA fragment having the base sequence as a probe.

[0037] In addition, a mutant form of the DNA encoding the peptide, which has the same function as before the mutation, can be synthesized by a site-specific mutagenesis method or the like. To introduce a mutation into the DNA encoding the peptide, known methods such as the Kunkel method, the Gapped duplex method, and the megaprimer PCR method can be used.

[0038] The DNA of the present invention is preferably one in which the codon usage frequency is optimized for the host, and more preferably a DNA in which the codon usage frequency is optimized for Escherichia coli.

[0039] As an index representing the codon usage frequency, the total of the host-optimal codon usage frequencies of each codon may be adopted. The optimal codon is defined as the codon with the highest usage frequency among the codons corresponding to the same amino acid. The codon usage frequency is not particularly limited as long as it is optimized for the host. For example, examples of the optimal codons of Escherichia coli include the following. 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).

[0040] Examples of the DNA of the present invention include DNA containing the nucleotide sequences shown in SEQ ID NOs: 3 and 13 to 17. The DNA consisting of the nucleotide sequence shown in SEQ ID NO: 3 encodes a polypeptide in which the 120th amino acid residue of the amino acid sequence of the polypeptide shown in SEQ ID NO: 1 described in (1) above is substituted with a glycine residue. The DNAs consisting of the nucleotide sequences shown in SEQ ID NOs: 13, 14, 15, 16, and 17 each encode a polypeptide in which the 88th amino acid residue of the amino acid sequence of the polypeptide shown in SEQ ID NO: 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.

[0041] As another example of the DNA of the present invention, there is provided DNA encoding a polypeptide having improved substrate specificity for L-menthol and / or L-menthol ester as compared to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, and containing a nucleotide sequence complementary to the nucleotide sequence consisting of each of SEQ ID NOs: 3 and 13 to 17, and DNA that hybridizes under stringent conditions.

[0042] Here, "under stringent conditions" refers to conditions of incubating at 50°C to 65°C for 4 hours to overnight in 6×SSC (1×SSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) containing 0.5% SDS, 5×Denhartz's (0.1% bovine serum albumin (BSA), 0.1% polyvinylpyrrolidone, 0.1% ficoll 400), and 100 μg / ml salmon sperm DNA.

[0043] Hybridization under stringent conditions is specifically carried out by the following method. That is, a nylon membrane immobilizing a DNA library or a cDNA library 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 salmon sperm DNA. Then, 32Add each probe labeled with P and incubate overnight at 65°C. After washing this nylon membrane 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, autoradiography can be performed to detect the DNA that specifically hybridizes with the probe.

[0044] As a further example of the DNA of the present invention, there is provided a DNA that encodes a polypeptide having improved substrate specificity for L-menthol and / or L-menthol ester as compared to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, and that has a homology of 80% or more with the DNA consisting of the nucleotide sequences shown in SEQ ID NOs: 3, 13 to 17, respectively. The homology is preferably 85% or more, 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.

[0045] Here, the "homology" of DNA is calculated using publicly available or commercially available software having an algorithm for comparing a reference sequence as a query sequence. Specifically, BLAST, FASTA, or GENETYX (manufactured by Software Development Co., Ltd.) can be used, and these may be used with the default parameters set.

[0046] 3. Recombinant vector A recombinant vector containing the DNA encoding the peptide of the present invention (hereinafter, may also be 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.

[0047] The recombinant vector of the present invention contains control factors such as a promoter operably linked to the DNA of the present invention. Representative control factors include a promoter, and further, transcription elements such as an enhancer, a CCAAT box, a TATA box, and an SPI site may be included as needed. Also, operably linked means that various control factors such as a promoter and an enhancer that regulate the DNA of the present invention are linked to the DNA of the present invention in a state where they can function in a host cell.

[0048] As the expression vector, those constructed for gene recombination from a phage, plasmid, or virus that can autonomously proliferate in a host are suitable. Such expression vectors are known. For example, commercially available expression vectors include pQE series vectors (Qiagen), pDR540, pRIT2T (GE Healthcare Biosciences), pET series vectors (Merck), and the like. The expression vector may be selected and used in an appropriate combination with a host cell. For example, when Escherichia coli is used as the host cell, a combination of a pET series vector and an Escherichia coli strain DH5α, a combination of a pET series vector and an Escherichia coli strain BL21(DE3), or a combination of a pDR540 vector and an Escherichia coli strain JM109 is preferably mentioned.

[0049] 4. Transformant A transformant (hereinafter, sometimes referred to as "the transformant of the present invention") can be obtained by transforming a host using the recombinant vector of the present invention.

[0050] The host used for the production of the transformant is not particularly limited as long as the recombinant vector is stable, capable of autonomous growth, and can express the traits of the foreign gene. For example, bacteria belonging to the genus Escherichia such as Escherichia coli, the genus Bacillus such as Bacillus subtilis, the genus Pseudomonas such as Pseudomonas putida; yeast, etc. are preferred examples, but others such as animal cells, insect cells, plants, etc. may also be used. Among these, Escherichia coli is particularly preferred.

[0051] 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 may be appropriately set according to the type of the host, etc. If the host is a bacterium, for example, methods using competent cells by calcium ion treatment and electroporation method, etc. can be mentioned. If the host is yeast, for example, electroporation method, spheroplast method, and lithium acetate method, etc. can be mentioned. If the host is an animal cell, for example, electroporation method, calcium phosphate method, and lipofection method, etc. can be mentioned. If the host is an insect cell, for example, calcium phosphate method, lipofection method, and electroporation method, etc. can be mentioned. If the host is a plant cell, for example, electroporation method, Agrobacterium method, particle gun method, and PEG method, etc. can be mentioned.

[0052] Whether the recombinant vector of the present invention has been incorporated into the host can be confirmed by methods such as PCR method, Southern hybridization method, and Northern hybridization method, etc.

[0053] When confirming whether the recombinant vector of the present invention has been incorporated into the host by the PCR method, for example, the recombinant vector can be separated and purified from the transformant.

[0054] The separation and purification of the recombinant vector is carried out, for example, when the host is a bacterium, based on the lysate obtained by lysing the bacterium. As a method of lysis, for example, treatment is performed with a lytic enzyme such as lysozyme, and a protease, other enzymes, and a surfactant such as sodium lauryl sulfate (SDS) are used in combination as necessary.

[0055] Furthermore, physical disruption methods such as freeze-thaw and French press treatment may be combined. The separation and purification of DNA from the lysate can be carried out, for example, by appropriately combining deproteinization treatment by phenol treatment and protease treatment, ribonuclease treatment, alcohol precipitation treatment, and commercially available kits.

[0056] Cleavage of DNA can be carried out according to a conventional method, for example, using restriction enzyme treatment. As the restriction enzyme, for example, a type II restriction enzyme that acts on a specific nucleotide sequence is used. The binding of DNA and the expression vector is carried out, for example, using DNA ligase.

[0057] Thereafter, using the separated and purified DNA as a template, primers specific to the DNA of the present invention are designed and PCR is performed. For the amplification product obtained by PCR, agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc. are performed, stained with ethidium bromide, SYBR Green solution, etc., and the amplification product is detected as a band, whereby it can be confirmed that transformation has occurred.

[0058] Also, PCR can be performed using primers previously labeled with a fluorescent dye or the like, and the amplification product can be detected. Furthermore, a method of binding the amplification product to a solid phase such as a microplate and confirming the amplification product by fluorescence and enzyme reaction may also be employed.

[0059] 5. Method for producing a polypeptide The polypeptide of the present invention can be obtained by a production method including the step of culturing the transformant of the present invention.

[0060] The culture conditions of the transformant may be appropriately set in consideration of the nutritional physiological properties of the host, but liquid culture is preferably used. In the case of industrial production, aeration and agitation culture is preferred.

[0061] As the nutrient source of the medium, those necessary for the growth of the transformant can be used. As the carbon source, any carbon compound that can be assimilated may be used, and examples include glucose, sucrose, lactose, maltose, molasses, pyruvic acid, and the like.

[0062] As the nitrogen source, any nitrogen compound that can be assimilated may be used, and examples include peptone, meat extract, yeast extract, casein hydrolyzate, and alkali extract of soybean meal.

[0063] In addition to the carbon source and nitrogen source, for example, salts such as phosphates, carbonates, sulfates, magnesium, calcium, potassium, iron, manganese, and zinc, specific amino acids, and specific vitamins may be used as needed.

[0064] The culture temperature can be appropriately set within the range in which the transformant of the present invention can grow and the transformant of the present invention can produce the polypeptide of the present invention, but is preferably about 15 to 37°C. The culture may be completed at an appropriate time taking into account the time when the polypeptide of the present invention reaches the highest yield, and usually the culture time is about 12 to 48 hours.

[0065] The transformant of the present invention is cultured, and the culture supernatant or the cells are recovered by a method such as centrifugation. The cells are treated by mechanical methods such as ultrasonic waves and French press or lytic enzymes such as lysozyme, and solubilized by using enzymes such as protease and surfactants such as sodium lauryl sulfate (SDS) as needed, whereby a water-soluble fraction containing the polypeptide of the present invention can be obtained.

[0066] Also, by selecting an appropriate expression vector and host, the expressed polypeptide of the present invention can be secreted into the culture solution.

[0067] The water-soluble fraction containing the polypeptide of the present invention obtained as described above may be directly subjected to purification treatment, or may be subjected to purification treatment after concentrating the polypeptide of the present invention in the water-soluble fraction.

[0068] Concentration can be carried out, for example, by concentration under reduced pressure, membrane concentration, salting-out treatment, fractional precipitation method using a hydrophilic organic solvent (e.g., methanol, ethanol, and acetone), etc.

[0069] The purification treatment of the polypeptide of the present invention can be carried out, for example, by appropriately combining methods such as gel filtration, adsorption chromatography, ion exchange chromatography, affinity chromatography, etc.

[0070] The above purification treatment is already known and can be carried out by referring to appropriate literature, magazines, textbooks, etc. The polypeptide of the present invention purified in this way can be powdered by freeze-drying, vacuum drying, spray drying, etc. and distributed on the market as needed.

[0071] 6. Enzyme composition The polypeptide of the present invention may be provided, for example, in the form of a composition in which other components coexist. Examples of the form of the enzyme composition include a culture solution containing the polypeptide obtained in the production process of the polypeptide of the present invention, a water-soluble fraction containing the polypeptide obtained from the culture solution, or a composition in which the degree of purification of the polypeptide from the water-soluble fraction is increased to an arbitrary level; an enzyme agent shown in "7. Enzyme agent" described below; a reaction mixture containing the unreacted polypeptide obtained by using the polypeptide of the present invention in the production of L-menthol and / or its ester, etc.

[0072] Other components included in the enzyme composition include any components added, produced, or mixed in during the preparation process of the enzyme composition. For example, contaminating protein components and / or components other than proteins derived from the medium used in the production of the polypeptide of the present invention; additives or bases shown in "7. Enzyme agents" described below; unreacted raw materials, products, etc. contained in the reaction mixture obtained in the production of L-menthol and / or its esters. include the following.

[0073] The enzyme composition may also contain other enzymes. Examples of other enzymes include, for example, amylase (α-amylase, β-amylase, glucoamylase), glucosidase (α-glucosidase, β-glucosidase), galactosidase (α-galactosidase, β-galactosidase), protease (acid protease, neutral protease, alkaline protease), peptidase (leucine peptidase, aminopeptidase), lipase, esterase, cellulase, phosphatase (acid phosphatase, alkaline phosphatase), nuclease, deaminase, oxidase, dehydrogenase, glutaminase, pectinase, catalase, dextranase, transglutaminase, protein deamidase, pullulanase, etc.

[0074] The content of the polypeptide of the present invention in the enzyme composition is not particularly limited, but is preferably 10% by mass or more, more preferably 30% by mass or more, based on the total protein of the enzyme composition. The form of the enzyme composition is not particularly limited, and examples include liquid, powder, granules, etc. The enzyme composition can be prepared by generally known methods or the methods shown in "8-3. Method for Producing L-Menthol Ester" described below.

[0075] 7. Enzyme agent The polypeptide of the present invention, or the enzyme composition containing the polypeptide of the present invention, may be provided, for example, in the form of an enzyme agent. The enzyme agent is an enzyme composition prepared for the purpose of using the polypeptide of the present invention in the "8. Use" described below, and contains the polypeptide of the present invention as an active ingredient. The enzyme agent may contain, in addition to the polypeptide of the present invention, additives or bases such as excipients, buffers, suspending agents, stabilizers, preservatives, antiseptics, physiological saline, and solvents. As excipients, starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, sucrose, 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, methylparaben, etc. can be used. As antiseptics, ethanol, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol, etc. can be used. Furthermore, the enzyme agent may contain other components (for example, any components added, produced, or mixed during the production process of the polypeptide as an active ingredient) to such an extent that it does not affect the effects of the present invention. The content of the polypeptide in the enzyme agent is appropriately set within the range in which the effects of the polypeptide are exhibited.

[0076] 8. Use The polypeptide of the present invention can be used in applications that require an esterification treatment for L-menthol and a hydrolysis treatment for the L-menthol ester. Examples of applications that require an esterification treatment for L-menthol include the production of L-menthol esters, and examples of applications that require a hydrolysis treatment for the L-menthol ester include the production of L-menthol. More specific examples of these applications include the production of flavor compounds; the production of additives for foods and drinks, cosmetics, pharmaceuticals, or quasi-drugs; the production of active ingredients for cosmetics, pharmaceuticals, or quasi-drugs; the production of intermediates for active ingredients for cosmetics, pharmaceuticals, or quasi-drugs, etc.

[0077] 8-1. Substrate L-menthol and / or L-menthol ester, which is a substrate of the polypeptide of the present invention, is known as a fragrance compound; an additive for food and drink, cosmetics, pharmaceuticals, or quasi-drugs; an active ingredient of cosmetics, pharmaceuticals, or quasi-drugs; an intermediate of an active ingredient of cosmetics, pharmaceuticals, or quasi-drugs, etc.

[0078] L-menthol is (1R,2S,5R)-5-methyl-2-(1-methylethyl)cyclohexanol. The L-menthol ester is not particularly limited as long as it is an ester of L-menthol and a carboxylic acid, and specifically, compounds represented by the following formula (1) can be mentioned.

[0079] [Chemical formula]

[0080] In formula (1), R 1 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 alkyl group of 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. When substituted, examples of the substituent 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.

[0081] Preferred examples of the L-menthol ester include L-menthyl acetate, L-menthyl benzoate, L-menthyl isovalerate, L-menthyl lactate, L-menthyl succinate, L-menthyl propionate, L-menthyl butyrate, etc., and preferably L-menthyl acetate.

[0082] 8-2. Mode of use of the polypeptide The usage forms of the polypeptide of the present invention are not particularly limited, and include the form of a free polypeptide and the form of an immobilized polypeptide. The immobilized polypeptide may be one in which the polypeptide of the present invention is immobilized on a carrier (for example, an ion exchange resin, a porous resin, ceramics, calcium carbonate, etc.) according to a conventional method.

[0083] In addition, as the polypeptide of the present invention, one kind may be used alone, or a plurality of kinds may be used in combination.

[0084] 8-3. Method for producing L-menthol ester The method for producing the L-menthol ester of the present invention includes a step of esterifying L-menthol by allowing the polypeptide of the present invention, the enzyme composition of the present invention, or the enzyme agent of the present invention (hereinafter referred to as "the polypeptide of the present invention, etc.") to act on a mixture containing L-menthol and D-menthol.

[0085] In the method for producing the L-menthol ester of the present invention, the polypeptide of the present invention, etc. is preferably used in the form of an immobilized polypeptide in which the polypeptide is immobilized on a carrier.

[0086] In addition, the polypeptide of the present invention, etc. can not only improve the substrate specificity for L-menthol, but also exhibit an excellent ester conversion rate of L-menthol relative to its transesterification activity value. Therefore, even if the transesterification activity value of the polypeptide of the present invention, etc. used in the method for producing the L-menthol ester is less than the transesterification activity value of the wild-type lipase of SEQ ID NO: 1, it can effectively achieve a high ester conversion rate of L-menthol. From such a viewpoint, the transesterification activity value of the polypeptide of the present invention, etc. is, for example, 0.3 to 0.8 times, preferably 0.45 to 0.7 times, more preferably 0.55 to 0.6 times, as the ratio (transesterification activity value ratio) to the transesterification activity value of the wild-type lipase of SEQ ID NO: 1 of the same mass. (Method for deriving the transesterification activity value ratio) Using phenylethyl alcohol (20 parts by weight) and vinyl acetate (80 parts by weight) as substrates, the wild-type lipase of SEQ ID NO: 1 or the modified lipase of the present invention is allowed to act at 30°C for 20 minutes to conduct a transesterification reaction. The obtained phenylethyl alcohol acetate ester is quantified by HPLC analysis. The amount of phenylethyl alcohol acetate ester obtained by the wild-type lipase (the transesterification activity value of the wild-type lipase) is set to 1, and the amount of phenylethyl alcohol acetate ester obtained by the modified lipase of the present invention (the ester activity value by the modified lipase of the present invention) is taken as the ratio of the transesterification activity values.

[0087] For the polypeptide and the like of the present invention, for example, 0.01 to 200 mg, preferably 0.03 to 20 mg, more preferably 0.05 to 1 mg can be used per 1 g of L-menthol.

[0088] Examples of the acylating agent include, for example, a carboxylic acid represented by the general formula R 2 COOH and its esters. Examples of the ester include a carboxylic acid alkyl ester represented by the general formula R 2 COOR 3 , a carboxylic acid vinyl ester represented by the general formula R 2 COOCH=CH2, and the like.

[0089] In the above general formula, R 2 represents a linear or branched alkyl group having 1 to 20 carbon atoms, preferably an alkyl group having 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 alkyl group of the above alkoxy group, and the alkyl group of the above alkylamino group may be unsubstituted or substituted. When substituted, examples of the substituent 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. These acylating agents may be used alone or in combination of two or more.

[0090] In the method for producing the L-menthol ester of the present invention, the polypeptide of the present invention or the like can be allowed to act on a mixture containing L-menthol and D-menthol together with an acylating agent. Preferred examples of the acylating agent include esters of carboxylic acids, more preferably vinyl esters of carboxylic acids, and still more preferably vinyl acetate, vinyl propionate, vinyl butyrate, vinyl caproate, vinyl caprylate, vinyl caprate, vinyl laurate, vinyl myristate, vinyl palmitate, vinyl stearate, vinyl pivalate, vinyl octylate, vinyl benzoate, vinyl isovalerate, acetic anhydride, vinyl butyrate, vinyl chloroacetate, propionic anhydride, and the like. Even more preferably, vinyl acetate is mentioned.

[0091] The acylating agent can be used in an amount of, for example, 0.5 to 3 moles, preferably 0.8 to 2 moles, more preferably 1 to 1.5 moles, and still more preferably 1.1 to 1.3 moles per mole of L-menthol.

[0092] In the method for producing an L-menthol ester of the present invention, the solvent used for the mixture containing L-menthol and D-menthol is a non-aqueous solvent. The non-aqueous solvent is an organic solvent substantially free of water. Examples of such organic solvents include hydrocarbon solvents such as pentane, hexane, heptane, and octane; ether solvents such as diethyl ether, methyl-t-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 preferably used, and heptane is more preferably used. These organic solvents may be used alone or in combination of two or more. Note that "substantially free of water" means that in addition to the case of completely free of water, it also allows the case of containing a trace amount of water that does not affect the esterification reaction. The specific amount of the trace amount of water is, for example, the amount of trace moisture that can be mixed due to moisture in the storage environment of the solvent. Specific examples include 1000 ppm or less, preferably 500 ppm or less. Among these organic solvents, hydrocarbon solvents are preferably used, and heptane is more preferably used. These organic solvents may be used alone or in combination of two or more. Note that "substantially free of water" means that in addition to the case of completely free of water, it also allows the case of containing a trace amount of water that does not affect the esterification reaction. The specific amount of the trace amount of water is, for example, the amount of trace moisture that can be mixed due to moisture in the storage environment of the solvent. Specific examples include 1000 ppm or less, preferably 500 ppm or less. These organic solvents may be used alone or in combination of two or more. Among these organic solvents, hydrocarbon solvents are preferably used, and heptane is more preferably used.

[0093] The amount of these solvents used may be any amount that can completely dissolve the mixture containing L-menthol and D-menthol. For example, per 1 g of the total weight of L-menthol and D-menthol, it may be, for example, 0.3 to 3 ml, preferably 0.5 to 2 ml, more preferably 0.8 to 1.5 ml.

[0094] The specific operation in the method for producing an L-menthol ester of the present invention is not particularly limited as long as a transesterification system in which a mixture containing L-menthol and D-menthol, an acylating agent, and the polypeptide of the present invention coexist can be constructed. For example, L-menthol and D-menthol (DL-menthol (racemate)) are dissolved in the above solvent, an acylating agent is added thereto and mixed, and further, the polypeptide of the present invention can be mixed. In the transesterification system, among L-menthol and D-menthol, L-menthol is specifically transesterified.

[0095] Since the polypeptide of the present invention has excellent substrate specificity for L-menthol, in the method for producing an L-menthol ester of the present invention, even when the transesterification rate of L-menthol is relatively high (that is, even when a large amount of D-menthol is present in the transesterification system), an L-menthol ester with high optical purity can be obtained. From this perspective, in the method for producing an L-menthol ester of the present invention, as the timing for terminating the reaction in the above transesterification system, the timing can be such that the transesterification rate of L-menthol is 80% or more, 85% or more, 90% or more, 93% or more, or 95% or more. Further, although the upper limit of the range of the transesterification rate of L-menthol is not particularly limited, from the perspective of obtaining high optical purity, for example, the timing can be such that it is 99% or less, preferably 97% or less, and more preferably 96% or less.

[0096] The obtained L-menthol ester can be purified by fractional extraction, fractional distillation, column chromatography, etc.

[0097] The L-menthol ester thus obtained may be used as a flavor compound as an L-menthol ester; an additive for foods and drinks, cosmetics, pharmaceuticals, or quasi-drugs; an active ingredient of cosmetics, pharmaceuticals, or quasi-drugs; an intermediate of an active ingredient of cosmetics, pharmaceuticals, or quasi-drugs, etc., and further, it may be used as a raw material for L-menthol. When the L-menthol ester obtained with the L-menthol ester of the present invention is used as a raw material for L-menthol, L-menthol can be produced by chemically hydrolyzing it with an acid or an alkali.

[0098] 8-4. Method for producing L-menthol The method for producing L-menthol of the present invention includes a step of hydrolyzing an L-menthol ester by allowing the polypeptide of the present invention, the enzyme composition of the present invention, or the enzyme agent of the present invention (such as the polypeptide of the present invention) to act on a mixture containing an L-menthol ester and a D-menthol ester.

[0099] In the method for producing L-menthol of the present invention, the polypeptide etc. of the present invention is preferably used in the form of a free polypeptide.

[0100] In addition, the polypeptide etc. of the present invention can not only improve the substrate specificity for L-menthol ester, but also exhibit an excellent L-menthol conversion rate considering its lipase activity value. For this reason, the polypeptide etc. of the present invention used in the method for producing L-menthol can exhibit an effectively high L-menthol conversion rate even if its lipase activity value is less than the lipase activity value of the wild-type lipase of SEQ ID NO: 1. From such a viewpoint, the lipase activity value of the polypeptide etc. of the present invention is, as a ratio (ratio of lipase activity values) to the lipase activity value of the wild-type lipase of SEQ ID NO: 1 of the same mass, for example, 0.1 to 0.95 times, preferably 0.2 to 0.9 times, more preferably 0.3 to 0.8 times, still more preferably 0.4 to 0.7 times, and even more preferably 0.5 to 0.6 times. (Method for deriving the ratio of lipase activity values) The lipase activity value is measured by the following procedure using Lipase Kit S (DS Pharma Biomedical Co., Ltd.). Prepare an activity measurement solution by mixing 1 mL of the chromogenic solution attached to the kit, 20 μL of the esterase inhibitor solution attached to the kit, 1 mL of the buffer solution attached to the kit, 100 μL of the substrate solution attached to the kit, and 8 mL of water. Add 10 μL of an enzyme solution obtained by diluting the wild-type lipase of SEQ ID NO: 1 or the polypeptide of the present invention to an appropriate concentration with 20 mM potassium phosphate buffer (pH 7.0) to 100 μL of the activity measurement solution, and measure the absorbance at 412 nm after reacting at 37°C for 15 minutes. For the blank, use 20 mM potassium phosphate buffer (pH 7.0) instead of the enzyme solution. Calculate the value obtained by multiplying the difference in absorbance 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 as the lipase activity value (U / mL), and calculate the ratio of the value obtained by the modified lipase of the present invention when the value obtained by the wild-type lipase is set to 1.

[0101] For the polypeptide etc. of the present invention, for example, 0.1 to 1000 mg, preferably 1 to 100 mg can be used per 1 g of L-menthol ester. Further, the polypeptide of the present invention can be used such that the lipase activity is, for example, 500 to 50000 U, preferably 1000 to 30000 U per 1 g of L-menthol ester.

[0102] In the method for producing L-menthol of the present invention, the solvent used for the mixture containing L-menthol ester and D-menthol ester contains at least water. Further, an organic solvent may be mixed in addition to water in the solvent, and examples of such an organic solvent include hydrocarbon solvents such as pentane, hexane, heptane, and octane; ether solvents such as diethyl ether, methyl-t-butyl ether, dibutyl 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 of plural kinds.

[0103] Examples of the reaction temperature include, for example, 10 to 50°C, preferably 20 to 45°C, more preferably 30 to 40°C, and still more preferably 33 to 38°C.

[0104] The specific operation in the method for producing L-menthol of the present invention is not particularly limited as long as a hydrolysis system in which a mixture containing L-menthol ester and D-menthol ester together with water and the polypeptide etc. of the present invention coexist can be constructed. For example, L-menthol ester and D-menthol ester (DL-menthol ester (racemate)) can be mixed in the above solvent, and further, the polypeptide etc. of the present invention can be mixed. In the hydrolysis system, among L-menthol ester and D-menthol ester, L-menthol ester is specifically hydrolyzed.

[0105] Since the polypeptide of the present invention has excellent substrate specificity for L-menthol ester, in the method for producing L-menthol of the present invention, even if the exchange rate to L-menthol is relatively high, L-menthol with high optical purity can be obtained.

[0106] The obtained L-menthol can be purified by removing unreacted substances by fractional extraction, fractional distillation, column chromatography, etc.

Examples

[0107] Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not construed as being limited to the following examples.

[0108] [Test Example 1: Substrate specificity for L-menthol ester in hydrolysis - 1] [1-1. When using acetic acid D-menthyl and acetic acid L-menthyl as substrates respectively] In this test example, as substrates, acetic acid D-menthyl (purity 98% or more, manufactured by Tokyo Chemical Industry Co., Ltd., oily, d = 0.9250 - 0.9280) and acetic acid L-menthyl (purity 98% or more, manufactured by Tokyo Chemical Industry Co., Ltd., oily, d = 0.9250 - 0.9280) were separately reacted with Burkholderia cepacia-derived lipase (PS lipase) or various mutants thereof (free enzyme), and each obtained reaction product was analyzed by gas chromatography to examine the substrate specificity for L-menthol ester in hydrolysis.

[0109] As mutants of Burkholderia cepacia-derived lipase, those described in Table 1 were prepared. Specifically, enzyme extracts containing each mutant were prepared by the following method.

[0110] (Construction of plasmid for E. coli expression) When constructing the E. coli expression system, each gene of B. cepacia M12-33 (LipA; lipase LipA gene (E. coli codon-optimized): SEQ ID NO: 4, LipX; chaperone gene (LipX) wild type: SEQ ID NO: 5) was fully synthesized with codon-optimized genes for E. coli expression.

[0111] PCR amplification (PrimeSTAR GXL DNA Polymerase (TaKaRa)) using the fully synthesized structural gene (LipA; SEQ ID NO: 4) as a template, and a linker sequence (Nco I, Hind III) was added by primers (forward primer: 5'-TTTTCCATGGCTCGTTCTATGCGTTCTCG-3': SEQ ID NO: 6, reverse primer: 5'-AAAAAAGCTTAAACACCCGCCAGTTTCAGACGG-3': SEQ ID NO: 7), and then purified (NucleoSpin Gel and PCR Clean-up (MACHEREY-NAGEL)) to obtain a gene fragment (BCL-LipA).

[0112] The gene fragment (BCL-LipA) and pETDuet-1 (Novagen) were treated with restriction enzymes (Nco I (TaKaRa), Hind III (TaKaRa)), then ligated (DNA Ligation Kit <Mighty Mix> (TaKaRa)) and transformed into E. coli DH5α (TaKaRa) to obtain E. coli BCL-LipA. Plasmid extraction from E. coli BCL-LipA was performed by inoculating into LB Broth Base (invitrogen) + Amp: 100 μg / mL: 5 mL and shaking culture (37 °C, 16 h, 140 rpm), and then using NucleoSpin Plasmid EasyPure (MACHEREY-NAGEL), thereby obtaining a plasmid (pETBCL-LipA).

[0113] The same procedure was also performed for the chaperone gene (LipX). That is, the fully synthesized chaperone gene (LipX; the chaperone gene LipX (E. coli codon optimization: SEQ ID NO: 8) was used as a template for PCR amplification (primers (forward primer: 5'-TTTTCATATGACCGCACGTGAAGGTCGCGC-3': SEQ ID NO: 9, reverse primer: 5'-AAAACTCGAGTTACTGTGCAGAACCCGCACCG-3': SEQ ID NO: 10). After adding the linker sequence (Nde I, Xho I), it was purified (NucleoSpin Gel and PCR Clean-up (MACHEREY-NAGEL)) to obtain a gene fragment (BCL-LipX).

[0114] After treating the gene fragment (BCL-LipX) and pETBCL-LipA with restriction enzymes (NdeI (TaKaRa), Xho I (TaKaRa)), they were ligated and transformed into E. coli DH5α to obtain E. coli BCL-LipAX. Plasmid extraction from E. coli BCL-LipAX was performed by inoculating it into LB Broth Base + Amp: 100 μg / mL: 5 mL and culturing it with shaking (37 °C, 16 h, 140 rpm), and then using NucleoSpin Plasmid EasyPure (MACHEREY-NAGEL). As a result, an E. coli expression plasmid (pETBCL-LipAX) was obtained.

[0115] (Construction of E. coli expression system) The obtained E. coli expression plasmid (pETBCL-LipAX) was transformed into E. coli BL21(DE3) (Nippongene) to obtain an E. coli expression strain: E. coli BL21(BCL-LipAX).

[0116] (Preparation of random mutant strains) Primers were designed to create a saturation mutation library at the mutation-introducing site.

[0117] · A120X primer: (Forward primer: 5'-NNKGATTTCGTTCAGGGCGTTCTGGC-3': SEQ ID NO: 11, Reverse primer: 5'-GAATTCAGAACCGCGATGCGGAGTG-3': SEQ ID NO: 12)

[0118] Introduction of mutations at the mutation sites was performed by PCR amplification using Primer (PrimeSTAR GXL DNA Polymerase (TaKaRa)) with plasmid (pETBCL-LipAX) as a template. After PCR amplification, treatment of the template plasmid with Dpn I (TaKaRa) (37 °C, 16 h), and ligation reaction using T4 polymerase (Toyobo) and Ligation High (Toyobo) (16 °C, o / n) were carried out, followed by transformation into E. coli BL21(DE3) to obtain a random mutant strain (E. coli BL21(BCL-A120X)) with random mutations introduced at the mutation sites.

[0119] (Preparation of mutant library using Terrific Broth (Amp: 100 μg / mL)) To prepare a mutant library for the mutation sites, the mutant strain selected above was inoculated into 1 mL of Terrific Broth (invitrogen) (Amp: 100 μg / mL) and then cultured with shaking in a shaking incubator (Taitec) (33 °C, 48 h). Induction of enzyme expression was carried out by adding IPTG to the culture solution to a final concentration of 0.1 mM at 24 h of culture. After culture, the cells were collected by centrifugation (3,300 g × 15 min, 4 °C), followed by cell lysis treatment using B-PER (ThermoFisher) (25 °C, 1,000 rpm), and then centrifugation (3,300 g × 15 min, 4 °C) to collect the supernatant, obtaining an enzyme extract containing the modified lipase.

[0120] 200 μL of the enzyme extract containing the obtained modified lipase (used so that the lipase activity was in the range of 10 to 200 U. The solvent was water. The protein concentration was about 20 mg / mL.) and 12 μL of the substrate were placed in a reaction vessel (96-well plate) and mixed, and hydrolysis was carried out using a plate shaker at 35 °C, 1,000 rpm for 72 hours. For the wild-type lipase, 20 mg / mL of the protein concentration and 12 μL of the substrate were placed in a reaction vessel (96-well plate) and mixed, and hydrolysis was carried out using a plate shaker at 35 °C, 1,000 rpm for 72 hours.

[0121] (Method for measuring the ratio of lipase activity values) The lipase activity value was measured using a lipase kit S (DS Pharma Biomedical Co., Ltd.) according to the following procedure. 1 mL of the chromogenic solution attached to the kit, 20 μL of the esterase inhibitor solution attached to the kit, 1 mL of the buffer solution attached to the kit, 100 μL of the substrate solution attached to the kit, and 8 mL of water were mixed to prepare an activity measurement solution. 10 μL of an enzyme solution obtained by diluting the wild-type lipase of SEQ ID NO: 1 or the polypeptide of the present invention to an appropriate concentration with 20 mM potassium phosphate buffer (pH 7.0) was added to 100 μL of the activity measurement solution, and the absorbance at 412 nm after reacting at 37 °C for 15 minutes was measured. For the blank, 20 mM potassium phosphate buffer (pH 7.0) was 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 was calculated as the lipase activity value (U / mL), and the ratio of the value obtained by the modified lipase of the present invention to the value obtained by the wild-type lipase with the value obtained by the wild-type lipase being set to 1 was calculated.

[0122] The reaction mixture was transferred to a 1.5 mL Eppendorf tube, 12 μL of 6 M hydrochloric acid solution and 200 μL of heptane were added and mixed by vortexing, followed by centrifugation (15,000 rpm, 10 min, 25 °C), and 150 μL of the heptane layer (upper layer) was collected in a vial for instrumental analysis to extract the reaction product. The obtained heptane layer was further diluted by adding 150 μL of heptane to prepare 300 μL of an analytical sample.

[0123] 300 μL of the analysis sample was subjected to gas chromatography under the following conditions to analyze the amounts of the residual raw materials and reaction products based on the chromatographic peak areas. (Gas chromatography analysis conditions) Column: CP-Chiral-DEX CB (0.25 mm ID × 25 m, J&W) Injection volume: 1 μL Inlet temperature: 200 °C Injection method: Split 1:100 Carrier gas: He Flow rate: 1.3 mL / min Oven: 130 °C, 8 minutes Detector: FID, 300 °C (H2: 40 mL / min, O2: 400 mL / min)

[0124] Based on the following formula, the conversion rate of L-menthol was calculated when L-menthyl acetate was used as the substrate. The results are shown in Table 1.

[0125]

Equation

[0126] For the case where D-menthyl acetate was used as the substrate, the conversion rate of D-menthol (%) 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.

[0127]

Equation

[0128]

Table 1

[0129] As is clear from Table 1, among the 120th position mutants of PS lipase (wild type; Comparative Example 1), only the A120G mutant (Example 1) showed higher substrate specificity for the L-form at each step than the wild type. Also, it was found that the conversion rate of L-menthol by the A120G mutant was excellent.

[0130] [1-2. When using acetic acid-DL-menthyl as the substrate] Using wild-type lipase (Comparative Example 1) or the modified lipase of the present invention (Example 1) as the enzyme, hydrolysis was carried out in the same manner as in Item 1-1 above, except that the substrate was changed to acetic acid-DL-menthyl (purity 98% or more, manufactured by Tokyo Chemical Industry Co., Ltd., oily, d = 0.9250 - 0.9280).

[0131] Also, the lipase activity values of the same mass of wild-type lipase (Comparative Example 1) and the modified lipase of the present invention (Example 1) were measured, and the ratio was derived. The results are shown in Table 2. as shown in

[0132]

Table 2

[0133] When using acetic acid-DL-menthyl as the substrate, the optical purity of L-menthol was calculated based on the following formula. The results are shown in Table 3.

[0134]

Equation

[0135]

Table 3

[0136] As is clear from Table 3, according to the modified lipase of the present invention (Example 1), although the L-form conversion rate was high compared to the wild-type lipase (Comparative Example 1), an improvement in optical purity was observed. Further, as shown in Table 2, although the lipase activity itself of the modified lipase of the present invention (Example 1) was significantly decreased compared to the wild-type lipase (Comparative Example 1), an improvement in the L-form conversion rate as shown in Table 3 was observed.

[0137] [Test Example 2: Substrate Specificity for L-Menthol in Transesterification Reaction] In this test example, as a substrate, DL-menthol (purity 98% or more, manufactured by Tokyo Chemical Industry Co., Ltd., racemic form, solid) was reacted with a mutant (immobilized enzyme) in which the 120th A of Burkholderia cepacia-derived lipase (PS lipase) was converted to G, and the resulting reaction product was analyzed by gas chromatography to examine the substrate specificity for L-menthol in the transesterification reaction.

[0138] The modified lipase in which the 120th A of Burkholderia cepacia-derived lipase (PS lipase) was converted to G was immobilized on a carrier (silica particles) according to a conventional method to obtain an immobilized enzyme (Example 2). Further, the wild-type lipase was also immobilized in the same manner to obtain an immobilized enzyme (Comparative Example 20). Note that about 0.2 to 2% by weight of the obtained immobilized enzyme is occupied by the wild-type lipase or the modified lipase.

[0139] The ratio of the transesterification activity values of the immobilized enzyme of the wild-type lipase (Comparative Example 20) and the immobilized enzyme of the modified lipase (Example 2) of the same mass was measured by the following method. The results are shown in Table 4. [Method for Measuring Transesterification Activity Value Ratio] Using phenylethyl alcohol (20 parts by weight) and vinyl acetate (80 parts by weight) as substrates, each immobilized enzyme was allowed to act at 30 °C for 20 minutes to conduct a transesterification reaction. The obtained phenylethyl alcohol acetyl ester was quantified by HPLC analysis. When the amount of phenylethyl alcohol acetyl ester obtained by the wild-type lipase (the transesterification activity value of the wild-type lipase) was set to 1, the amount of phenylethyl alcohol acetyl ester obtained by the modified lipase of the present invention (the ester activity value by the modified lipase of the present invention) was taken as the ratio of the transesterification activity values.

[0140]

Table 4

[0141] 50 g of DL-menthol was dissolved in 52.5 ml of heptane, and further 15.4 g of vinyl acetate as an acylating agent was added and mixed (before the enzyme reaction). 2 g of the immobilized enzyme was added, and the reaction was started by stirring at 25 °C. After reacting for 18 hours, the immobilized enzyme was removed by filtration with filter paper to obtain a reaction filtrate. 20 μl of an internal standard solution was added to 100 μl of the reaction filtrate, and 25 μl thereof was diluted with 1 ml of heptane to prepare an analytical sample.

[0142] The analytical sample was subjected to gas chromatography under the following conditions to analyze the reaction product. (Gas Chromatography Analysis Conditions) Column: CP-Chiral-DEX CB (0.25 mm ID × 25 m, J&W) Injection volume: 1 μL Inlet temperature: 25 °C Injection method: Split 1:100 Carrier gas: Flow rate: 1.3 mL / min Oven: 110 °C, 25 minutes Detector: FID, 300 °C

[0143] Based on the following formula, the conversion rate of L-menthyl acetate (L-form conversion rate) in the reaction product was calculated. The results are shown in Table 5.

[0144]

Number

[0145] Based on the following formula, the optical purity of the reaction product was calculated. The results are shown in Table 3.

[0146]

Number

[0147]

Table 5

[0148] As is clear from Table 5, according to the modified lipase of the present invention (Example 2), although the L-form conversion rate is equivalent to that of the wild-type lipase (Comparative Example 20), an improvement in optical purity was observed. Also, as shown in Table 4, although the ester activity value itself of the modified lipase of the present invention (Example 2) is significantly decreased compared to the wild-type lipase (Comparative Example 20), almost no decrease in the L-form conversion rate was observed as shown in Table 5.

[0149] [Test Example 3: Substrate specificity for L-menthol ester in hydrolysis - 2] As mutants of lipase derived from Burkholderia cepacia, the same operations as in Test Example 1 were carried out, except that the polypeptides described in Table 6 were prepared using appropriately designed primers, and the substrate specificity for L-menthol ester in hydrolysis of the mutants was examined. The results are shown in Table 6.

[0150]

Table 6

[0151] As is clear from Table 6, among the 88th position 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 higher substrate specificity for the L-form than the wild type. Further, it was found that the Q88A mutant, Q88G mutant, Q88D mutant, Q88M mutant, and Q88L mutant were also excellent in the conversion rate of L-menthol at each stage.

Sequence Listing Free-Text

[0152] SEQ ID NO: 3 is DNA encoding the A120G mutant of lipase derived from Burkholderia cepacia. SEQ ID NO: 6 is the forward primer for LipA. SEQ ID NO: 7 is the reverse primer for LipA. SEQ ID NO: 9 is the forward primer for LipX. SEQ ID NO: 10 is the reverse primer for LipX. SEQ ID NO: 11 is the forward primer for A120X. SEQ ID NO: 12 is the reverse primer for A120X. SEQ ID NO: 13 is DNA encoding the Q88A mutant of lipase derived from Burkholderia cepacia. SEQ ID NO: 14 is DNA encoding the Q88G mutant of lipase derived from Burkholderia cepacia. SEQ ID NO: 15 is DNA encoding the Q88D mutant of lipase derived from Burkholderia cepacia. SEQ ID NO: 16 is DNA encoding the Q88M mutant of lipase derived from Burkholderia cepacia. SEQ ID NO: 17 is DNA encoding the Q88L mutant of lipase derived from Burkholderia cepacia.

Claims

1. A polypeptide shown in any one of the following (1) to (3): (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) In the 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, 1 to 20 amino acid residues other than the amino acid residue into which the substitution has been introduced are substituted, added, inserted or deleted, having esterification activity against L-menthol and / or hydrolysis activity against L-menthol ester, and having improved substrate specificity for L-menthol and / or L-menthol ester as compared to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; (3) In the 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, the sequence identity excluding the amino acid residue into which the substitution has been introduced with respect to the amino acid sequence shown in SEQ ID NO: 1 is 90% or more, having esterification activity against L-menthol and / or hydrolysis activity against L-menthol ester, and having improved substrate specificity for L-menthol and / or L-menthol ester as compared to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO:

1.

2. A polypeptide shown in any one of the following (4) to (6): (4) 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; (5) In the 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, 1 to 20 amino acid residues other than the amino acid residue into which the substitution has been introduced are substituted, added, inserted or deleted, having esterification activity against L-menthol and / or hydrolysis activity against L-menthol ester, and having improved substrate specificity for L-menthol and / or L-menthol ester as compared to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; A polypeptide having an amino acid sequence in which the 88th amino acid 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, having a sequence identity of 90% or more excluding the amino acid residue into which the substitution with respect to the amino acid sequence shown in SEQ ID NO: 1 has been introduced, having esterification activity with respect to L-menthol and / or hydrolysis activity with respect to an L-menthol ester, and having improved substrate specificity with respect to L-menthol and / or an L-menthol ester as compared to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO:

1.

3. DNA encoding the polypeptide according to claim 1 or 2.

4. A recombinant vector containing the DNA according to claim 3.

5. A transformant obtained by transforming a host with the recombinant vector according to claim 4.

6. 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 containing the polypeptide according to claim 1 or 2.

8. An enzyme agent containing 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 reacting the polypeptide according to claim 1 or 2, the enzyme composition according to claim 7, or the enzyme agent according to claim 8 with a mixture containing L-menthol and D-menthol to esterify L-menthol.

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

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