Modified chrysanthemic acid esterase

Abstract

To provide an esterase useful for the production of (1R,3S)-chrysanthemic acid.SOLUTION: A modified esterase comprises a specific amino acid sequence comprising a specific amino acid substitution, or another amino acid sequence having at least 90% identity with the specific amino acid sequence (where differences in the amino acid sequences occur in parts other than the position of the amino acid substitution). The modified esterase has improved reactivity with (1R,3S)-ethyl chrysanthemate as compared to an esterase comprising the specific amino acid sequence.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a modified enzyme (chrysanthemic acid esterase) that exhibits hydrolytic activity against ethyl chrysanthemic acid. More specifically, the present invention relates to a modified chrysanthemic acid esterase with altered stereoselectivity and its uses. This application claims priority based on Japanese Patent Application No. 2018-229479, filed on December 6, 2018, the entire contents of which are incorporated by reference. [Background technology]

[0002] Chrysanthemum acid is widely used as a raw material for the synthesis of synthetic pyrethroid insecticides. There are four types of isomers of chrysanthemum acid ((1R,3R)-chrysanthemum acid, (1R,3S)-chrysanthemum acid, (1S,3S)-chrysanthemum acid, and (1S,3R)-chrysanthemum acid), of which (1R,3R)-chrysanthemum acid has the highest insecticidal activity. In industrial production, chrysanthemum acid is produced by chemical synthesis. Meanwhile, chrysanthemum acid is also produced by enzymatic reaction using chrysanthemic acid esterase. Previous reports on chrysanthemic acid esterase are shown below (Patent Document 1, Non-Patent Documents 1-3). Patent Document 1 describes that esterase derived from Arthrobacter globiformis specifically decomposes chrysanthemic acid ethyl ester in a 1R,3R ((+)-trans) manner (Table 1). Furthermore, Non-Patent Document 1 describes that 1R,3R ((+)-trans)-specific or 1S,3R ((-)-trans)-specific decomposition of chrysanthemic acid ethyl ester is performed by esterase derived from Arthrobacter globiformis or esterase derived from other sources. Non-Patent Document 2 describes that, as the insecticidal effect of each isomer of chrysanthemic acid, 1R,3R ((+)-trans) and 1R,3S ((+)-cis) show different insecticidal effects. Non-Patent Document 3 is a research report on the decomposition of chrysanthemic acid ethyl ester by esterase derived from Arthrobacter globiformis, and shows that a specific mutation (K62R mutation) changes stereoselectivity (reactivity to (1S,3R)-ethyl chrysanthemic acid is shown). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Publication No. 5-56787 [Non-patent literature]

[0004] [Non-Patent Document 1] Agric.Biol.Chem.,55(11),2865-2870,1991 [Non-Patent Document 2] Chemistry and Function of Recent Synthetic Pyrethroids and Their Stereoisomers, Kosuke Yoshioka, Organic Synthesis Chemistry Vol. 38, No. 12 (1980), 1151-1162 [Non-Patent Document 3] Research on the production of optically active pyrethroid intermediates using microbial enzymes, Graduate School of Materials Science, Nara Institute of Science and Technology, Masako Nishizawa, March 2011 Summary of the Invention [Problem to be solved by the invention]

[0005] (1R,3S)-chrysanthemum acid, one of the isomers of chrysanthemum acid, also has insecticidal activity and is useful in itself. An enzyme that efficiently produces (1R,3S)-chrysanthemum acid (i.e., an esterase highly selective for (1R,3S)-ethyl chrysanthemate) is not known, and (1R,3S)-chrysanthemum acid is currently obtained by chemical hydrolysis of a mixture of ethyl chrysanthemate isomers and subsequent optical resolution (purification by chiral column). From the standpoint of yield, cost, or wastewater regulations, it is desirable to produce (1R,3S)-chrysanthemum acid using an enzyme, but no enzyme that acts selectively or specifically on (1R,3S)-ethyl chrysanthemate has been reported. Therefore, an object of the present invention is to provide an esterase useful for producing (1R,3S)-chrysanthemum acid. [Means for solving the problem]

[0006] In order to solve the above problems, the present inventors have attempted to modify the stereoselectivity of an esterase derived from Arthrobacter globiformis by protein engineering techniques. This esterase is specific to (1R,3R)-ethyl chrysanthemic acid, and is practically unusable for the production of (1R,3S)-chrysanthemic acid. Through trial and error, they have succeeded in identifying multiple mutations (amino acid residues) that are effective in improving the reactivity or selectivity to (1R,3S)-ethyl chrysanthemic acid, and have obtained a highly useful mutant (modified esterase). In particular, a mutant (A328G / A221F) in which two amino acids have been substituted is an enzyme that acts specifically on (1R,3S)-ethyl chrysanthemic acid, and is extremely useful and valuable. The mutation A328G, which is also included in this mutant, is an important mutation that can be said to determine the stereoselectivity of the substrate.

[0007] The following invention is based on the above results and considerations. [1] A modified esterase having an amino acid sequence containing any one of the following amino acid substitutions (1) to (23) in the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence showing 90% or more identity to said amino acid sequence (with the proviso that the amino acid sequence differs in a portion other than the position of the amino acid substitution), and having improved reactivity to (1R,3S)-ethyl chrysanthemate compared to an esterase consisting of the amino acid sequence of SEQ ID NO: 1: (1) The mutation site is F58, and the substituted amino acid is E, I, C, A, M, or Y; (2) The mutation site is D227, and the substituted amino acid is I, S, A, W, H, G, F, or V; (3) The mutation site is I228, and the amino acid after substitution is Y, S, T, or W; (4) The mutation site is L229, and the amino acid after substitution is Y, F, or V; (5) The mutation site is L231, and the amino acid after substitution is M, A, or G; (6) The mutation site is S244 and the substituted amino acid is F; (7) The mutation site is A245, and the amino acid after substitution is N or G; (8) The mutation site is V297, and the substituted amino acid is R, W, Q, A, or I; (9) The mutation site is F298 and the substituted amino acid is S; (10) The mutation site is A328 and the substituted amino acid is N; (11) The mutation site is A328 and the substituted amino acid is G; (12) The mutation sites are A328 and F58, the amino acid substituted at the mutation site A328 is G, and the amino acid substituted at the mutation site F58 is L or E; (13) The mutation sites are A328 and L151, the amino acid substituted at A328 is G, and the amino acid substituted at L151 is Q, R, H, W, A, V, M, N, C, T, or I; (14) The mutation sites are A328 and F217, the amino acid substituted at the mutation site A328 is G, and the amino acid substituted at the mutation site F217 is W, G, A, D, T, R, E, V, or Y; (15) The mutation sites are A328 and S220, the amino acid after the substitution of the mutation site A328 is G, and the amino acid after the substitution of the mutation site S220 is Q, L, N, or G; (16) The mutation sites are A328 and A221, the amino acid after the substitution of the mutation site A328 is G, and the amino acid after the substitution of the mutation site A221 is F, M, L, V, E, R, G, or S; (17) The mutation sites are A328 and D227, the amino acid substituted at A328 is G, and the amino acid substituted at D227 is E or C; (18) The mutation sites are A328 and I228, the amino acid substituted at the mutation site A328 is G, and the amino acid substituted at the mutation site I228 is L or G; (19) The mutation sites are A328 and L229, the amino acid substituted at the mutation site A328 is G, and the amino acid substituted at the mutation site L229 is R, V, I, or H; (20) The mutation sites are A328 and V297, the amino acid substituted at A328 is G, and the amino acid substituted at V297 is C or L; (21) The mutation sites are A328, A221, and D326, the amino acid substituted at mutation site A328 is G, the amino acid substituted at mutation site A221 is F, and the amino acid substituted at mutation site D326 is V; (22) The mutation sites are A328, A221, D326, and F298, the amino acid substituted at mutation site A328 is G, the amino acid substituted at mutation site A221 is F, the amino acid substituted at mutation site D326 is V, and the amino acid substituted at mutation site F298 is L; (23) The mutation sites are A328, A221, D326, F298, and N222. The amino acid substituted after mutation site A328 is G, the amino acid substituted after mutation site A221 is F, the amino acid substituted after mutation site D326 is V, the amino acid substituted after mutation site F298 is L, and the amino acid substituted after mutation site N222 is D. [2] (1) wherein the amino acid after substitution is E, I, C, A, or M; (2) The amino acid after substitution is I, S, A, W, H, or G; (3) The amino acid after substitution is Y or S, (4) The amino acid after substitution is Y or F, (5) The amino acid after substitution is M or A, (8) The amino acid after substitution is R, W or Q; (12) The amino acid after substitution at the mutation site F58 is L; (13) The amino acid after substitution at the mutation site L151 is Q or R; (14) The amino acid after substitution at the mutation point F217 is W, G, A, D, T, R or E; (15) The amino acid after substitution at the mutation site S220 is Q or L; (16) The amino acid after substitution at the mutation site A221 is F, M, L, V, E, R or G; The modified esterase according to [1], wherein the amino acid after substitution at mutation point L229 in (19) is R, V or I. [3] The amino acid after substitution in (4) is Y, (5) The amino acid after substitution is M; (8) The amino acid after substitution is R or W; (13) The amino acid after substitution at the mutation site L151 is Q; (14) The amino acid after substitution at the mutation point F217 is W, G, A, D, or T; The modified esterase according to [1], wherein the amino acid after substitution at mutation point A221 in (16) is F, M, L, V, E or R. [4] The modified esterase according to [1], wherein the amino acid substitution is any one of (11) to (23), and the selectivity for (1R,3S)-ethyl chrysanthemate is improved. [5] The modified esterase according to [4], wherein the amino acid substitution is A328G, A328G / F217W, A328G / A221M, A328G / A221L, A328G / A221V, or A328G / I228L. [6] The modified esterase described in [4], which has an amino acid substitution of A328G / A221F and acts specifically on (1R,3S)-ethyl chrysanthemate. [7] The modified esterase according to [4], wherein the amino acid substitutions are A328G / A221F / D326V. [8] The modified esterase according to [4], wherein the amino acid substitutions are A328G / A221F / D326V / F298L. [9] The modified esterase described in [4], wherein the amino acid substitutions are A328G / A221F / D326V / F298L / N222D.

[10] The modified esterase according to any one of [1] to [9], wherein the identity is 95% or more.

[11] The modified esterase according to [1], which consists of any one of the amino acid sequences of SEQ ID NOs: 2 to 11.

[12] A gene encoding the modified esterase described in any one of [1] to

[11] .

[13] The gene according to

[12] , comprising any one of the base sequences of SEQ ID NOs: 13 to 22.

[14] A recombinant DNA comprising the gene described in

[12] or

[13] .

[15] A microorganism carrying the recombinant DNA described in

[14] .

[16] An enzyme preparation comprising the modified esterase described in any one of [1] to

[11] .

[17] A method for preparing a modified esterase, comprising the following steps (I) to (III): (I) preparing a nucleic acid encoding the amino acid sequence of the modified esterase according to any one of [1] to

[11] ; (II) expressing the nucleic acid; and (III) recovering the expression product.

[18] The preparation method described in

[17] , wherein the amino acid sequence is any one of the amino acid sequences of SEQ ID NOs: 2 to 11.

[19] The preparation method described in

[17] , wherein the nucleic acid comprises any one of the base sequences of SEQ ID NOs: 13 to 22.

[20] A method for producing (1R,3S)-chrysanthemic acid, comprising allowing the modified esterase according to any one of [1] to

[11] to act on (1R,3S)-ethyl chrysanthemic acid.

[21] The method according to

[20] , wherein (1R,3S)-ethyl chrysanthemate coexists with (1R,3R)-ethyl chrysanthemate, (1S,3S)-ethyl chrysanthemate and (1S,3R)-ethyl chrysanthemate. [Brief description of the drawings]

[0008] [Figure 1] Evaluation results for the mutant F58X. The "reactivity to (1R,3S)-ethyl chrysanthemic acid" was evaluated as the value (B / A) obtained by dividing the "ratio (%) of the amount of (1R,3S)-chrysanthemic acid produced in the reaction using (1R,3S)-ethyl chrysanthemic acid (single substrate) relative to the wild-type enzyme" (B) by the "ratio (%) of the amount of (1R,3R)-chrysanthemic acid produced in the reaction using mixed substrates relative to the wild-type enzyme" (A) (the same applies to the following mutations). [Diagram 2] Evaluation results of mutations F217X, S220X, and D227X. [Diagram 3] Evaluation results for mutations I228X, L229X, and L231X. [Figure 4] Evaluation results for mutations S244X, A245X, and V297X. [Diagram 5] Evaluation results of mutations F298X and A328X. [Figure 6]Evaluation results of the combination mutations A328G / F58X, A328G / L151X, and A328G / F217X. The "selectivity for (1R,3S)-ethyl chrysanthemic acid" was evaluated by dividing the "ratio (%) of the amount of (1R,3S)-chrysanthemic acid produced in the reaction with mixed substrates to the A328G mutant" (C) by the "ratio (%) of the amount of (1R,3R)-chrysanthemic acid produced in the reaction with mixed substrates to the A328G mutant" (D) (the same applies to the following combination mutations). The value of the amount of (1R,3S)-chrysanthemic acid produced / the amount of (1R,3R)-chrysanthemic acid produced is also shown. [Figure 7] Evaluation results for combination mutations A328G / S220X, A328G / A221X, and A328G / D227X. [Figure 8] Evaluation results for combination mutations A328G / I228X, A328G / L229X, and A328G / V297X. [Figure 9] Evaluation results of combination mutations A328G / A221F, A328G / A221F / D326V, A328G / A221F / D326V / F298L, and A328G / A221F / D326V / F298L / N222D. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] For ease of explanation, some of the terms used in connection with the present invention are defined below. (term) The term "modified esterase" refers to an enzyme obtained by modifying or mutating a reference esterase (hereinafter referred to as "reference esterase"). The reference esterase is typically an esterase derived from Arthrobacter globiformis having the amino acid sequence of SEQ ID NO: 1.

[0010] In the present invention, the modification or mutation is "amino acid substitution". Therefore, when the modified esterase is compared with the reference esterase, differences are observed in some amino acid residues. In this specification, the modified esterase is also called a modified enzyme or a mutant.

[0011] In accordance with the convention used herein, each amino acid is represented by a single letter as follows: Methionine: M, Serine: S, Alanine: A, Threonine: T, Valine: V, Tyrosine: Y, Leucine: L, Asparagine: N, Isoleucine: I, Glutamine: Q, Proline: P, Aspartic Acid: D, Phenylalanine: F, Glutamic Acid: E, Tryptophan: W, Lysine: K, Cysteine: C, Arginine: R, Glycine: G, Histidine: H

[0012] In the present specification, the position of the mutation site is identified by numbers starting from the N-terminus toward the C-terminus, with the N-terminus being the first amino acid residue.

[0013] In this specification, a mutation due to amino acid substitution is expressed by a combination of one letter representing the amino acid residue before substitution, a number representing the mutation point (the position of the amino acid residue where the amino acid substitution occurs), and one letter representing the amino acid residue after substitution. For example, a mutation in which alanine at position 328 is replaced with glycine is expressed as "A328G". In addition, when expressing a combination (combination) of two mutations, the symbol " / " is used. For example, a combination of a mutation in which alanine at position 328 is replaced with glycine and a mutation in which alanine at position 221 is replaced with phenylalanine is expressed as "A328G / A221F".

[0014] 1. Modified esterase A first aspect of the present invention relates to a modified esterase (modified enzyme). The modified enzyme of the present invention typically has an amino acid sequence containing one or more specific amino acid substitutions (mutations) in the amino acid sequence of SEQ ID NO: 1. Due to this characteristic, the reactivity or selectivity, or both, to (1R,3S)-ethyl chrysanthemate are improved compared to an esterase consisting of the amino acid sequence of SEQ ID NO: 1. The amino acid sequence of SEQ ID NO: 1 is the sequence of an esterase derived from Arthrobacter globiformis.

[0015] "Reactivity towards (1R,3S)-ethyl chrysanthemate" is an expression that includes reactivity (relative reactivity) compared with the reactivity towards the optical isomers of (1R,3S)-ethyl chrysanthemate ((1R,3R)-ethyl chrysanthemate, (1S,3S)-ethyl chrysanthemate, (1S,3R)-ethyl chrysanthemate). Therefore, "improved reactivity towards (1R,3S)-ethyl chrysanthemate" is brought about by improved selectivity towards (1R,3S)-ethyl chrysanthemate, increased activity towards (1R,3S)-ethyl chrysanthemate, decreased activity towards the optical isomers of (1R,3S)-ethyl chrysanthemate ((1R,3R)-ethyl chrysanthemate, (1S,3S)-ethyl chrysanthemate, (1S,3R)-ethyl chrysanthemate), etc.

[0016] Here, the esterase derived from Arthrobacter globiformis (wild-type enzyme) has high selectivity for (1R,3R)-ethyl chrysanthemate, and when the four optical isomers ((1R,3R)-ethyl chrysanthemate, (1R,3S)-ethyl chrysanthemate, (1S,3S)-ethyl chrysanthemate, and (1S,3R)-ethyl chrysanthemate) coexist, it preferentially hydrolyzes (1R,3R)-ethyl chrysanthemate. Therefore, only (1R,3R)-chrysanthemic acid is usually detected as the product. In addition, the wild-type enzyme also has hydrolytic activity for (1R,3S)-ethyl chrysanthemate, but the reaction rate is overwhelmingly slower than the reaction rate of the hydrolytic activity for (1R,3R)-chrysanthemic acid, so that it does not apparently react with (1R,3S)-ethyl chrysanthemate in the coexistence of (1R,3R)-ethyl chrysanthemate. On the other hand, when (1R,3S)-ethyl chrysanthemic acid is reacted alone as a substrate, the product (1R,3S)-chrysanthemic acid is detected.

[0017] In the present application, taking into consideration the above-mentioned characteristics of the wild-type enzyme, "reactivity toward (1R,3S)-ethyl chrysanthemate" is evaluated based on the amount of (1R,3S)-chrysanthemic acid produced in a reaction using only (1R,3S)-ethyl chrysanthemate as a substrate (single substrate) and the amount of (1R,3R)-ethyl chrysanthemate produced in a reaction using a mixed substrate, which is a mixture of (1R,3R)-ethyl chrysanthemate, (1R,3S)-ethyl chrysanthemate, (1S,3S)-ethyl chrysanthemate, and (1S,3R)-ethyl chrysanthemate. Specifically, the "reactivity to (1R,3S)-ethyl chrysanthemic acid" is evaluated by dividing the "ratio (%) of the amount of (1R,3S)-chrysanthemic acid produced in the reaction using (1R,3S)-ethyl chrysanthemic acid (single substrate) to that of the wild-type enzyme" by the "ratio (%) of the amount of (1R,3R)-chrysanthemic acid produced in the reaction using mixed substrates to that of the wild-type enzyme", and when this value exceeds 1, it is determined that the "reactivity to (1R,3S)-ethyl chrysanthemic acid" is improved. In the preferred modified enzyme, this value is 2.0 or more, indicating a high degree of reactivity improvement. In the more preferred modified enzyme, this value is 3.0 or more, indicating even further improved reactivity. The wild-type enzyme is the enzyme before modification, i.e., the reference esterase.

[0018] As described above, the wild-type enzyme has high selectivity for (1R,3R)-ethyl chrysanthemic acid, and when reacted with a mixed substrate, (1R,3S)-chrysanthemic acid is not detected (substantially not produced). On the other hand, as shown in the Examples below, a mutant in which the 328th amino acid alanine of the wild-type enzyme is replaced with glycine (A328G mutant) has improved selectivity for (1R,3S)-ethyl chrysanthemic acid, and even when reacted with a mixed substrate, it exhibits hydrolysis activity for (1R,3S)-ethyl chrysanthemic acid, and the production of (1R,3S)-chrysanthemic acid was observed. Therefore, in the present application, the A328G mutant is used to evaluate "selectivity for (1R,3S)-ethyl chrysanthemic acid". Specifically, the "selectivity for (1R,3S)-ethyl chrysanthemic acid" is evaluated by dividing the "ratio (%) of the amount of (1R,3S)-chrysanthemic acid produced in the reaction using mixed substrates to the A328G mutant" by the "ratio (%) of the amount of (1R,3R)-chrysanthemic acid produced in the reaction using mixed substrates to the A328G mutant." When the value is 1 or more, the selectivity is equal to or higher than that of the A328G mutant, and it is determined that the "selectivity for (1R,3S)-ethyl chrysanthemic acid" is improved. In a preferred modified enzyme, the value is 1.5 or more, indicating a high degree of selectivity improvement. In a more preferred modified enzyme, the value is 2.0 or more, indicating an even higher level of selectivity. Note that when the amount of (1R,3R)-chrysanthemic acid produced is below the detection limit, i.e., when (1R,3R)-chrysanthemic acid is not substantially produced, the "ratio of the amount of (1R,3R)-chrysanthemic acid produced in the reaction using mixed substrates to the A328G mutant" is zero, and the above value cannot be calculated. The modified enzyme that produces such results (corresponding to the A328G / A221F mutant) can be said to be specific for (1R,3S)-ethyl chrysanthemate.

[0019] As used herein, "containing an amino acid substitution" means that the mutation point (i.e., the position of the amino acid residue where a specific amino acid substitution occurs) is the substituted amino acid. Therefore, when an amino acid sequence containing an amino acid substitution (mutated amino acid sequence) is compared with the amino acid sequence of SEQ ID NO: 1 that does not contain an amino acid substitution (reference amino acid sequence), a difference in amino acid residue is found at the position of the amino acid substitution.

[0020] The following lists the amino acid substitutions (mutation sites and amino acids after substitution) contained in the modified enzyme of the present invention that improve the reactivity to ethyl (1R,3S)-chrysanthemate. Note that (11) to (23) are more preferred because they improve not only the reactivity to ethyl (1R,3S)-chrysanthemate but also the selectivity to ethyl (1R,3S)-chrysanthemate.

[0021] (1) The mutation site is F58, and the substituted amino acid is E, I, C, A, M, or Y. Preferably, the substituted amino acid is E, I, C, A, or M. (2) The mutation site is D227, and the substituted amino acid is I, S, A, W, H, G, F, or V. Preferably, the substituted amino acid is I, S, A, W, H, or G. More preferably, the substituted amino acid is I, S, A, W, or H. (3) The mutation site is I228, and the amino acid after substitution is Y, S, T, or W. Preferably, the amino acid after substitution is Y or S. (4) The mutation site is L229, and the substituted amino acid is Y, F, or V. Preferably, the substituted amino acid is Y or F. More preferably, the substituted amino acid is Y. (5) The mutation site is L231, and the amino acid after substitution is M, A, or G. Preferably, the amino acid after substitution is M or A. More preferably, the amino acid after substitution is M. (6) The mutation site is S244 and the substituted amino acid is F. (7) The mutation site is A245, and the amino acid after substitution is N or G. (8) The mutation site is V297, and the substituted amino acid is R, W, Q, A, or I. Preferably, the substituted amino acid is R, W, or Q. More preferably, the substituted amino acid is R or W. (9) The mutation site is F298 and the substituted amino acid is S. (10) The mutation site is A328 and the substituted amino acid is N. (11) The mutation site is A328 and the substituted amino acid is G. (12) The mutation sites are A328 and F58, the amino acid following substitution at the mutation site A328 is G, and the amino acid following substitution at the mutation site F58 is L or E. Preferably, the amino acid following substitution at the mutation site F58 is L. (13) The mutation sites are A328 and L151, the amino acid substituted at the mutation site A328 is G, and the amino acid substituted at the mutation site L151 is Q, R, H, W, A, V, M, N, C, T, or I. Preferably, the amino acid substituted at the mutation site L151 is Q or R. More preferably, the amino acid substituted at the mutation site L151 is Q. (14) The mutation sites are A328 and F217, the amino acid substituted at the mutation site A328 is G, and the amino acid substituted at the mutation site F217 is W, G, A, D, T, R, E, V, or Y. Preferably, the amino acid substituted at the mutation site F217 is W, G, A, D, T, R, or E. More preferably, the amino acid substituted at the mutation site F217 is W, G, A, D, or T. (15) The mutation sites are A328 and S220, the amino acid after substitution at the mutation site A328 is G, and the amino acid after substitution at the mutation site S220 is Q, L, N, or G. Preferably, the amino acid after substitution at the mutation site S220 is Q or L. (16) The mutation sites are A328 and A221, the amino acid after substitution at the mutation site A328 is G, and the amino acid after substitution at the mutation site A221 is F, M, L, V, E, R, G, or S. Preferably, the amino acid after substitution at the mutation site A221 is F, M, L, V, E, R, or G. More preferably, the amino acid after substitution at the mutation site A221 is F, M, L, V, E, or R. (17) The mutation sites are A328 and D227, the amino acid substituted at the mutation site A328 is G, and the amino acid substituted at the mutation site D227 is E or C. (18) The mutation sites are A328 and I228, the amino acid substituted at the mutation site A328 is G, and the amino acid substituted at the mutation site I228 is L or G. (19) The mutation sites are A328 and L229, the amino acid after substitution at the mutation site A328 is G, and the amino acid after substitution at the mutation site L229 is R, V, I, or H. Preferably, the amino acid after substitution at the mutation site L229 is R, V, or I. (20) The mutation sites are A328 and V297, the amino acid substituted at the mutation site A328 is G, and the amino acid substituted at the mutation site V297 is C or L. (21) The mutation sites are A328, A221, and D326. The amino acid substituted after mutation site A328 is G. The amino acid substituted after mutation site A221 is F. The amino acid substituted after mutation site D326 is V. (22) The mutation sites are A328, A221, D326, and F298. The amino acid substituted after mutation site A328 is G, the amino acid substituted after mutation site A221 is F, the amino acid substituted after mutation site D326 is V, and the amino acid substituted after mutation site F298 is L. (23) The mutation sites are A328, A221, D326, F298, and N222. The amino acid substituted after mutation site A328 is G, the amino acid substituted after mutation site A221 is F, the amino acid substituted after mutation site D326 is V, the amino acid substituted after mutation site F298 is L, and the amino acid substituted after mutation site N222 is D.

[0022] Among the above amino acid substitutions, (14) and (15) are noteworthy because, although the mutation site F217 and the mutation site S220 alone did not improve reactivity, the combination with the A328G mutation improved the selectivity for (1R,3S)-ethyl chrysanthemate. In addition, the mutation sites A328 and A221, the amino acid after the substitution of the A328 mutation site with G, and the amino acid after the substitution of the A221 mutation site with F dramatically improved the selectivity for (1R,3S)-ethyl chrysanthemate, making it possible to prepare a modified enzyme that acts specifically on (1R,3S)-ethyl chrysanthemate, and are therefore extremely preferable amino acid substitutions.

[0023] Specific examples of the modified enzyme of the present invention include esterases having any one of the amino acid sequences of SEQ ID NOs: 2 to 11 (respectively, a mutant containing the amino acid substitution A328G of (11), a mutant containing one of the amino acid substitutions A328G / F217W of (14), a mutant containing one of the amino acid substitutions A328G / A221F of (16), a mutant containing one of the amino acid substitutions A328G / A221M of (16), a mutant containing one of the amino acid substitutions A328G / A221L of (16), and an esterase having the amino acid substitution A328G / A221L of (16). Examples of the corresponding mutants include a mutant containing A328G / A221V, which is one of the amino acid substitutions in (18), a mutant containing A328G / I228L, which is one of the amino acid substitutions in (21), a mutant containing A328G / A221F / D326V, which is one of the amino acid substitutions in (22), and a mutant containing A328G / A221F / D326V / F298L / N222D, which is one of the amino acid substitutions in (23). As shown in the Examples below, these mutants have particularly improved (1R,3S)-ethyl chrysanthemate selectivity. Among these, the A328G / A221F mutant acts specifically on (1R,3S)-ethyl chrysanthemate and is particularly useful. In addition, the A328G / A221F / D326V mutant, the A328G / A221F / D326V / F298L mutant, and the A328G / A221F / D326V / F298L / N222D mutant are extremely useful because they have very high reactivity as well as selectivity for (1R,3S)-ethyl chrysanthemate.

[0024] In general, when a part of the amino acid sequence of a protein is mutated, the mutated protein may have the same function as the protein before the mutation. That is, the mutation of the amino acid sequence may not substantially affect the function of the protein, and the function of the protein may be maintained before and after the mutation. On the other hand, when the identity of the amino acid sequences of two proteins is high, the probability that both proteins will show the same characteristics is high. Considering these technical common sense, if an enzyme has an amino acid sequence that is not completely identical (i.e., 100% identity) to the amino acid sequence of the modified enzyme, that is, "the amino acid sequence of SEQ ID NO: 1 containing any of the amino acid substitutions (1) to (23) above (specific examples of the amino acid sequence are the amino acid sequences of SEQ ID NOs: 2 to 11)" but has an amino acid sequence that shows high identity and exhibits a desired change in characteristics, it can be considered to be an enzyme that is substantially the same as the modified enzyme (substantially the same esterase). The identity here is 60% or more, 70% or more, 80% or more, 82% or more, 85% or more, 90% or more, 93% or more, 95% or more, 98% or more, or 99% or more. The higher the identity, the more preferred, thus in the most preferred embodiment the identity is 99% or greater.

[0025] When the above modified enzyme is compared with a substantially identical esterase, slight differences in amino acid sequence are observed. However, the differences in amino acid sequence are assumed to occur at positions other than the positions where the above amino acid substitutions have been made. Thus, for example, when the identity standard is the amino acid sequence of SEQ ID NO:2, positions other than G at position 328; when the identity standard is the amino acid sequence of SEQ ID NO:3, positions other than G at position 328 and W at position 217; when the identity standard is the amino acid sequence of SEQ ID NO:4, positions other than G at position 328 and F at position 221; when the identity standard is the amino acid sequence of SEQ ID NO:5, positions other than G at position 328 and M at position 221; when the identity standard is the amino acid sequence of SEQ ID NO:6, positions other than G at position 328 and L at position 221; when the identity standard is the amino acid sequence of SEQ ID NO:7, positions other than G at position 328 and M at position 221; The amino acid sequence differs at a position other than V at position 221, at a position other than G at position 328 and L at position 228 when the identity standard is the amino acid sequence of SEQ ID NO: 8, at a position other than G at position 328, F at position 221, and V at position 326 when the identity standard is the amino acid sequence of SEQ ID NO: 9, at a position other than G at position 328, F at position 221, V at position 326, and L at position 298 when the identity standard is the amino acid sequence of SEQ ID NO: 10, and at a position other than G at position 328, F at position 221, V at position 326, L at position 298, and D at position 222 when the identity standard is the amino acid sequence of SEQ ID NO: 11. In other words, the amino acid at position 328 is G in amino acid sequences that show the above identities (60% or more, 70% or more, 80% or more, 82% or more, 85% or more, 90% or more, 93% or more, 95% or more, 98%, or 99% or more) to the amino acid sequence of SEQ ID NO: 2.Similarly, in the amino acid sequence showing the above-mentioned identity with the amino acid sequence of SEQ ID NO:3, the amino acid at position 328 is G and the amino acid at position 217 is W; in the amino acid sequence showing the above-mentioned identity with the amino acid sequence of SEQ ID NO:4, the amino acid at position 328 is G and the amino acid at position 221 is F; in the amino acid sequence showing the above-mentioned identity with the amino acid sequence of SEQ ID NO:5, the amino acid at position 328 is G and the amino acid at position 221 is M; in the amino acid sequence showing the above-mentioned identity with the amino acid sequence of SEQ ID NO:6, the amino acid at position 328 is G and the amino acid at position 221 is L; in the amino acid sequence showing the above-mentioned identity with the amino acid sequence of SEQ ID NO:7, the amino acid at position 328 is G and the amino acid at position 221 is V; In the amino acid sequence showing the above-mentioned identity with the amino acid sequence of SEQ ID NO:8, the amino acid at position 328 is G and the amino acid at position 228 is L; in the amino acid sequence showing the above-mentioned identity with the amino acid sequence of SEQ ID NO:9, the amino acid at position 328 is G, the amino acid at position 221 is F, and the amino acid at position 326 is V; in the amino acid sequence showing the above-mentioned identity with the amino acid sequence of SEQ ID NO:10, the amino acid at position 328 is G, the amino acid at position 221 is F, the amino acid at position 326 is V, and the amino acid at position 298 is L; and in the amino acid sequence showing the above-mentioned identity with the amino acid sequence of SEQ ID NO:11, the amino acid at position 328 is G, the amino acid at position 221 is F, the amino acid at position 326 is V, the amino acid at position 298 is L, and the amino acid at position 222 is D.

[0026] The "slight difference in amino acid sequence" here is caused by deletion, substitution, addition, insertion, or a combination thereof of amino acids. Typically, it refers to a mutation (change) in the amino acid sequence caused by deletion or substitution of one to several (upper limit, for example, 3, 5, 7, 10) amino acids constituting the amino acid sequence, or addition or insertion of one to several (upper limit, for example, 3, 5, 7, 10) amino acids, or a combination thereof. The "slight difference in amino acid sequence" is preferably caused by conservative amino acid substitution. The "conservative amino acid substitution" here refers to the substitution of a certain amino acid residue with an amino acid residue having a side chain with similar properties. Amino acid residues are classified into several families according to their side chains, such as basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Conservative amino acid substitutions are preferably substitutions between amino acid residues in the same family. It is known that the active residues of the esterase derived from Arthrobacter globiformis (SEQ ID NO: 1) are the amino acid serine at position 59, the amino acid histidine at position 325, and the amino acid aspartic acid at position 326. Therefore, when making a mutation, it is preferable to avoid affecting the acid residues.

[0027] Incidentally, the identity (%) of two amino acid sequences or two base sequences (hereinafter, the term "two sequences" is used to include both) can be determined, for example, by the following procedure. First, the two sequences are aligned to enable optimal comparison (for example, gaps may be introduced into the first sequence to optimize the alignment with the second sequence). When a molecule (amino acid residue or nucleotide) at a specific position in the first sequence is the same as a molecule at the corresponding position in the second sequence, the molecule at that position can be said to be identical. The identity of two sequences is a function of the number of identical positions shared by the two sequences (i.e., identity (%)=number of identical positions / total number of positions×100), and preferably, the number and size of gaps required for optimization of the alignment are also taken into account.

[0028] Comparison of two sequences and determination of identity can be achieved using a mathematical algorithm. A non-limiting example of a mathematical algorithm that can be used for sequence comparison is the algorithm described in Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68 and modified in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into the NBLAST program and XBLAST program (version 2.0) described in Altschul et al. (1990) J. Mol. Biol. 215:403-10. To obtain equivalent nucleotide sequences, for example, BLAST nucleotide searches can be performed with the NBLAST program with score = 100 and wordlength = 12. Equivalent amino acid sequences can be obtained by, for example, performing a BLAST polypeptide search with the XBLAST program with score = 50 and wordlength = 3. To obtain gapped alignments for comparison, Gapped BLAST, as described in Altschul et al. (1997) Amino Acids Research 25(17):3389-3402, can be used. When using BLAST and Gapped BLAST, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used. For details, see http: / / www.ncbi.nlm.nih.gov. Examples of other mathematical algorithms that can be used for comparing sequences include the algorithm described in Myers and Miller (1988) ComputAppl Biosci. 4:11-17. Such algorithms are incorporated, for example, in the ALIGN program available on the GENESTREAM network server (IGH Montpellier, France) or the ISREC server. When using the ALIGN program to compare amino acid sequences, for example, the PAM120 residue mass table can be used, with gap length penalty=12 and gap penalty=4.

[0029] The identity of two amino acid sequences can be determined using the GAP program in the GCG software package, using a Blossom 62 matrix or a PAM250 matrix with a gap weight of 12, 10, 8, 6, or 4, and a gap length weight of 2, 3, or 4. The identity of two nucleotide sequences can also be determined using the GAP program in the GCG software package (available at http: / / www.gcg.com), with a gap weight of 50 and a gap length weight of 3.

[0030] Typically, an esterase having the amino acid sequence of SEQ ID NO: 1, i.e., an esterase derived from Arthrobacter globiformis, is mutated (by any of the amino acid substitutions (1) to (23) above) to obtain the modified enzyme of the present invention. The substantially identical esterase can be obtained by further mutating an esterase having the amino acid sequence of SEQ ID NO: 1 mutated (by any of the amino acid substitutions (1) to (23) above), by adding an equivalent mutation to an esterase having an amino acid sequence highly identical to the amino acid sequence of SEQ ID NO: 1, such as an esterase derived from the same species or genus as the Arthrobacter globiformis strain that produces the esterase having the amino acid sequence of SEQ ID NO: 1, or by further mutating an ester obtained by the above mutation. Here, the "equivalent mutation" refers to the substitution of an amino acid residue corresponding to the amino acid residue at the mutation site in the present invention (the mutation site in any of the amino acid substitutions (1) to (23) above) in an amino acid sequence highly identical to the amino acid sequence of SEQ ID NO: 1. An example of an esterase having an amino acid sequence highly identical to the amino acid sequence of SEQ ID NO: 1 is an esterase derived from Arthrobacter sp. Rue61a and having the amino acid sequence of SEQ ID NO: 23 (amino acid sequence identity: 81%).

[0031] The term "corresponding" when used herein with respect to amino acid residues means that the amino acid residues make an equivalent contribution to the exertion of the function between the proteins (enzymes) being compared. For example, when the amino acid sequence to be compared with the amino acid sequence of the reference esterase (the amino acid sequence of SEQ ID NO: 1) is aligned to allow optimal comparison while taking into account partial homology of the primary structure (amino acid sequence) (at this time, gaps may be introduced as necessary to optimize the alignment), an amino acid at a position corresponding to a specific amino acid in the reference amino acid sequence can be identified as a "corresponding amino acid". Instead of or in addition to the comparison between primary structures, the "corresponding amino acid" can also be identified by comparing three-dimensional structures (three-dimensional structures). By using three-dimensional structure information, a highly reliable comparison result can be obtained. In this case, a method of performing alignment while comparing the atomic coordinates of the three-dimensional structures of multiple enzymes can be adopted. The three-dimensional structure information of the target enzyme to be mutated can be obtained, for example, from the Protein Data Bank (http: / / www.pdbj.org / index_j.html).

[0032] An example of a method for determining the three-dimensional structure of a protein by X-ray crystallography is shown below. (1) Crystallize the protein. Crystallization is essential for determining the three-dimensional structure of the protein, but it is also useful industrially as a method for purifying the protein to a high degree of purity and for preserving the protein in a stable manner at a high density. In this case, it is recommended to crystallize the protein bound to a substrate or its analog compound as a ligand. (2) The prepared crystals are irradiated with X-rays to collect diffraction data. In many cases, protein crystals are damaged by X-ray irradiation, resulting in a deterioration of their diffraction ability. In such cases, a low-temperature measurement technique has recently become widespread, in which the crystals are rapidly cooled to around -173°C and diffraction data is collected in this state. Finally, highly brilliant synchrotron radiation is used to collect high-resolution data for structure determination. (3) In order to perform crystal structure analysis, in addition to diffraction data, phase information is required. If the crystal structure of a protein related to the target protein is unknown, it is impossible to determine the structure by molecular replacement, and the phase problem must be solved by heavy atom isomorphous replacement. Heavy atom isomorphous replacement is a method of obtaining phase information by introducing metal atoms with large atomic numbers such as mercury and platinum into a crystal and utilizing the contribution of the metal atoms' large X-ray scattering ability to the X-ray diffraction data. The determined phase can be improved by smoothing the electron density in the solvent region of the crystal. Since water molecules in the solvent region have a large fluctuation and almost no electron density is observed, approximating the electron density in this region to 0 can bring it closer to the true electron density, and thus improve the phase. In addition, if the asymmetric unit contains multiple molecules, the phase can be further improved by averaging the electron densities of these molecules. A model of the protein is fitted to the electron density map calculated using the improved phase in this way. This process is carried out on computer graphics using a program such as QUANTA by MSI (USA). After this, the structure is refined using a program such as X-PLOR by MSI, and the structure analysis is completed. If the crystal structure of a protein related to the target protein is known, the atomic coordinates of the known protein can be used to determine the structure by molecular replacement. Molecular replacement and structure refinement can be performed using a program such as CNS_SOLVE ver.11.

[0033] 2. Nucleic acid encoding modified esterase A second aspect of the present invention provides nucleic acids related to the modified enzymes of the present invention, i.e., genes encoding the modified enzymes, nucleic acids that can be used as probes for identifying the nucleic acids encoding the modified enzymes, and nucleic acids that can be used as primers for amplifying or mutating the nucleic acids encoding the modified enzymes.

[0034] A gene encoding a modified enzyme is typically used for preparing the modified enzyme. A genetic engineering preparation method using a gene encoding the modified enzyme makes it possible to obtain a modified enzyme in a more homogeneous state. This method is also suitable for preparing a large amount of modified enzyme. The use of the gene encoding the modified enzyme is not limited to the preparation of modified enzymes. For example, the nucleic acid can be used as an experimental tool for elucidating the mechanism of action of the modified enzyme, or as a tool for designing or producing further modified forms of the enzyme.

[0035] As used herein, the term "gene encoding a modified enzyme" refers to a nucleic acid that, when expressed, gives the modified enzyme, and includes not only a nucleic acid having a base sequence corresponding to the amino acid sequence of the modified enzyme, but also a nucleic acid in which a sequence that does not code for an amino acid sequence is added to such a nucleic acid. Codon degeneracy is also taken into consideration.

[0036] Examples of gene sequences (base sequences) encoding the modified enzymes are shown in SEQ ID NOs: 13 to 22. These sequences encode the mutants shown in the Examples below. SEQ ID NO: 13: A328G mutant SEQ ID NO: 14: A328G / A217W mutant SEQ ID NO: 15: A328G / A221F mutant SEQ ID NO: 16: A328G / A221M mutant SEQ ID NO: 17: A328G / A221L mutant SEQ ID NO: 18: A328G / A221V mutant SEQ ID NO: 19: A328G / I228L mutant SEQ ID NO: 20: A328G / A221F / D326V mutant SEQ ID NO: 21: A328G / A221F / D326V / F298L mutant SEQ ID NO: 22: A328G / A221F / D326V / F298L / N222D mutant

[0037] When the gene of the present invention is expressed in a host, a sequence encoding a signal peptide (signal sequence) may be added to the 5'-terminus of the above sequence (any of SEQ ID NOs: 13 to 22). The signal sequence may be selected depending on the host. Any signal sequence that can express the desired mutant can be used in the present invention. Available signal sequences include a sequence encoding the signal peptide of α-factor (Protein Engineering, 1996, vol. 9, p. 1055-1061), a sequence encoding the signal peptide of α-factor receptor, a sequence encoding the signal peptide of SUC2 protein, a sequence encoding the signal peptide of PHO5 protein, a sequence encoding the signal peptide of BGL2 protein, a sequence encoding the signal peptide of AGA2 protein, a sequence encoding the signal peptide of TorA (trimethylamine N-oxide reductase), a sequence encoding the signal peptide of PhoD (phosphoesterase) derived from Bacillus subtilis, a sequence encoding the signal peptide of LipA (lipase) derived from Bacillus subtilis, a sequence encoding the signal peptide of Taka-amylase derived from Aspergillus oryzae (JP Patent Publication 2009-60804), and a sequence encoding the signal peptide of α-amylase derived from Bacillus amyloliquefaciens (Eur. J. Biochem. 155, 577-581). (1986)), a sequence encoding the signal peptide of neutral protease derived from Bacillus subtilis (APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1995, pp. 1610-1613 Vol. 61, No. 4), a sequence encoding the signal peptide of cellulase derived from bacteria of the genus Bacillus (JP Patent Publication No. 2007-130012), and a sequence encoding pelB used in the pET system can be mentioned as examples.

[0038] The nucleic acids of the present invention can be prepared in an isolated state by using standard genetic engineering techniques, molecular biological techniques, biochemical techniques, chemical synthesis, etc., with reference to the sequence information disclosed in this specification or the attached sequence listing.

[0039] In another aspect of the present invention, there is provided a nucleic acid (hereinafter also referred to as "homologous nucleic acid"; a base sequence defining a homologous nucleic acid is also referred to as "homologous base sequence") that has a part of a different base sequence, although the function of the protein it encodes is equivalent when compared with the base sequence of a gene encoding the modified enzyme of the present invention. An example of a homologous nucleic acid is a DNA that has a base sequence containing one or more base substitutions, deletions, insertions, additions, or inversions based on the base sequence of a nucleic acid encoding the modified enzyme of the present invention, and encodes a protein having an enzyme activity characteristic of the modified enzyme (i.e., esterase activity). Base substitutions or deletions may occur at multiple sites. Here, "multiple" refers to, for example, 2 to 40 bases, preferably 2 to 20 bases, and more preferably 2 to 10 bases, although this varies depending on the position and type of amino acid residues in the three-dimensional structure of the protein encoded by the nucleic acid.

[0040] A homologous nucleic acid has, for example, 60% or more, preferably 70% or more, more preferably 80% or more, even more preferably 85% or more, even more preferably about 90% or more, even more preferably 95% or more, and most preferably 99% or more identity to a reference base sequence.

[0041] Such homologous nucleic acids can be obtained, for example, by restriction enzyme treatment, treatment with exonuclease or DNA ligase, or introduction of mutations by site-directed mutagenesis (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New York) or random mutagenesis (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New York). Homologous nucleic acids can also be obtained by other methods such as ultraviolet irradiation.

[0042] Another aspect of the present invention relates to a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of a gene encoding the modified enzyme of the present invention. Yet another aspect of the present invention provides a nucleic acid having a nucleotide sequence at least about 60%, 70%, 80%, 90%, 95%, 99%, or 99.9% identical to the nucleotide sequence of a gene encoding the modified enzyme of the present invention or a complementary nucleotide sequence thereto.

[0043] Yet another aspect of the present invention relates to a nucleic acid having a base sequence that hybridizes under stringent conditions to a base sequence of a gene encoding the modified enzyme of the present invention or a base sequence complementary to the homologous base sequence. Here, "stringent conditions" refers to conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed. Such stringent conditions are known to those skilled in the art and can be set with reference to, for example, Molecular Cloning (Third Edition, Cold Spring Harbor Laboratory Press, New York) or Current protocols in molecular biology (edited by Frederick M. Ausubel et al., 1987). An example of stringent conditions includes incubation at about 42°C to about 50°C using a hybridization solution (50% formamide, 10xSSC (0.15M NaCl, 15mM sodium citrate, pH 7.0), 5xDenhardt's solution, 1% SDS, 10% dextran sulfate, 10µg / ml denatured salmon sperm DNA, 50mM phosphate buffer (pH 7.5)), followed by washing at about 65°C to about 70°C using 0.1xSSC, 0.1% SDS. More preferred stringent conditions include, for example, conditions using a hybridization solution containing 50% formamide, 5xSSC (0.15M NaCl, 15mM sodium citrate, pH 7.0), 1xDenhardt's solution, 1% SDS, 10% dextran sulfate, 10μg / ml denatured salmon sperm DNA, and 50mM phosphate buffer (pH 7.5).

[0044] Yet another aspect of the present invention provides a nucleic acid (nucleic acid fragment) having a part of the base sequence of a gene encoding the modified enzyme of the present invention, or a complementary base sequence thereto. Such a nucleic acid fragment can be used for detecting, identifying, and / or amplifying a nucleic acid having the base sequence of a gene encoding the modified enzyme of the present invention. For example, the nucleic acid fragment is designed to include at least a portion that hybridizes to a continuous nucleotide portion (e.g., about 10 to about 100 bases long, preferably about 20 to about 100 bases long, more preferably about 30 to about 100 bases long) in the base sequence of a gene encoding the modified enzyme of the present invention. When used as a probe, the nucleic acid fragment can be labeled. For example, a fluorescent substance, an enzyme, or a radioisotope can be used for labeling.

[0045] Yet another aspect of the present invention relates to a recombinant DNA containing the gene of the present invention (a gene encoding a modified enzyme). The recombinant DNA of the present invention is provided, for example, in the form of a vector. As used herein, the term "vector" refers to a nucleic acid molecule that can transport a nucleic acid inserted therein into a target such as a cell.

[0046] An appropriate vector is selected depending on the purpose of use (cloning, protein expression) and the type of host cell. Examples of vectors using E. coli as a host include pET21 or modified versions thereof, M13 phage or modified versions thereof, λ phage or modified versions thereof, pBR322 or modified versions thereof (pB325, pAT153, pUC8, etc.), etc. Examples of vectors using yeast as a host include pYepSec1, pMFa, pYES2, pPIC3.5K, etc., examples of vectors using insect cells as a host include pAc, pVL, etc., and examples of vectors using mammalian cells as a host include pCDM8, pMT2PC, etc.

[0047] The vector of the present invention is preferably an expression vector. An "expression vector" refers to a vector that can introduce a nucleic acid inserted therein into a target cell (host cell) and can express the nucleic acid in the cell. An expression vector usually contains a promoter sequence necessary for the expression of the inserted nucleic acid, an enhancer sequence that promotes the expression, and the like. An expression vector containing a selection marker can also be used. When such an expression vector is used, the presence or absence (and the degree of introduction) of the expression vector can be confirmed using the selection marker.

[0048] Insertion of the nucleic acid of the present invention into a vector, insertion of a selection marker gene (if necessary), insertion of a promoter (if necessary), etc. can be carried out using standard recombinant DNA techniques (for example, see Molecular Cloning, Third Edition, 1.84, Cold Spring Harbor Laboratory Press, New York; well-known methods using restriction enzymes and DNA ligase).

[0049] As host cells, microorganisms such as Aspergillus oryzae, Bacillus bacteria (e.g., Bacillus subtilis), Escherichia coli, and Saccharomyces cerevisiae can be used from the viewpoint of ease of handling, but any host cell capable of replicating recombinant DNA and expressing the gene for the modified enzyme can be used. Preferably, Escherichia coli and Saccharomyces cerevisiae can be used. Examples of Escherichia coli include E. coli BL21(DE3) when a T7 promoter is used, and E. coli JM109 when not. Examples of budding yeast include SHY2, AH22, and INVSc1 (Invitrogen).

[0050] Another aspect of the present invention relates to a microorganism carrying the recombinant DNA of the present invention (i.e., a transformant). The microorganism of the present invention can be obtained by transfection or transformation using the above-mentioned vector of the present invention. For example, the calcium chloride method (J. Mol. Biol., vol. 53, p. 159 (1970)), the Hanahan method (J. Mol. Biol., vol. 166, p. 557 (1983)), the SEM method (Gene, vol. 96, p. 23 (1990)), the method of Chung et al. (Proceedings of the National Academy of Sciences of the USA, vol. 86, p. 2172 (1989)), the calcium phosphate coprecipitation method, electroporation (Potter, H. et al., Proc. Natl. Acad. Sci. USA 81, 7161-7165 (1984)), lipofection (Felgner, PL et al., Proc. Natl. Acad. Sci. USA 84, 7413-7417 (1984)) etc. The microorganism of the present invention can be used to produce the modified enzyme of the present invention.

[0051] 3. Enzyme preparations containing modified esterase The modified enzyme of the present invention is provided in the form of, for example, an enzyme preparation. The enzyme preparation may contain, in addition to the active ingredient (the modified enzyme of the present invention), an excipient, a buffer, a suspending agent, a stabilizer, a preservative, a physiological saline solution, and the like. Examples of the excipient that can be used include starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, sucrose, glycerol, and the like. Examples of the buffer that can be used include phosphates, citrates, acetates, and the like. Examples of the stabilizer that can be used include propylene glycol, ascorbic acid, and the like. Examples of the preservative that can be used include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like. Examples of the preservative that can be used include ethanol, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol, and the like.

[0052] 4. Method for preparing modified esterase A further aspect of the present invention relates to a method for preparing a modified enzyme. In one embodiment of the preparation method of the present invention, the modified enzyme that the present inventors have succeeded in obtaining is prepared by genetic engineering techniques. In this embodiment, a nucleic acid encoding any one of the amino acid sequences of SEQ ID NOs: 2 to 11 is prepared (step (I)). Here, the "nucleic acid encoding a specific amino acid sequence" is a nucleic acid that, when expressed, gives a polypeptide having the amino acid sequence, and may be a nucleic acid consisting of a base sequence corresponding to the amino acid sequence, or may have an extra sequence (which may be a sequence encoding an amino acid sequence or a sequence not encoding an amino acid sequence) added to such a nucleic acid. Codon degeneracy is also taken into consideration. The "nucleic acid encoding any one of the amino acid sequences of SEQ ID NOs: 2 to 11" can be prepared in an isolated state by using standard genetic engineering techniques, molecular biological techniques, biochemical techniques, etc., with reference to the sequence information disclosed in this specification or the attached sequence listing. Here, all of the amino acid sequences of SEQ ID NOs: 2 to 11 are obtained by mutating the amino acid sequence of an esterase derived from Arthrobacter globiformis. Therefore, a nucleic acid (gene) encoding any one of the amino acid sequences of SEQ ID NOs: 2 to 11 can be obtained by adding necessary mutations to a gene encoding an esterase derived from Arthrobacter globiformis. Many methods for site-specific base sequence substitution are known in the art (see, for example, Molecular Cloning, Third Edition, Cold Spring Harbor Laboratory Press, New York), and an appropriate method can be selected and used from among them. As a site-specific mutagenesis method, a site-specific amino acid saturation mutagenesis method can be adopted. The site-specific amino acid saturation mutagenesis method is a "semi-rational, semi-random" method in which a position involved in a desired function is predicted based on the three-dimensional structure of a protein, and an amino acid saturation mutation is introduced (J. Mol. Biol. 331, 585-592 (2003)).For example, it is possible to introduce site-specific amino acid saturation mutations using a kit such as Quick Change (Stratagene) or overlap extension PCR (Nucleic Acid Res. 16, 7351-7367 (1988)). DNA polymerases used in PCR include Taq polymerase. However, it is preferable to use highly accurate DNA polymerases such as KOD-PLUS- (Toyobo) and Pfu turbo (Stratagene).

[0053] Following step (I), the prepared nucleic acid is expressed (step (II)). For example, an expression vector into which the nucleic acid is inserted is first prepared, and then a host cell is transformed with the vector.

[0054] Next, the transformant is cultured under conditions in which the modified enzyme, which is the expression product, is produced. The transformant may be cultured according to a conventional method. The carbon source used in the medium may be any assimilable carbon compound, such as glucose, sucrose, lactose, maltose, molasses, or pyruvic acid. The nitrogen source may be any usable nitrogen compound, such as peptone, meat extract, yeast extract, casein hydrolysate, or soybean meal alkaline extract. In addition, salts such as phosphates, carbonates, sulfates, magnesium, calcium, potassium, iron, manganese, and zinc, specific amino acids, and specific vitamins may be used as necessary.

[0055] Meanwhile, the culture temperature can be set within the range of 30°C to 40°C (preferably around 37°C). The culture time can be set in consideration of the growth characteristics of the transformant to be cultured and the production characteristics of the modified enzyme. The pH of the medium is adjusted within a range in which the transformant grows and the enzyme is produced. The pH of the medium is preferably about 6.0 to 9.0 (preferably around pH 7.0).

[0056] Next, the expression product (modified enzyme) is recovered (step (III)). The culture solution containing the cells after the culture can be used as an enzyme solution as is or after concentration, removal of impurities, etc., but the expression product is generally first recovered from the culture solution or cells. If the expression product is a secretory protein, it can be recovered from the culture solution, and if not, it can be recovered from the cells. When recovering from the culture medium, for example, the culture supernatant is filtered and centrifuged to remove insoluble matter, and then vacuum concentration, membrane concentration, salting out using ammonium sulfate or sodium sulfate, fractional precipitation using methanol, ethanol, acetone, or the like, dialysis, heat treatment, isoelectric focusing, gel filtration, adsorption chromatography, ion exchange chromatography, affinity chromatography, and other types of chromatography (for example, gel filtration using Sephadex gel (GE Healthcare Biosciences), DEAE Sepharose CL-6B (GE Healthcare Biosciences), Octyl Sepharose CL-6B (GE Healthcare Biosciences), CM Sepharose CL-6B (GE Healthcare Biosciences)), and the like are combined to obtain a purified product of the modified enzyme. On the other hand, when recovering from within the cells, the cells are collected by filtering, centrifuging, or the like, and then the cells are destroyed by mechanical methods such as pressurization, ultrasonication, and physical disruption, or by enzymatic methods using lysozyme, and then the modified enzyme is separated and purified in the same manner as described above to obtain a purified product.

[0057] The purified enzyme obtained as described above can be provided in a powder form, for example, by freeze-drying, vacuum drying, or spray drying. In this case, the purified enzyme may be dissolved in advance in a phosphate buffer, triethanolamine buffer, Tris-HCl buffer, or Good's buffer. Preferably, a phosphate buffer or triethanolamine buffer can be used. Examples of Good's buffer include PIPES, MES, and MOPS.

[0058] Usually, gene expression and expression products (modified enzymes) are collected using an appropriate host-vector system as described above, but a cell-free synthesis system may also be used. Here, the term "cell-free synthesis system (cell-free transcription system, cell-free transcription / translation system)" refers to the in vitro synthesis of mRNA and proteins encoded by template nucleic acids (DNA and mRNA) using ribosomes and transcription / translation factors derived from living cells (or obtained by genetic engineering techniques), rather than using living cells. In cell-free synthesis systems, cell extracts obtained by purifying cell lysates as necessary are generally used. Cell extracts generally contain ribosomes, various factors such as initiation factors, and various enzymes such as tRNA, which are necessary for protein synthesis. When synthesizing proteins, various amino acids, energy sources such as ATP and GTP, and other substances necessary for protein synthesis, such as creatine phosphate, are added to the cell extract. Of course, during protein synthesis, ribosomes, various factors, and / or various enzymes prepared separately may be supplemented as necessary.

[0059] The development of a transcription / translation system in which each molecule (factor) required for protein synthesis is reconstituted has also been reported (Shimizu, Y. et al.: Nature Biotech., 19, 751-755, 2001). In this synthesis system, the genes of 31 types of factors, consisting of three types of initiation factors, three types of elongation factors, four types of factors involved in termination, 20 types of aminoacyl-tRNA synthetases that bind each amino acid to tRNA, and methionyl-tRNA formyltransferase, which constitute the bacterial protein synthesis system, are amplified from the E. coli genome, and the protein synthesis system is reconstituted in vitro using these. Such a reconstituted synthesis system may be used in the present invention.

[0060] The term "cell-free transcription / translation system" is used interchangeably with cell-free protein synthesis system, in vitro translation system, or in vitro transcription / translation system. In an in vitro translation system, RNA is used as a template to synthesize proteins. Total RNA, mRNA, in vitro transcription products, etc. are used as template RNA. On the other hand, in an in vitro transcription / translation system, DNA is used as a template. The template DNA should contain a ribosome binding region, and preferably contains an appropriate terminator sequence. In an in vitro transcription / translation system, conditions are set in which factors required for each reaction are added so that the transcription reaction and the translation reaction proceed consecutively.

[0061] 5. Uses of modified esterases A further aspect of the present invention relates to the use of the modified enzyme, and provides a method for producing (1R,3S)-chrysanthemic acid using the modified enzyme of the present invention. In the production method of the present invention, the modified enzyme of the present invention is allowed to act on (1R,3S)-ethyl chrysanthemic acid to hydrolyze it and produce (1R,3S)-chrysanthemic acid. The reaction conditions are not particularly limited as long as the desired hydrolysis reaction is achieved. For example, the reaction can be carried out using a buffer solution such as glycine-NaOH or PIPES under conditions of pH 6 to 11 and temperature 40°C to 60°C. As the substrate, a highly pure (1R,3S)-ethyl chrysanthemic acid (e.g., purity of 98% or more) may be used, or a substrate in which (1R,3R)-ethyl chrysanthemic acid, (1S,3S)-ethyl chrysanthemic acid and (1S,3R)-ethyl chrysanthemic acid coexist (e.g., a mixed ethyl chrysanthemic acid of (1R,3R):(1S,3S):(1R,3S):(1S,3R)=3:3:2:2, etc.) may be used. The reaction product is used for various purposes after optical resolution as necessary. Column chromatography can be used for optical resolution. The modified enzyme may be immobilized. EXAMPLES

[0062] The wild-type esterase derived from Arthrobacter globiformis (amino acid sequence shown in SEQ ID NO: 1 and gene sequence shown in SEQ ID NO: 12) has high selectivity for ethyl (1R,3R)-chrysanthemate. Modification of the stereoselectivity of the esterase by protein engineering techniques was attempted.

[0063] 1. Preparation of mutant libraries Mutant libraries (CASTing Library and two-point combinatorial mutants) were prepared by the following method. (1) Introduction of mutations and transformation PCR primers for introducing mutations were designed, and PCR was performed under the following conditions to introduce mutations. <Template plasmid> pET21b (introduced with wild-type enzyme gene) approximately 30 ng / μL <PCR reaction composition> (total volume 20 μL) 5× Prime STAR GXL Buffer (Takara Bio): 4 μL dNTP Mixture (2.5 mM each): 1.6 μL Template (approximately 30 ng / μL): 0.25 μL F-prime: 0.2 μL R-prime: 0.2 μL Prime STAR GXL DNA Polymerase (Takara Bio): 0.8 μL The above were mixed and adjusted to 20 μL with ultrapure water (Milli Q water)

[0064] <PCR conditions> Reaction at 98°C for 1 minute Reactions at 98°C for 10 seconds, 60°C for 15 seconds, and 68°C for 2 minutes for 20 cycles Reaction at 68°C for 5 minutes Left standing at 4°C

[0065] 1.5 μL of DpnI was added to 15 μL of the PCR reaction solution and treated (37°C, for 3 hours or more). Next, using 2 μL of the DpnI-treated solution, ligation treatment (16°C, O / N) was performed with the following ligation reaction composition. <Ligation reaction composition> (total volume 5.5 μL) DpnI-treated PCR product: 2 μL T4 Polynucleotide Kinase: 1μL Ligation High (Toyobo): 2.5μL

[0066] 5.5 μL of the ligation reaction solution was used to transform 25 μL of E. coli BL21(DE3) (treated at 42°C for 45 seconds, then rapidly cooled on ice). Approximately 30 μL of the transformation solution was applied to an LB agar medium plate supplemented with ampicillin (final concentration 0.1 mg / mL) and cultured at 37°C overnight (preculture).

[0067] (2) Obtaining enzyme extract After preculture, each mutant strain was inoculated into 1 mL of TB medium (Invitrogen) supplemented with ampicillin (final concentration 0.1 mg / mL). Culture was performed at 33°C for 48 hours (IPTG (final concentration 0.1 mM) was added 24 hours after the start of culture), followed by centrifugation (3,000 g x 10 minutes). The supernatant was removed to collect the bacterial cells, which were then lysed with a lysing agent (25°C, 2 hours or more) to extract the enzyme. The lysate was centrifuged (3,000 g x 10 minutes), and the supernatant was collected to obtain the enzyme extract.

[0068] (3) Generation of combination mutants Using the A328G mutant as a template, PCR was performed in the same manner as in (1) above. The PCR product was collected, and an enzyme extract was obtained in the same manner as in (2) above. Based on the results of the CASTing Library evaluation and the results of docking simulations using MOE (MOLSIS), candidates for the mutation site to be combined with A328G were selected.

[0069] (4) Generation of multiple mutants Using the A328G / A221F mutant as a template, random mutagenesis was performed by error-prone PCR. Using trans-pNP chrysanthemic acid, screening was performed based on the amount of released pNP. Using the obtained mutant as a template, similar mutagenesis and screening were repeated multiple times to produce multiple mutants with increased reactivity to (1R,3S)-ethyl chrysanthemic acid.

[0070] 2. Mutant Evaluation 2-1. Evaluation of CASTing Library The CASTing Library was evaluated by the following method. To evaluate the change in reactivity to (1R,3S)-ethyl chrysanthemate, the following two types of substrates were used. Substrate 1 (mixed substrate): Ethyl chrysanthemum acid (purity 95% or higher, mixture of (1R,3R), (1R,3S), (1S,3R), and (1S,3S) ((1R,3R):(1S,3S):(1R,3S):(1S,3R)=3:3:2:2), Tokyo Chemical Industry) Substrate 2 (single substrate): (1R,3S)-ethyl chrysanthemate 1 μL of substrate (Substrate 1 or Substrate 2) was dispensed into a reaction vessel, and 99 μL of the measurement sample (enzyme extract prepared in 1.(2) above) was added and reacted (40° C., 24 hours). After the reaction, 10 μL of 6N HCl and 150 μL of hexane were added. After thorough stirring, the mixture was centrifuged and 100 μL of the organic layer was collected. The extract was dried and redissolved in an analytical solvent, and then analyzed by supercritical fluid chromatography (SFC). The analytical conditions are shown below. <Supercritical fluid chromatography (SFC) analysis conditions> Equipment: ACQUITY UPC2 PDA System (Waters) Column: ACQUITY UPC2 Trefoil AMY1 (Waters) 2.5 μm, 3.0 × 150 mm Mobile phase: CO2 / 10mM ammonium acetate containing 2-propanol (0.5% acetic acid) = 92 / 8 Flow rate: 2.0 mL / min Injection volume: 1μL Analysis time: 1.5 minutes PDA detector: 205-400 nm Resolution: 1.2 nm

[0071] The change in reactivity to (1R,3S)-ethyl chrysanthemic acid was evaluated based on the reactivity of the wild-type enzyme. Specifically, the value of "the ratio (%) of the amount of (1R,3S)-chrysanthemic acid produced in the reaction using (1R,3S)-ethyl chrysanthemic acid (single substrate) to the wild-type enzyme" / "the ratio (%) of the amount of (1R,3R)-chrysanthemic acid produced in the reaction using mixed substrates to the wild-type enzyme" was calculated from the SFC area, and if this value exceeded 1, it was determined that the reactivity to (1R,3S)-ethyl chrysanthemic acid had increased.

[0072] The evaluation results are shown in Figures 1 to 5. The following mutations were identified as having increased reactivity to (1R,3S)-ethyl chrysanthemate. F58E, F58I, F58C, F58A, F58M, F58Y D227I, D227S, D227A, D227W, D227H, D227G, D227F, D227V I228Y, I228S, I228T, I228W L229Y, L229F, L229V L231M, L231A, L231G S244F A245N, A245G V297R, V297W, V297Q, V297A, V297I F298S A328N, A328G

[0073] The following mutations show values ​​of 2.0 or more and are therefore preferred for improving the reactivity to (1R,3S)-ethyl chrysanthemate. F58E, F58I, F58C, F58A, F58M D227I, D227S, D227A, D227W, D227H, D227G I228Y, I228S L229Y, L229F L231M, L231A V297R, V297W, V297Q A328G

[0074] The following mutations have values ​​of 3.0 or more, which are more preferable. Note that A328G is a particularly effective mutation because it produces (1R,3S)-chrysanthemic acid even in the reaction using substrate 1 (mixed substrate). F58E, F58I, F58C, F58A, F58M D227I, D227S, D227A, D227W, D227H I228Y, I228S L229Y L231M V297R, V297W A328G

[0075] 2-2. Evaluation of combination mutants 1 μL of Substrate 1 was dispensed into a reaction vessel, and 99 μL of the measurement sample (enzyme extract prepared in 1.(3) above) was added and reacted (40°C, 24 hours). After the reaction, 10 μL of 6N HCl and 150 μL of hexane were added. After thorough stirring and centrifugation, 100 μL of the organic layer was collected. The extract was dried and redissolved in an analytical solvent, and then analyzed by supercritical fluid chromatography (SFC). The analytical conditions were the same as those for the evaluation of the CASTing Library.

[0076] The change in selectivity for (1R,3S)-ethyl chrysanthemic acid was evaluated based on the reactivity / selectivity of the mutant enzyme (A328G mutant) that had the mutation (A328G) that improved the selectivity for (1R,3S)-ethyl chrysanthemic acid. Specifically, the value of "the ratio (%) of the amount of (1R,3S)-chrysanthemic acid produced in the reaction with mixed substrates to the amount of A328G mutant produced in the reaction with mixed substrates" / "the ratio (%) of the amount of (1R,3R)-chrysanthemic acid produced in the reaction with mixed substrates to the amount of A328G mutant" was calculated from the SFC area, and if the value was 1 or more, it was determined that the mutation was effective in improving the selectivity for (1R,3S)-ethyl chrysanthemic acid.

[0077] The evaluation results are shown in Figures 6 to 8. The following combinations of mutations were identified as effective in improving the selectivity for (1R,3S)-ethyl chrysanthemate. Surprisingly, A328G / F217E, A328G / F217V, A328G / F217Y, A328G / S220Q, A328G / S220L, A328G / S220N, and A328G / S220G did not improve reactivity by themselves, but when combined with the A328G mutation, the selectivity for (1R,3S)-ethyl chrysanthemate was improved. A328G / F58L, A328G / F58E A328G / L151Q, A328G / L151R, A328G / L151H, A328G / L151W, A328G / L151A, A328G / L151V, A328G / L151M, A328G / L151N, A328G / L151C, A328G / L151T, A328G / L151I A328G / F217W, A328G / F217G, A328G / F217A, A328G / F217D, A328G / F217T, A328G / F217R, A328G / F217E, A328G / F217V, A328G / F217Y A328G / S220Q, A328G / S220L, A328G / S220N, A328G / S220G A328G / A221F, A328G / A221M, A328G / A221L, A328G / A221V, A328G / A221E, A328G / A221R, A328G / A221G, A328G / A221S A328G / D227E, A328G / D227C A328G / I228L, A328G / I228G A328G / L229R, A328G / L229V, A328G / L229I, A328G / L229H A328G / V297C, A328G / V297L

[0078] The following combinations of mutations show values ​​of 1.5 or more and are therefore preferred for improving selectivity for (1R,3S)-ethyl chrysanthemate. A328G / F58L A328G / L151Q, A328G / L151R A328G / F217W, A328G / F217G, A328G / F217A, A328G / F217D, A328G / F217T, A328G / F217R, A328G / F217E A328G / S220Q, A328G / S220L A328G / A221F, A328G / A221M, A328G / A221L, A328G / A221V, A328G / A221E, A328G / A221R, A328G / A221G A328G / I228L, A328G / I228G A328G / L229R, A328G / L229V, A328G / L229I

[0079] The following combinations of mutations have values ​​of 2.0 or more, and are therefore more preferable. Of particular note is the mutation A328G / A221F, which shows specific reactivity to (1R,3S)-ethyl chrysanthemate. A328G / L151Q A328G / F217W, A328G / F217G, A328G / F217A, A328G / F217D, A328G / F217T A328G / A221F, A328G / A221M, A328G / A221L, A328G / A221V, A328G / A221E, A328G / A221R A328G / I228L, A328G / I228G

[0080] 2-3. Evaluation of multiple mutants 2 μL of substrate 1 was dispensed into a reaction vessel, and 98 μL of the measurement sample (enzyme extract prepared in 1.(3) above) was added and reacted (40°C, 24 hours). After the reaction, 10 μL of 6N HCl and 150 μL of hexane were added. After thorough mixing and centrifugation, 100 μL of the organic layer was collected. The extract was dried and redissolved in an analytical solvent, and then analyzed by supercritical fluid chromatography (SFC). The analytical conditions were the same as those for the evaluation of CASTingLibrary.

[0081] The change in selectivity for (1R,3S)-ethyl chrysanthemate was evaluated based on the reactivity / selectivity of the mutant enzyme (A328G mutant) having the mutation (A328G) that improved the selectivity for (1R,3S)-ethyl chrysanthemate. Specifically, the value of "the ratio (%) of the amount of (1R,3S)-chrysanthemate produced in the reaction with mixed substrates to the A328G mutant" / "the ratio (%) of the amount of (1R,3R)-chrysanthemate produced in the reaction with mixed substrates to the A328G mutant" was calculated from the SFC area. If the value was 1 or more, the mutation was effective in improving the selectivity for (1R,3S)-ethyl chrysanthemate, and the higher the ratio of the amount of (1R,3S)-ethyl chrysanthemate produced in the A328G mutant, the more useful the mutation was for improving the productivity of (1R,3S)-ethyl chrysanthemate.

[0082] The evaluation results are shown in Figure 9. The following mutation combinations were identified as having very high selectivity and reactivity to (1R,3S)-ethyl chrysanthemate. A328G / A221F / D326V A328G / A221F / D326V / F298L A328G / A221F / D326V / F298L / N222D

[0083] 3. Summary We have succeeded in identifying several mutations that are effective in improving reactivity / selectivity to (1R,3S)-ethyl chrysanthemate. The A328G / A221F mutant exhibits specific reactivity to (1R,3S)-ethyl chrysanthemate and is highly distinctive. The A328G / A221F / D326V / F298L / N222D mutant is extremely useful as it has significantly improved reactivity as well as selectivity to (1R,3S)-ethyl chrysanthemate. The amino acid sequences of mutants containing particularly effective mutations, including these mutants, and the genes encoding them (examples) are shown below. A328G mutant: SEQ ID NO: 2 (amino acid sequence), SEQ ID NO: 13 (gene sequence) A328G / F217W mutant: SEQ ID NO:3 (amino acid sequence), SEQ ID NO:14 (gene sequence) A328G / A221F mutant: SEQ ID NO: 4 (amino acid sequence), SEQ ID NO: 15 (gene sequence) A328G / A221M mutant: SEQ ID NO:5 (amino acid sequence), SEQ ID NO:16 (gene sequence) A328G / A221L mutant: SEQ ID NO:6 (amino acid sequence), SEQ ID NO:17 (gene sequence) A328G / A221V mutant: SEQ ID NO: 7 (amino acid sequence), SEQ ID NO: 18 (gene sequence) A328G / I228L mutant: SEQ ID NO: 8 (amino acid sequence), SEQ ID NO: 19 (gene sequence) A328G / A221F / D326V mutant: SEQ ID NO: 9 (amino acid sequence), SEQ ID NO: 20 (gene sequence) A328G / A221F / D326V / F298L mutant: SEQ ID NO: 10 (amino acid sequence), SEQ ID NO: 21 (gene sequence) A328G / A221F / D326V / F298L / N222D mutant: SEQ ID NO: 11 (amino acid sequence), SEQ ID NO: 22 (gene sequence) [Industrial Applicability]

[0084] The modified esterase of the present invention has high reactivity with (1R,3S)-ethyl chrysanthemic acid and is useful for producing (1R,3S)-chrysanthemic acid. By using the modified esterase of the present invention, it is possible to efficiently produce (1R,3S)-chrysanthemic acid by an enzymatic method, and the problems associated with the conventional production method (chemical hydrolysis) can be solved.

[0085] The present invention is not limited to the above-mentioned embodiment and examples. Various modifications within the scope of the claims and within the scope that can be easily conceived by a person skilled in the art are also included in the present invention. The contents of papers, published patent publications, patent publications, etc. specified in this specification are all incorporated by reference. [Sequence List Free Text]

[0086] SEQ ID NO: 2: Description of the artificial sequence: A328G mutant SEQ ID NO: 3: Description of the artificial sequence: A328G / F217W mutant SEQ ID NO: 4: Description of the artificial sequence: A328G / A221F mutant SEQ ID NO: 5: Description of the artificial sequence: A328G / A221M mutant SEQ ID NO: 6: Description of the artificial sequence: A328G / A221L mutant SEQ ID NO: 7: Description of the artificial sequence: A328G / A221V mutant SEQ ID NO: 8: Description of the artificial sequence: A328G / I228L mutant SEQ ID NO: 9: Description of the artificial sequence: A328G / A221F / D326V mutant SEQ ID NO: 10: Description of the artificial sequence: A328G / A221F / D326V / F298L mutant SEQ ID NO: 11: Description of the artificial sequence: A328G / A221F / D326V / F298L / N222D mutant SEQ ID NO: 13: Description of the artificial sequence: A328G mutant SEQ ID NO: 14: Description of the artificial sequence: A328G / F217W mutant SEQ ID NO: 15: Description of the artificial sequence: A328G / A221F mutant SEQ ID NO: 16: Description of the artificial sequence: A328G / A221M mutant SEQ ID NO: 17: Description of the artificial sequence: A328G / A221L mutant SEQ ID NO: 18: Description of the artificial sequence: A328G / A221V mutant SEQ ID NO: 19: Description of the artificial sequence: A328G / I228L mutant SEQ ID NO: 20: Description of the artificial sequence: A328G / A221F / D326V mutant SEQ ID NO: 21: Description of the artificial sequence: A328G / A221F / D326V / F298L mutant SEQ ID NO: 22: Description of the artificial sequence: A328G / A221F / D326V / F298L / N222D mutant

Claims

[Claim 1] A method for producing (1R,3S)-chrysanthemic acid, comprising reacting any one of the following modified esterases with (1R,3S)-ethyl chrysanthemic acid: A modified esterase having an amino acid sequence of SEQ ID NO: 1, in which the mutation site is F58 and the amino acid after the substitution is A, or an amino acid sequence that is 90% or more identical to said amino acid sequence (however, the amino acid sequence differs in parts other than the site of the amino acid substitution), and which has improved reactivity to (1R,3S)-ethyl chrysanthemate compared to an esterase consisting of the amino acid sequence of SEQ ID NO: 1.

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