Modified chrysanthemic acid esterase

Abstract

The present invention addresses the problem of providing an esterase which is useful for the production of (1R, 3S)-chrysanthemic acid. In esterase enzymes from microorganisms of the genus Arthrobacter, a plurality of amino acid substitutions are disclosed that are effective to increase reactivity, which is reactivity to ethyl (1R, 3S)-chrysanthemumate.

Description

[0001] This application is a divisional application of the invention application with application number 201980080721.9, application date November 29, 2019, and invention name “Modified chrysanthemic acid esterase”. Technical Field

[0002] The present invention relates to the modification of an enzyme (chrysanthemic acid esterase) that exhibits hydrolysis activity on chrysanthemic acid ethyl ester. In detail, it relates to a modified chrysanthemic acid esterase in which stereoselectivity has changed and its use. This application claims priority based on Japanese patent application No. 2018-229479 filed on December 6, 2018, and the patent application is cited here by reference. Background Art

[0003] Chrysanthemic acid is widely used as a synthetic raw material for synthetic pyrethroid insecticides. Chrysanthemic acid exists in four isomers ((1R,3R)-chrysanthemic acid, (1R,3S)-chrysanthemic acid, (1S,3S)-chrysanthemic acid and (1S,3R)-chrysanthemic acid), but (1R,3R)-chrysanthemic acid has the highest insecticidal activity. Chrysanthemic acid is produced industrially by chemical synthesis. On the other hand, chrysanthemic acid is also produced by enzyme reaction using chrysanthemic acid esterase. Past reports on chrysanthemic acid esterase are shown below (Patent Document 1, Non-Patent Documents 1 to 3). Patent Document 1 records that the esterase from Arthrobacter globiformis specifically decomposes 1R,3R ((+)-trans) chrysanthemic acid ethyl ester (Table 1). In addition, non-patent document 1 describes the specific decomposition of 1R,3R((+)-trans) chrysanthemic acid ethyl ester or 1S,3R((-)-trans) chrysanthemic acid ethyl ester by esterases from Arthrobacter sphaeroides or esterases from other sources. Non-patent document 2 describes the insecticidal effects of each isomer of chrysanthemic acid, with 1R,3R((+)-trans) and 1R,3S((+)-cis) showing different insecticidal effects. Non-patent document 3 is a research report on the decomposition of chrysanthemic acid ethyl ester by esterases from Arthrobacter sphaeroides, which shows a change in stereoselectivity (reactivity to (1S,3R)-chrysanthemic acid ethyl ester) through a specific mutation (K62R mutation).

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: Japanese Patent Application Laid-Open No. 5-56787

[0007] Non-patent literature

[0008] Non-patent document 1: Agric. Biol. Chem., 55(11), 2865-2870, 1991

[0009] Non-patent document 2: Chemistry and action of recent synthetic pyrethroids and their stereoisomers, Yoshioka Kosuke, Organic Synthetic Chemistry, Vol. 38, No. 12 (1980), 1151-1162

[0010] Non-patent document 3: Research on the production of optically active pyrethroid intermediates using microbial enzymes, Graduate School of Material Science, Nara Institute of Science and Technology, Masako Nishizawa, March 2011 Summary of the invention

[0011] (1R,3S)-chrysanthemic acid, one of the isomers of chrysanthemic acid, also has insecticidal activity and is useful in itself. An enzyme that efficiently produces (1R,3S)-chrysanthemic acid (i.e., an esterase with high selectivity for (1R,3S)-chrysanthemic acid ethyl ester) is unknown, and (1R,3S)-chrysanthemic acid is currently obtained by chemical hydrolysis of a mixture of chrysanthemic acid ethyl ester isomers and subsequent optical resolution (purification using a chiral column). From the perspective of yield, cost, or drainage restrictions, it is desirable to produce (1R,3S)-chrysanthemic acid using an enzyme, but no enzyme that selectively or specifically acts on (1R,3S)-chrysanthemic acid ethyl ester has been reported. Therefore, the subject of the present invention is to provide an esterase useful for the production of (1R,3S)-chrysanthemic acid.

[0012] In order to solve the above-mentioned problems, the inventors of the present invention have attempted to modify the stereoselectivity of the esterase from the microorganism of the genus Arthrobacter (Arthrobacter sphaeroides) by protein engineering methods. The esterase is specific for (1R, 3R)-chrysanthemic acid ethyl ester and cannot be used for the production of (1R, 3S)-chrysanthemic acid. After repeated experiments, multiple mutation points (amino acid residues) that are effective in improving the reactivity or selectivity for (1R, 3S)-chrysanthemic acid ethyl ester were successfully determined, and a highly useful variant (modified esterase) was obtained. In particular, the variant (A328G / A221F) in which the amino acids at two positions are substituted is an enzyme that specifically acts on (1R, 3S)-chrysanthemic acid ethyl ester, and its usefulness and utilization value are extremely high. It should be noted that the mutation A328G also contained in the variant can also be said to be an important mutation that determines the stereoselectivity of the substrate.

[0013] The following inventions are based on the above findings and observations.

[0014] [1] A modified esterase having an amino acid sequence in which any one of the following (1) to (23) is substituted in the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence showing 90% or more homology with the amino acid sequence (wherein a difference in the amino acid sequence occurs in a portion other than the position of the amino acid substitution), and having improved reactivity to (1R,3S)-chrysanthemic acid ethyl ester compared to an esterase consisting of the amino acid sequence of SEQ ID NO: 1,

[0015] (1) The mutation point is F58, and the substituted amino acid is E, I, C, A, M or Y,

[0016] (2) The mutation point is D227, and the substituted amino acid is I, S, A, W, H, G, F or V,

[0017] (3) The mutation point is I228, and the substituted amino acid is Y, S, T or W,

[0018] (4) The mutation point is L229, and the substituted amino acid is Y, F or V,

[0019] (5) The mutation point is L231, and the substituted amino acid is M, A or G.

[0020] (6) The mutation point is S244, and the substituted amino acid is F.

[0021] (7) The mutation point is A245, and the substituted amino acid is N or G.

[0022] (8) The mutation point is V297, and the substituted amino acid is R, W, Q, A or I,

[0023] (9) The mutation point is F298, and the substituted amino acid is S.

[0024] (10) The mutation point is A328, and the substituted amino acid is N.

[0025] (11) The mutation point is A328, and the substituted amino acid is G.

[0026] (12) The mutation points are A328 and F58, the amino acid after substitution of mutation point A328 is G, and the amino acid after substitution of mutation point F58 is L or E,

[0027] (13) The mutation points are A328 and L151, the amino acid after the mutation point A328 is G, and the amino acid after the mutation point L151 is Q, R, H, W, A, V, M, N, C, T or I,

[0028] (14) The mutation points are A328 and F217, the amino acid after substitution of the mutation point A328 is G, and the amino acid after substitution of the mutation point F217 is W, G, A, D, T, R, E, V or Y,

[0029] (15) The mutation points are A328 and S220, the amino acid after substitution of the mutation point A328 is G, and the amino acid after substitution of the mutation point S220 is Q, L, N or G,

[0030] (16) The mutation points are A328 and A221, the amino acid after substitution of mutation point A328 is G, and the amino acid after substitution of mutation point A221 is F, M, L, V, E, R, G or S,

[0031] (17) The mutation points are A328 and D227, the amino acid after substitution of mutation point A328 is G, and the amino acid after substitution of mutation point D227 is E or C,

[0032] (18) The mutation points are A328 and I228, the amino acid after substitution of the mutation point A328 is G, and the amino acid after substitution of the mutation point I228 is L or G,

[0033] (19) The mutation points are A328 and L229, the amino acid after substitution of the mutation point A328 is G, and the amino acid after substitution of the mutation point L229 is R, V, I or H,

[0034] (20) The mutation points are A328 and V297, the amino acid after substitution of the mutation point A328 is G, and the amino acid after substitution of the mutation point V297 is C or L,

[0035] (21) The mutation points are A328, A221 and D326. The amino acid after substitution at mutation point A328 is G, the amino acid after substitution at mutation point A221 is F, and the amino acid after substitution at mutation point D326 is V.

[0036] (22) The mutation points are A328, A221, D326 and F298. The amino acid after substitution at mutation point A328 is G, the amino acid after substitution at mutation point A221 is F, the amino acid after substitution at mutation point D326 is V, and the amino acid after substitution at mutation point F298 is L.

[0037] (23) The mutation points are A328, A221, D326, F298 and N222. The amino acid after substitution of mutation point A328 is G, the amino acid after substitution of mutation point A221 is F, the amino acid after substitution of mutation point D326 is V, the amino acid after substitution of mutation point F298 is L, and the amino acid after substitution of mutation point N222 is D.

[0038] [2] The modified esterase according to [1], wherein the substituted amino acid in (1) is E, I, C, A or M,

[0039] (2) The substituted amino acid is I, S, A, W, H or G,

[0040] (3) wherein the substituted amino acid is Y or S,

[0041] (4) wherein the substituted amino acid is Y or F,

[0042] (5) wherein the substituted amino acid is M or A,

[0043] (8) wherein the substituted amino acid is R, W or Q,

[0044] The amino acid after substitution at mutation point F58 of (12) is L,

[0045] The amino acid after substitution at the mutation point L151 of (13) is Q or R,

[0046] The amino acid after substitution at the mutation point F217 of (14) is W, G, A, D, T, R or E,

[0047] The amino acid after substitution at mutation point S220 of (15) is Q or L,

[0048] The amino acid after substitution at the mutation point A221 of (16) is F, M, L, V, E, R or G,

[0049] The amino acid after substitution at the mutation point L229 of (19) is R, V or I.

[0050] [3] The modified esterase according to [1], wherein the substituted amino acid of (4) is Y,

[0051] The amino acid after substitution in (5) is M,

[0052] (8) wherein the substituted amino acid is R or W,

[0053] The amino acid after substitution at the mutation point L151 of (13) is Q,

[0054] The amino acid after substitution at the mutation point F217 of (14) is W, G, A, D or T,

[0055] The amino acid after substitution at the mutation point A221 of (16) is F, M, L, V, E or R.

[0056] [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)-chrysanthemic acid ethyl ester is improved.

[0057] [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.

[0058] [6] The modified esterase according to [4], wherein the amino acid substitution is A328G / A221F, and the modified esterase specifically acts on (1R,3S)-ethyl chrysanthemate.

[0059] [7] The modified esterase according to [4], wherein the amino acid substitution is A328G / A221F / D326V.

[0060] [8] The modified esterase according to [4], wherein the amino acid substitution is A328G / A221F / D326V / F298L.

[0061] [9] The modified esterase according to [4], wherein the amino acid substitution is A328G / A221F / D326V / F298L / N222D.

[0062]

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

[0063]

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

[0064]

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

[11] .

[0065]

[13] The gene according to

[12] , comprising any one of the base sequences of sequence numbers 13 to 22.

[0066]

[14] A recombinant DNA comprising the gene described in

[12] or

[13] .

[0067]

[15] A microorganism that retains the recombinant DNA described in

[14] .

[0068]

[16] An enzyme agent comprising the modified esterase according to any one of [1] to

[11] .

[0069]

[17] A method for preparing a modified esterase, comprising the following steps (I) to (III):

[0070] (I) preparing a nucleic acid encoding the amino acid sequence of the modified esterase according to any one of [1] to

[11] ,

[0071] (II) a step of expressing the nucleic acid, and

[0072] (III) A step of recovering the expression product.

[0073]

[18] The preparation method according to

[17] , wherein the amino acid sequence is any one of the amino acid sequences Nos. 2 to 11.

[0074]

[19] The preparation method according to

[17] , wherein the nucleic acid comprises any base sequence of sequence numbers 13 to 22.

[0075]

[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 ethyl (1R, 3S)-chrysanthemic acid.

[0076]

[21] The production method according to

[20] , wherein (1R,3S)-chrysanthemic acid ethyl ester is in a state of coexistence with (1R,3R)-chrysanthemic acid ethyl ester, (1S,3S)-chrysanthemic acid ethyl ester and (1S,3R)-chrysanthemic acid ethyl ester. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 This is the evaluation result of the variant F58X. The "reactivity to ethyl (1R,3S)-chrysanthemate" was evaluated by dividing the "ratio (%) of the amount of (1R,3S)-chrysanthemate produced in the reaction using ethyl (1R,3S)-chrysanthemate (single substrate) to the wild-type enzyme" (B) by the "ratio (%) of the amount of (1R,3R)-chrysanthemate produced in the reaction using the mixed substrate to the wild-type enzyme" (A) (B / A) (the same applies to the following mutations).

[0078] Figure 2 These are the evaluation results for the variants F217X, S220X, and D227X.

[0079] Figure 3 These are the evaluation results for mutations I228X, L229X, and L231X.

[0080] Figure 4 These are the evaluation results for the variants S244X, A245X, and V297X.

[0081] Figure 5 These are the evaluation results for variants F298X and A328X.

[0082] Figure 6 The results of the evaluation of the combined variants A328G / F58X, A328G / L151X, and A328G / F217X are shown. The "selectivity for ethyl (1R, 3S)-chrysanthemic acid ester" was evaluated by dividing the "ratio (%) of the amount of (1R, 3S)-chrysanthemic acid produced in the reaction using a mixed substrate to the A328G variant" (C) by the "ratio (%) of the amount of (1R, 3R)-chrysanthemic acid produced in the reaction using a mixed substrate to the A328G variant" (D) (C / D). The same applies to the following combined variants. The value of the amount of (1R, 3S)-chrysanthemic acid produced / the amount of (1R, 3R)-chrysanthemic acid produced is also shown.

[0083] Figure 7These are the evaluation results for the combined variants A328G / S220X, A328G / A221X, and A328G / D227X.

[0084] Figure 8 These are the evaluation results for the combined variants A328G / I228X, A328G / L229X, and A328G / V297X.

[0085] Fig. 9 These are the evaluation results for the combined variants A328G / A221F, A328G / A221F / D326V, A328G / A221F / D326V / F298L, and A328G / A221F / D326V / F298L / N222D. DETAILED DESCRIPTION

[0086] For the convenience of explanation, some of the terms used in connection with the present invention are defined below.

[0087] (the term)

[0088] 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 an Arthrobacter microorganism (Arthrobacter sphaeroides) having the amino acid sequence of SEQ ID NO:1.

[0089] In the present invention, "amino acid substitution" is performed as modification or variation. Therefore, when the modified esterase and the reference esterase are compared, some amino acid residues are confirmed to be different. It should be noted that in this specification, the modified esterase is also referred to as a modified enzyme or a variant.

[0090] In this specification, according to convention, each amino acid is represented by a single letter as shown below.

[0091] 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

[0092] In the present specification, the amino acid residue at the N-terminus is regarded as the first position, and the position of the mutation point is identified by numbering from the N-terminus to the C-terminus.

[0093] In this specification, a variation based on 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, if the variation is that the alanine at position 328 is substituted with glycine, it is expressed as "A328G". In addition, in the case of representing a combination of two variations (used in combination), the symbol " / " is used. For example, if the variation is that the alanine at position 328 is substituted with glycine and the alanine at position 221 is substituted with phenylalanine, it is expressed as "A328G / A221F".

[0094] 1. Modified esterase

[0095] The 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 sequence number 1. According to this feature, the reactivity or selectivity or both of (1R, 3S)-chrysanthemic acid ethyl ester are improved compared to the esterase composed of the amino acid sequence of sequence number 1. It should be noted that the amino acid sequence of sequence number 1 is the sequence of an esterase from Arthrobacter sphaeroides.

[0096] "Reactivity to (1R,3S)-chrysanthemic acid ethyl ester" is an expression of reactivity (relative reactivity) including the reactivity to the optical isomers of (1R,3S)-chrysanthemic acid ethyl ester ((1R,3R)-chrysanthemic acid ethyl ester, (1S,3S)-chrysanthemic acid ethyl ester, (1S,3R)-chrysanthemic acid ethyl ester). Therefore, "improvement of reactivity to (1R,3S)-chrysanthemic acid ethyl ester" can also be achieved by improving the selectivity to (1R,3S)-chrysanthemic acid ethyl ester, increasing the activity to (1R,3S)-chrysanthemic acid ethyl ester, decreasing the activity to the optical isomers of (1R,3S)-chrysanthemic acid ethyl ester ((1R,3R)-chrysanthemic acid ethyl ester, (1S,3S)-chrysanthemic acid ethyl ester, (1S,3R)-chrysanthemic acid ethyl ester), and the like.

[0097] Here, the esterase (wild-type enzyme) from Arthrobacter sphaeroides has high selectivity for (1R,3R)-chrysanthemic acid ethyl ester, and preferentially hydrolyzes (1R,3R)-chrysanthemic acid ethyl ester when four optical isomers ((1R,3R)-chrysanthemic acid ethyl ester, (1R,3S)-chrysanthemic acid ethyl ester, (1S,3S)-chrysanthemic acid ethyl ester, and (1S,3R)-chrysanthemic acid ethyl ester) coexist. Therefore, usually, only (1R,3R)-chrysanthemic acid is detected as a product. In addition, the wild-type enzyme also has hydrolysis activity for (1R,3S)-chrysanthemic acid ethyl ester, but its reaction rate is overwhelmingly slower than the reaction rate of the hydrolysis activity for (1R,3R)-chrysanthemic acid, and therefore, it does not apparently react with (1R,3S)-chrysanthemic acid ethyl ester in the coexistence of (1R,3R)-chrysanthemic acid ethyl ester. On the other hand, when (1R, 3S)-chrysanthemic acid ethyl ester was reacted alone as a substrate, its product (1R, 3S)-chrysanthemic acid was detected.

[0098] In the present application, considering the above-mentioned characteristics of the wild-type enzyme, the "reactivity to (1R,3S)-chrysanthemic acid ethyl ester" is evaluated based on the amount of (1R,3S)-chrysanthemic acid produced in a reaction using only (1R,3S)-chrysanthemic acid ethyl ester as a substrate (single substrate) and the amount of (1R,3R)-chrysanthemic acid ethyl ester produced in a reaction using a mixture of (1R,3R)-chrysanthemic acid ethyl ester, (1R,3S)-chrysanthemic acid ethyl ester, (1S,3S)-chrysanthemic acid ethyl ester and (1S,3R)-chrysanthemic acid ethyl ester, i.e., a mixed substrate. Specifically, the "reactivity to (1R,3S)-chrysanthemic acid ethyl ester" is evaluated using the value obtained by dividing the "ratio (%) of the amount of (1R,3S)-chrysanthemic acid produced in the reaction using (1R,3S)-chrysanthemic acid ethyl ester (single substrate) to the wild-type enzyme" by the "ratio (%) of the amount of (1R,3R)-chrysanthemic acid produced in the reaction using the mixed substrate to the wild-type enzyme". When this value exceeds 1, it is judged that the "reactivity to (1R,3S)-chrysanthemic acid ethyl ester" is improved. In a preferred modified enzyme, this value is 2.0 or more, and the degree of improved reactivity is high. In a further preferred modified enzyme, this value is 3.0 or more, and the reactivity is further improved. It should be noted that the wild-type enzyme is the enzyme before modification, that is, the benchmark esterase.

[0099] As described above, the wild-type enzyme has high selectivity for (1R, 3R)-chrysanthemic acid ethyl ester, and when reacting with a mixed substrate, no (1R, 3S)-chrysanthemic acid is detected (substantially not generated). On the other hand, as shown in the examples described below, the variant (A328G variant) in which the amino acid alanine at position 328 of the wild-type enzyme is substituted with glycine has improved selectivity for (1R, 3S)-chrysanthemic acid ethyl ester, and even in the case of reacting with a mixed substrate, it shows hydrolysis activity for (1R, 3S)-chrysanthemic acid ethyl ester, and the generation of (1R, 3S)-chrysanthemic acid is confirmed. Therefore, in the present application, the evaluation of "selectivity for (1R, 3S)-chrysanthemic acid ethyl ester" uses the A328G variant. Specifically, the "selectivity for (1R,3S)-chrysanthemic acid ethyl ester" is evaluated using the value obtained by dividing the "ratio (%) of the amount of (1R,3S)-chrysanthemic acid produced in the reaction using a mixed substrate to the A328G variant" by the "ratio (%) of the amount of (1R,3R)-chrysanthemic acid produced in the reaction using a mixed substrate to the A328G variant". When this value is greater than 1, the selectivity is equal to or greater than that of the A328G variant, and it is judged that the "selectivity for (1R,3S)-chrysanthemic acid ethyl ester" is improved. In a preferred modified enzyme, this value is greater than 1.5, and the degree of improved selectivity is high. In a further preferred modified enzyme, this value is greater than 2.0, and the selectivity is further improved. It should be noted that when the amount of (1R,3R)-chrysanthemic acid produced is below the detection limit, that is, when (1R,3R)-chrysanthemic acid is substantially not produced, the "ratio of the amount of (1R,3R)-chrysanthemic acid produced in the reaction using the mixed substrate to the A328G variant" is zero, and the above value cannot be calculated. The modified enzyme that brings about such a result (A328G / A221F variant is consistent) can be said to be specific for (1R,3S)-chrysanthemic acid ethyl ester.

[0100] In this specification, "comprising an amino acid substitution" means that the variation point (i.e., the position of the amino acid residue producing a specific amino acid substitution) is the substituted amino acid. Therefore, if the amino acid sequence comprising an amino acid substitution (variant amino acid sequence) is compared with the amino acid sequence of sequence number 1 not containing an amino acid substitution (reference amino acid sequence), the difference in amino acid residues is confirmed at the position of the amino acid substitution.

[0101] The following lists the amino acid substitutions (mutation points and amino acids after substitution) contained in the modified enzyme of the present invention that improve the reactivity to (1R, 3S)-chrysanthemic acid ethyl ester. It should be noted that (11) to (23) are more preferred because they not only improve the reactivity to (1R, 3S)-chrysanthemic acid ethyl ester, but also improve the selectivity to (1R, 3S)-chrysanthemic acid ethyl ester.

[0102] (1) The mutation point is F58, and the amino acid after substitution is E, I, C, A, M or Y. Preferably, the amino acid after substitution is E, I, C, A or M.

[0103] (2) The mutation point is D227, and the amino acid after substitution is I, S, A, W, H, G, F or V. Preferably, the amino acid after substitution is I, S, A, W, H or G. More preferably, the amino acid after substitution is I, S, A, W or H.

[0104] (3) The mutation point is I228, and the amino acid after substitution is Y, S, T or W. Preferably, the amino acid after substitution is Y or S.

[0105] (4) The mutation point is L229, and the amino acid after substitution is Y, F or V. Preferably, the amino acid after substitution is Y or F. More preferably, the amino acid after substitution is Y.

[0106] (5) The mutation point 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.

[0107] (6) The mutation point is S244, and the substituted amino acid is F.

[0108] (7) The mutation point is A245, and the substituted amino acid is N or G

[0109] (8) The mutation point is V297, and the amino acid after substitution is R, W, Q, A or I. Preferably, the amino acid after substitution is R, W or Q. More preferably, the amino acid after substitution is R or W.

[0110] (9) The mutation point is F298, and the substituted amino acid is S.

[0111] (10) The mutation point is A328, and the substituted amino acid is N.

[0112] (11) The mutation point is A328, and the substituted amino acid is G.

[0113] (12) The mutation points are A328 and F58, the amino acid after substitution at mutation point A328 is G, and the amino acid after substitution at mutation point F58 is L or E. Preferably, the amino acid after substitution at mutation point F58 is L.

[0114] (13) The mutation points are A328 and L151, the amino acid after substitution at the mutation point A328 is G, and the amino acid after substitution at the mutation point L151 is Q, R, H, W, A, V, M, N, C, T, or I. Preferably, the amino acid after substitution at the mutation point L151 is Q or R. More preferably, the amino acid after substitution at the mutation point L151 is Q.

[0115] (14) The mutation points are A328 and F217, the amino acid after substitution at mutation point A328 is G, and the amino acid after substitution at mutation point F217 is W, G, A, D, T, R, E, V or Y. Preferably, the amino acid after substitution at mutation point F217 is W, G, A, D, T, R or E. More preferably, the amino acid after substitution at mutation point F217 is W, G, A, D or T.

[0116] (15) The mutation points are A328 and S220, the amino acid after substitution at the mutation point A328 is G, and the amino acid after substitution at the mutation point S220 is Q, L, N, or G. Preferably, the amino acid after substitution at the mutation point S220 is Q or L.

[0117] (16) The mutation points are A328 and A221, the amino acid after substitution at mutation point A328 is G, and the amino acid after substitution at mutation point A221 is F, M, L, V, E, R, G, or S. Preferably, the amino acid after substitution at mutation point A221 is F, M, L, V, E, R, or G. More preferably, the amino acid after substitution at mutation point A221 is F, M, L, V, E, or R.

[0118] (17) The mutation points are A328 and D227. The amino acid after substitution at mutation point A328 is G, and the amino acid after substitution at mutation point D227 is E or C.

[0119] (18) The mutation points are A328 and I228. The amino acid after substitution at mutation point A328 is G, and the amino acid after substitution at mutation point I228 is L or G.

[0120] (19) The mutation points are A328 and L229, the amino acid after substitution at the mutation point A328 is G, and the amino acid after substitution at the mutation point L229 is R, V, I or H. Preferably, the amino acid after substitution at the mutation point L229 is R, V or I.

[0121] (20) The mutation points are A328 and V297. The amino acid after substitution at mutation point A328 is G, and the amino acid after substitution at mutation point V297 is C or L.

[0122] (21) The mutation points are A328, A221 and D326. The amino acid after substitution at mutation point A328 is G, the amino acid after substitution at mutation point A221 is F, and the amino acid after substitution at mutation point D326 is V.

[0123] (22) The mutation points are A328, A221, D326 and F298. The amino acid after substitution of mutation point A328 is G, the amino acid after substitution of mutation point A221 is F, the amino acid after substitution of mutation point D326 is V, and the amino acid after substitution of mutation point F298 is L.

[0124] (23) The mutation points are A328, A221, D326, F298 and N222. The amino acid after substitution of mutation point A328 is G, the amino acid after substitution of mutation point A221 is F, the amino acid after substitution of mutation point D326 is V, the amino acid after substitution of mutation point F298 is L, and the amino acid after substitution of mutation point N222 is D.

[0125] Among the above amino acid substitutions, for (14) and (15), although mutation points F217 and S220 did not improve reactivity when alone, when combined with A328G, the selectivity for (1R, 3S)-chrysanthemic acid ethyl ester was improved, which is noteworthy. In addition, for mutation points A328 and A221, the amino acid after substitution of mutation point A328 is G, and the amino acid after substitution of mutation point A221 is F, the selectivity for (1R, 3S)-chrysanthemic acid ethyl ester is dramatically improved, and a modified enzyme that specifically acts on (1R, 3S)-chrysanthemic acid ethyl ester can be prepared, which is an extremely preferred amino acid substitution.

[0126] Specific examples of the modified enzyme of the present invention include esterases composed of any one of the amino acid sequences of SEQ ID NOs: 2 to 11 (corresponding in sequence to a variant comprising the amino acid substitution A328G of (11), a variant comprising one of the amino acid substitutions A328G / F217W of (14), a variant comprising one of the amino acid substitutions A328G / A221F of (16), a variant comprising one of the amino acid substitutions A328G / A221M of (16), a variant comprising one of the amino acid substitutions A328G / A221L of (16), A variant of A328G / A221V containing one of the amino acid substitutions of (16), a variant of A328G / I228L containing one of the amino acid substitutions of (18), a variant of A328G / A221F / D326V containing one of the amino acid substitutions of (21), a variant of A328G / A221F / D326V / F298L containing one of the amino acid substitutions of (22), and a variant of A328G / A221F / D326V / F298L / N222D containing one of the amino acid substitutions of (23). As shown in the examples described below, these variants have particularly improved selectivity for (1R, 3S)-chrysanthemic acid ethyl ester. Among them, the A328G / A221F variant acts specifically on (1R, 3S)-chrysanthemic acid ethyl ester and is particularly useful. In addition, the A328G / A221F / D326V variant, the A328G / A221F / D326V / F298L variant, and the A328G / A221F / D326V / F298L / N222D variant have very high selectivity for (1R,3S)-chrysanthemic acid ethyl ester and very high reactivity, and are extremely useful.

[0127] In general, when a part of the amino acid sequence of a certain protein is mutated, the mutated protein sometimes has the same function as the protein before the mutation. That is, sometimes the mutation of the amino acid sequence does not substantially affect the function of the protein, and the function of the protein is maintained before and after the mutation. On the other hand, when the homology of the amino acid sequences of two proteins is high, the possibility that the two show the same characteristics is high. Taking these technical common sense into consideration, although it is not completely identical (i.e., 100% homology) with the amino acid sequence of the above-mentioned modified enzyme, that is, "the amino acid sequence containing any of the amino acid substitutions in the amino acid sequence of sequence number 1 (a specific example of the amino acid sequence is the amino acid sequence of sequence numbers 2 to 11)", as long as it is a modified enzyme having an amino acid sequence showing high homology and confirming the desired characteristic change, it can be regarded as an enzyme substantially the same as the above-mentioned modified enzyme (substantially the same esterase). The homology 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 homology, the more preferred it is. Therefore, in the most preferred embodiment, the homology is 99% or more.

[0128] If the above-mentioned modified enzyme is compared with a substantially identical esterase, slight differences in the amino acid sequence are confirmed. However, the difference in the amino acid sequence occurs at positions other than the positions where the above-mentioned amino acid substitution is performed. Therefore, for example, when the basis of homology is the amino acid sequence of sequence number 2, the difference in amino acid sequence occurs at positions other than G at position 328, when the basis of homology is the amino acid sequence of sequence number 3, the difference in amino acid sequence occurs at positions other than G at position 328 and W at position 217, when the basis of homology is the amino acid sequence of sequence number 4, the difference in amino acid sequence occurs at positions other than G at position 328 and F at position 221, when the basis of homology is the amino acid sequence of sequence number 5, the difference in amino acid sequence occurs at positions other than G at position 328 and M at position 221, when the basis of homology is the amino acid sequence of sequence number 6, the difference in amino acid sequence occurs at positions other than G at position 328 and L at position 221, when the basis of homology is the amino acid sequence of sequence number 7, The amino acid sequence differs at positions other than position 328 G and position 221 V. When the basis of homology is the amino acid sequence of SEQ ID NO: 8, the amino acid sequence differs at positions other than position 328 G and position 228 L. When the basis of homology is the amino acid sequence of SEQ ID NO: 9, the amino acid sequence differs at positions other than position 328 G, position 221 F, and position 326 V. When the basis of homology is the amino acid sequence of SEQ ID NO: 10, the amino acid sequence differs at positions other than position 328 G, position 221 F, position 326 V, and position 298 L. When the basis of homology is the amino acid sequence of SEQ ID NO: 11, the amino acid sequence differs at positions other than position 328 G, position 221 F, position 326 V, position 298 L, and position 222 D. In other words, in the amino acid sequence that shows the above-mentioned homology (more than 60%, more than 70%, more than 80%, more than 82%, more than 85%, more than 90%, more than 93%, more than 95%, more than 98% or more than 99%) with the amino acid sequence of sequence number 2, the amino acid at position 328 is G.Similarly, in the amino acid sequence showing the above homology 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 homology 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 homology 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 homology 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 homology 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, and in the amino acid sequence showing the above homology with the amino acid sequence of SEQ ID NO: In the amino acid sequence showing the above homology with the amino acid sequence of sequence number 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 homology with the amino acid sequence of sequence number 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 homology with the amino acid sequence of sequence number 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. In the amino acid sequence showing the above homology with the amino acid sequence of sequence number 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.

[0129] The "slight differences in amino acid sequences" herein are produced by the deletion, substitution, addition, insertion or a combination thereof of amino acids. Typically, it refers to the mutation (change) in the amino acid sequence by the deletion, substitution, or addition, insertion or a combination thereof of 1 to more (the upper limit is, for example, 3, 5, 7, 10) amino acids constituting the amino acid sequence. "Slight differences in amino acid sequences" are preferably produced by conservative amino acid substitutions. "Conservative amino acid substitutions" herein refer to the substitution of a certain amino acid residue with an amino acid residue having a side chain of the same nature. Amino acid residues are classified into several groups according to their side chains, such as basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar 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 within the same group. It should be noted that the active residues of the esterase from Arthrobacter sphaeroides (SEQ ID NO. 1) are known to be amino acid serine at position 59, amino acid histidine at position 325, and amino acid aspartic acid at position 326, and therefore, when implementing the mutation, it is sufficient that there is no effect on the acid residues.

[0130] However, the homology (%) of two amino acid sequences or two base sequences (hereinafter, "two sequences" is used as a term including them) can be determined, for example, by the following steps. First, the two sequences are arranged in a manner that allows for optimal comparison (for example, gaps can be introduced into the first sequence to optimize the alignment with the second sequence). When the molecule (amino acid residue or nucleotide) at a specific position in the first sequence is the same as the molecule at the corresponding position in the second sequence, it can be said that the molecules at that position are the same. The homology of two sequences is a function of the number of identical positions in common in the two sequences (i.e., homology (%) = number of identical positions / total number of positions × 100), preferably also taking into account the number and size of gaps required for optimization of the alignment.

[0131] Comparison of two sequences and determination of homology can be accomplished using mathematical algorithms. Specific examples of mathematical algorithms that can be used for sequence comparison include, but are not limited to, 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 algorithms are incorporated into the NBLAST program and the XBLAST program (version 2.0) described in Altschul et al. (1990) J. Mol. Biol. 215: 403-10. To obtain equivalent nucleotide sequences, for example, a BLAST nucleotide search can be performed using the NBLAST program with score = 100 and wordlength = 12. To obtain equivalent amino acid sequences, a BLAST polypeptide search can be performed using 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, please refer to http: / / www.ncbi.nlm.nih.gov. As an example of another mathematical algorithm that can be used for sequence comparison, there is the algorithm described in Myers and Miller (1988) Comput Appl Biosci. 4:11-17. Such an algorithm is integrated into the ALIGN program that can be used, for example, in the GENESTREAM web server (IGH Montpellier, France) or the ISREC server. When the ALIGN program is used for comparison of amino acid sequences, for example, the PAM120 residue mass table is used, with gap length penalty = 12 and gap penalty = 4.

[0132] The homology of two amino acid sequences can be determined using the GAP program of the GCG software package, using Blossom62Matrix or PAM250 Matrix, with gap weight = 12, 10, 8, 6 or 4, and gap length weight = 2, 3 or 4. Alternatively, the homology of two base sequences can be determined using the GAP program of the GCG software package (available at http: / / www.gcg.com), with gap weight = 50, and gap length weight = 3.

[0133] Typically, the modified enzyme of the present invention is obtained by mutating an esterase consisting of the amino acid sequence of sequence number 1, i.e., an esterase derived from Arthrobacter sphaeroides (substitution of any of the amino acids in (1) to (23) above). The above-mentioned substantially identical esterase can be obtained by further adding a mutation to an esterase consisting of the amino acid sequence of sequence number 1 (substitution of any of the amino acids in (1) to (23) above), adding an equivalent mutation to an esterase consisting of an amino acid sequence having a high homology to the amino acid sequence of sequence number 1, etc., which is derived from an Arthrobacter sphaeroides strain of the same species and genus as the esterase that produces the esterase consisting of the amino acid sequence of sequence number 1, or further adding a mutation to the esterase obtained by the mutation. The "equivalent mutation" herein means that in an amino acid sequence having a high homology to the amino acid sequence of sequence number 1, an amino acid residue corresponding to the amino acid residue at the mutation point in the present invention (the mutation point of any of the amino acid substitutions in (1) to (23) above) is substituted. It should be noted that an example of an esterase composed of an amino acid sequence highly homologous 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 (homology in amino acid sequence: 81%).

[0134] The term "equivalent" when used in this specification with respect to amino acid residues refers to the fact that the proteins (enzymes) being compared make equal contributions to the performance of their functions. For example, when the amino acid sequence of the comparison object is arranged relative to the amino acid sequence of the benchmark esterase (amino acid sequence of sequence number 1) in a manner that takes into account the partial identity of the primary structure (amino acid sequence) and enables optimal comparison (at this time, gaps can also be introduced as needed to optimize the comparison), the amino acid at the position corresponding to the specific amino acid in the benchmark amino acid sequence can be determined as "equivalent amino acid". It is also possible to replace the comparison of the primary structures or to determine the "equivalent amino acid" by comparing the three-dimensional structures (three-dimensional structures) on this basis. By using the three-dimensional structure information, a comparison result with high reliability is obtained. At this time, a method of comparing the atomic coordinates of the three-dimensional structures of multiple enzymes can be adopted. The three-dimensional structure information of the mutated target enzyme can be obtained from ProteinData Bank (http: / / www.pdbj.org / index_j.html), for example.

[0135] An example of a method for determining the three-dimensional structure of a protein using X-ray crystal structure analysis is shown below.

[0136] (1) Crystallizing the protein. Crystallization is essential for determining the three-dimensional structure. In addition, it is also industrially useful as a method for purifying proteins to high purity and storing them stably at high density. In this case, the protein bound to the substrate or its analogous compound as a ligand can be crystallized.

[0137] (2) Irradiate the prepared crystals with X-rays to collect diffraction data. It should be noted that protein crystals are often damaged by X-ray irradiation, and their diffraction ability is often degraded. In this case, low-temperature measurement technology has recently become popular, in which the crystals are rapidly cooled to about -173°C and diffraction data are collected in this state. It should be noted that, in order to finally collect high-resolution data for structure determination, high-brightness synchrotron radiation is used.

[0138] (3) In the analysis of crystal structure, in addition to diffraction data, phase information is also required. For the target protein, when the crystal structure of the closely related protein is unknown, it is impossible to determine the structure by molecular substitution method, and the phase problem must be solved by heavy atom isomorphous substitution method. Heavy atom isomorphous substitution method is a method of introducing metal atoms with large atomic numbers such as mercury and platinum into crystals, and using the contribution of the large X-ray scattering ability of metal atoms to X-ray diffraction data to obtain phase information. The determined phase can be improved by smoothing the electron density in the solvent region of the crystal. Since the water molecules in the solvent region fluctuate greatly and the electron density is almost not observed, by approximating the electron density in this region to 0, it is possible to approach the true electron density, thereby improving the phase. In addition, when the asymmetric unit contains multiple molecules, the phase can be further greatly improved by averaging the electron density of these molecules. The protein model is fitted in the electron density map obtained by calculation using the improved phase. This process is performed on a computer map using programs such as QUANTA of MSI (USA). Then, the structure is refined using programs such as X-PLOR of MSI to complete the structure analysis. When the crystal structure of a related protein is known for the target protein, it can be determined by molecular substitution using the atomic coordinates of the known protein. Molecular substitution and structural refinement can be performed using a program such as CNS_SOLVE ver.11.

[0139] 2. Nucleic acid encoding modified esterase, etc.

[0140] The second aspect of the present invention provides a nucleic acid related to the modified enzyme of the present invention. That is, a gene encoding a modified enzyme, a nucleic acid that can be used as a probe for identifying a nucleic acid encoding a modified enzyme, and a nucleic acid that can be used as a primer for amplifying or mutating a nucleic acid encoding a modified enzyme are provided.

[0141] Genes encoding modified enzymes are typically used for the preparation of modified enzymes. According to the genetic engineering preparation method using genes encoding modified enzymes, a modified enzyme in a more homogeneous state can be obtained. In addition, this method can also be said to be a preferred method when preparing a large number of modified enzymes. It should be noted that the use of genes encoding modified enzymes is not limited to the preparation of modified enzymes. For example, as an experimental tool for the purpose of clarifying the mechanism of action of modified enzymes, or as a tool for designing or making further modified bodies of enzymes, the nucleic acid can also be used.

[0142] In this specification, "gene encoding modified enzyme" refers to a nucleic acid that can obtain the modified enzyme when expressed, and of course includes a nucleic acid having a base sequence corresponding to the amino acid sequence of the modified enzyme, and also includes a nucleic acid formed by adding a sequence that does not encode an amino acid sequence to such a nucleic acid. In addition, the degeneracy of codons is also considered.

[0143] Examples of sequences (base sequences) of genes encoding modified enzymes are shown in SEQ ID NOs: 13 to 22. These sequences encode variants shown in the Examples described below as follows.

[0144] SEQ ID NO: 13: A328G variant

[0145] SEQ ID NO. 14: A328G / A217W variant

[0146] SEQ ID NO. 15: A328G / A221F variant

[0147] SEQ ID NO. 16: A328G / A221M variant

[0148] SEQ ID NO: 17: A328G / A221L variant

[0149] Serial No. 18: A328G / A221V variant

[0150] SEQ ID NO: 19: A328G / I228L variant

[0151] Serial No. 20: A328G / A221F / D326V variant

[0152] SEQ ID NO: 21: A328G / A221F / D326V / F298L variant

[0153] SEQ ID NO. 22: A328G / A221F / D326V / F298L / N222D variant

[0154] When the gene of the present invention is expressed in a host, a sequence encoding a signal peptide (signal sequence) can be added to the 5' end of the above sequence (any one of sequence numbers 13 to 22). The signal sequence can be selected according to the host. As long as it is a signal sequence that can express the target variant, it can be used in the present invention. Examples of available signal sequences include a sequence encoding a signal peptide of an α-factor (Protein Engineering, 1996, vol9, p.1055-1061), a sequence encoding a signal peptide of an α-factor receptor, a sequence encoding a signal peptide of a SUC2 protein, a sequence encoding a signal peptide of a PHO5 protein, a sequence encoding a signal peptide of a BGL2 protein, a sequence encoding a signal peptide of an AGA2 protein, a sequence encoding a signal peptide of a TorA (trimethylamine N-oxidoreductase), and a sequence encoding a signal peptide of a toxin. The sequence of the signal peptide from PhoD (phosphatase) of Bacillus subtilis, the sequence encoding the signal peptide of LipA (lipase) from Bacillus subtilis, the sequence encoding the signal peptide of Aspergillus oryzae (Japanese Patent Application Laid-Open No. 2009-60804), the sequence encoding the signal peptide of α-amylase from Bacillus amyloliquefaciens (Eur. J. Biochem. 155, 577-581 (1986)), the sequence encoding the signal peptide of neutral protease from Bacillus subtilis (APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1995, p. 1610-1613 Vol. 61, No. 4), the sequence encoding the signal peptide of cellulase from Bacillus bacteria (Japanese Patent Application Laid-Open No. 2007-130012), and the sequence encoding pelB for pET system.

[0155] The nucleic acid of the present invention can be prepared in an isolated state by using standard genetic engineering methods, molecular biology methods, biochemical methods, chemical synthesis, etc., with reference to the sequence information disclosed in this specification or the attached sequence listing.

[0156] In another embodiment of the present invention, a nucleic acid is provided which has the same function as the protein encoded by the gene encoding the modified enzyme of the present invention but has a difference in a part of the base sequence (hereinafter also referred to as "the same nucleic acid". In addition, the base sequence of the specified same nucleic acid is referred to as "the same base sequence"). As an example of the same nucleic acid, a DNA encoding a protein having an enzyme activity (i.e., esterase activity) characteristic of the modified enzyme can be cited, which is composed of a base sequence containing substitutions, deletions, insertions, additions or inversions of one or more bases based on the base sequence of the nucleic acid encoding the modified enzyme of the present invention. Substitutions, deletions, etc. of bases can occur at multiple sites. The "multiple" here varies depending on the position and type of amino acid residues in the three-dimensional structure of the protein encoded by the nucleic acid, for example, 2 to 40 bases, preferably 2 to 20 bases, and more preferably 2 to 10 bases.

[0157] The identical nucleic acid has, for example, 60% or more, preferably 70% or more, more preferably 80% or more, further preferably 85% or more, further preferably about 90% or more, further preferably 95% or more, and most preferably 99% or more homology with respect to the base sequence serving as a reference.

[0158] The same nucleic acid as described above can be obtained by, for example, restriction endonuclease treatment, treatment with exonucleases, DNA ligases, etc., mutation introduction using position-specific mutation introduction method (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New York), random mutation introduction method (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New York), etc. In addition, the same nucleic acid can also be obtained by other methods such as ultraviolet irradiation.

[0159] Another embodiment of the present invention relates to a nucleic acid having a base sequence complementary to the base sequence of the gene encoding the modified enzyme of the present invention. Still another embodiment of the present invention provides a nucleic acid having a base sequence that is at least about 60%, 70%, 80%, 90%, 95%, 99%, or 99.9% identical to the base sequence of the gene encoding the modified enzyme of the present invention or a base sequence complementary thereto.

[0160] Another embodiment 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 a modified enzyme of the present invention or a base sequence complementary to the same base sequence. "Stringent conditions" herein refer to conditions that form so-called specific hybridization and do not form non-specific hybridization. Such stringent conditions are well known to those skilled in the art, and can be set, for example, with reference to Molecular Cloning (Third Edition, Cold Spring Harbor Laboratory Press, New York), Current protocols in molecular biology (edited by Frederick M. Ausubel et al., 1987). Examples of stringent conditions include a hybridization solution (50% formamide, 10×SSC (0.15 M NaCl, 15 mM sodium citrate, pH 7.0), 5×Denhardt's solution, 1% SDS, 10% dextran sulfate, 10 μg / ml denatured salmon sperm DNA, 50 mM phosphate buffer (pH 7.5)), incubation at about 42° C. to about 50° C., and then washing at about 65° C. to about 70° C. using 0.1×SSC, 0.1% SDS. Examples of more preferred stringent conditions include a hybridization solution (50% formamide, 5×SSC (0.15 M NaCl, 15 mM sodium citrate, pH 7.0), 1×Denhardt's solution, 1% SDS, 10% dextran sulfate, 10 μg / ml denatured salmon sperm DNA, 50 mM phosphate buffer (pH 7.5)).

[0161] Still another embodiment of the present invention provides a nucleic acid (nucleic acid fragment) having a base sequence of a gene encoding a modified enzyme of the present invention or a portion of a base sequence complementary thereto. Such a nucleic acid fragment can be used to detect, identify and / or amplify nucleic acids having a base sequence of a gene encoding a modified enzyme of the present invention, etc. The nucleic acid fragment is designed, for example, in a manner that contains at least a portion that hybridizes with a continuous nucleotide portion (e.g., about 10 to about 100 bases long, preferably about 20 to about 100 bases long, and more preferably about 30 to about 100 bases long) in the base sequence of the gene encoding the modified enzyme of the present invention. When used as a probe, the nucleic acid fragment can be labeled. Labeling can use, for example, fluorescent substances, enzymes, and radioactive isotopes.

[0162] Another aspect of the present invention relates to a recombinant DNA containing the gene of the present invention (gene encoding a modified enzyme). The recombinant DNA of the present invention is provided, for example, in the form of a vector. In this specification, the term "vector" refers to a nucleic acid molecule capable of transporting a nucleic acid inserted therein to a target such as a cell.

[0163] According to the purpose of use (cloning, protein expression), the type of host cell is also considered to select an appropriate vector. As a vector using Escherichia coli as a host, pET21 or its modified body, M13 phage or its modified body, lambda phage or its modified body, pBR322 or its modified body (pB325, pAT153, pUC8, etc.) can be exemplified, as a vector using yeast as a host, pYepSec1, pMFa, pYES2, pPIC3.5K, etc. can be exemplified, as a vector using insect cells as a host, pAc, pVL, etc. can be exemplified, as a vector using mammalian cells as a host, pCDM8, pMT2PC, etc. can be exemplified.

[0164] The vector of the present invention is preferably an expression vector. "Expression vector" refers to a vector that can introduce a nucleic acid inserted thereinto into a target cell (host cell) and can be expressed in the cell. An expression vector generally contains a promoter sequence required for the expression of the inserted nucleic acid, an enhancer sequence that promotes expression, and the like. An expression vector containing a selection marker can also be used. When using the expression vector, a selection marker can be used to confirm whether the expression vector has been introduced (and to what extent).

[0165] Insertion of the nucleic acid of the present invention into a vector, insertion of a selectable marker gene (when necessary), insertion of a promoter (when necessary), etc. can be carried out using standard recombinant DNA techniques (for example, refer to the well-known method using restriction endonucleases and DNA ligases in Molecular Cloning, Third Edition, 1.84, Cold Spring Harbor Laboratory Press, New York).

[0166] As host cell, from the aspect of handling ease, microorganisms such as Aspergillus (such as Aspergillus oryzae), Bacillus bacteria (such as Bacillus subtilis), Escherichia coli (Escherichia coli), budding yeast (Saccharomyces cerevisiae) can be used, as long as the host cell that the recombinant DNA can replicate and the gene of the modified enzyme can express can be utilized. Escherichia coli (Escherichia coli) and budding yeast (Saccharomyces cerevisiae) can be preferably used. As the example of Escherichia coli, when utilizing T7 system promoter, Escherichia coli BL21 (DE3) can be enumerated, and when it is not the case, Escherichia coli JM109 can be enumerated. In addition, as the example of budding yeast, budding yeast SHY2, budding yeast AH22 or budding yeast INVSc1 (Invitrogen company) can be enumerated.

[0167] Another aspect of the present invention relates to a microorganism (ie, a transformant) having the recombinant DNA of the present invention. The microorganism of the present invention can be obtained by transfection or transformation using the above-mentioned vector of the present invention. For example, it can be carried out by the calcium chloride method (Journal of Molecular Biology (J. Mol. Biol.), Vol. 53, p. 159 (1970)), the Hanahan method (Journal of Molecular Biology, 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 co-precipitation method, electroporation (Potter, H. et al., Proc. Natl. Acad. Sci. USA 81, 7161-7165 (1984)), lipofection (Felgner, P et al., Proc. Natl. Acad. Sci. USA 84, 7413-7417 (1984)), etc. It should be noted that the microorganism of the present invention can be used to produce the modified enzyme of the present invention.

[0168] 3. Enzyme containing modified esterase

[0169] The modified enzyme of the present invention can be provided in the form of an enzyme agent, for example. The enzyme agent can also contain an excipient, a buffer, a suspending agent, a stabilizer, a preservative, a preservative, a physiological saline, etc. in addition to the active ingredient (the modified enzyme of the present invention). As an excipient, starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, white sugar, glycerol, etc. can be used. As a buffer, phosphates, citrates, acetates, etc. can be used. As a stabilizer, propylene glycol, ascorbic acid, etc. can be used. As a preservative, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methyl parahydroxybenzoate, etc. can be used. As a preservative, ethanol, benzalkonium chloride, parahydroxybenzoic acid, chlorobutanol, etc. can be used.

[0170] 4. Preparation method of modified esterase

[0171] Another 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 successfully obtained by the inventors is prepared by a genetic engineering method. In the case of this embodiment, a nucleic acid encoding any one of the amino acid sequences in sequence numbers 2 to 11 is prepared (step (I)). Here, "nucleic acid encoding a specific amino acid sequence" is a nucleic acid that obtains a polypeptide having the amino acid sequence when it is expressed. A nucleic acid consisting of a base sequence corresponding to the amino acid sequence is of course possible, and an extra sequence may be added to such a nucleic acid (either a sequence encoding an amino acid sequence or a sequence that does not encode an amino acid sequence). In addition, the degeneracy of codons may also be considered. "Nucleic acid encoding any one of the amino acid sequences in sequence numbers 2 to 11" can be prepared into an isolated state using standard genetic engineering methods, molecular biology methods, biochemical techniques, etc. by referring to the sequence information disclosed in this specification or the attached sequence table. Here, the amino acid sequences of sequence numbers 2 to 11 are all amino acid sequences obtained by mutating the amino acid sequence of the esterase from Arthrobacter sphaeroides. Therefore, by applying the necessary mutation to the gene encoding the esterase from Arthrobacter sphaeroides, a nucleic acid (gene) encoding any of the amino acid sequences in sequence numbers 2 to 11 can also be obtained. There are many methods for position-specific base sequence substitution known in the art (for example, refer to Molecular Cloning, Third Edition, Cold Spring Harbor Laboratory Press, New York), and an appropriate method can be selected and used. As a position-specific mutation introduction method, a position-specific amino acid saturation mutation method can be used. The position-specific amino acid saturation mutation method is based on the three-dimensional structure of the protein, and the position related to the required function is inferred, and the amino acid saturation mutation is introduced. "Semi-rational, semi-random" method (J.Mol.Biol.331,585-592 (2003)). For example, a kit such as Quickchange (Stratagene) or Overlap extentionPCR (Nucleic Acid Res.16,7351-7367 (1988)) can be used to introduce position-specific amino acid saturation mutation. The DNA polymerase used in PCR can use Taq polymerase, etc. Among them, it is preferable to use a DNA polymerase having high precision, such as KOD-PLUS- (Toyobo Co., Ltd.) and Pfuturbo (Stratagene).

[0172] After step (I), the prepared nucleic acid is expressed (step (II)). For example, an expression vector having the above nucleic acid inserted therein is first prepared, and a host cell is transformed using the expression vector.

[0173] Next, the transformant is cultured under conditions that produce the expression product, i.e., the modified enzyme. The culture of the transformant can be carried out according to conventional methods. As the carbon source used in the culture medium, any assimilable carbon compound can be used, for example, glucose, sucrose, lactose, maltose, molasses, pyruvic acid, etc. can be used. In addition, as the nitrogen source, any available nitrogen compound can be used, for example, peptone, meat extract, yeast extract, casein hydrolyzate, soybean meal alkaline extract, etc. can be used. In addition, salts such as phosphates, carbonates, sulfates, magnesium, calcium, potassium, iron, manganese, zinc, specific amino acids, specific vitamins, etc. can be used as needed.

[0174] On the other hand, 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 of the culture object, the production characteristics of the modified enzyme, etc. The pH of the culture medium is prepared within the range in which the transformant grows and produces the enzyme. The pH of the culture medium is preferably about 6.0 to 9.0 (preferably around pH 7.0).

[0175] Next, the expression product (modified enzyme) is recovered (step (III)). The culture solution containing the cultured bacterial cells may be used as an enzyme solution directly or after concentration, impurity removal, etc., but the expression product is usually temporarily recovered from the culture solution or bacterial cells. If the expression product is a secretory protein, it can be recovered from the culture solution, and if it is a protein other than that, it can be recovered from the bacterial body. When recovering from the culture solution, for example, the culture supernatant is filtered, centrifuged to remove insoluble matter, and then concentrated under reduced pressure, membrane concentrated, salted out with ammonium sulfate or sodium sulfate, separate precipitation with methanol, ethanol or acetone, etc., dialysis, heat treatment, isoelectric point treatment, gel filtration, adsorption chromatography, ion exchange chromatography, affinity chromatography and other various chromatography methods (for example, gel filtration using Sephadex gel (GE Healthcare Science), DEAE agarose CL-6B (GE Healthcare Science), octyl agarose CL-6B (GE Healthcare Science), CM agarose CL-6B (GE Healthcare Science)) and the like are combined to separate and purify, thereby obtaining a purified product of the modified enzyme. On the other hand, when recovering from the bacterial body, the bacterial body is collected by filtering the culture solution, centrifuging, etc., and then the bacterial body is disrupted by a mechanical method such as pressure treatment, ultrasonic treatment, physical disruption treatment or an enzymatic method such as lysozyme, and then separated and purified in the same manner as above, thereby obtaining a purified product of the modified enzyme.

[0176] The purified enzyme obtained as above can also be provided by powdering such as freeze drying, vacuum drying or spray drying. At this time, the purified enzyme can be dissolved in phosphate buffer, triethanolamine buffer, Tris hydrochloric acid buffer, GOOD buffer in advance. Phosphate buffer and triethanolamine buffer can be preferably used. It should be noted that, as GOOD buffer, PIPES, MES or MOPS can be enumerated.

[0177] Usually, an appropriate host-vector system is used as described above for gene expression and recovery of the expression product (modified enzyme), but a cell-free synthesis system can also be used. Here, the "cell-free synthesis system (cell-free transcription system, cell-free transcription / translation system)" means that no living cells are used, and ribosomes, transcription / translation factors, etc. from living cells (or obtained by genetic engineering methods) are used to synthesize the mRNA and protein encoded by them in vitro based on the nucleic acid (DNA, mRNA) as a template. In the cell-free synthesis system, a cell extract obtained by purifying the cell disrupted liquid as needed is usually used. The cell extract usually contains ribosomes, initiation factors and other factors required for protein synthesis, and various enzymes such as tRNA. When synthesizing proteins, various amino acids, energy sources such as ATP and GTP, and other substances required for protein synthesis such as creatine phosphate are added to the cell extract. Of course, when synthesizing proteins, ribosomes, various factors and / or various enzymes prepared separately can be supplemented as needed.

[0178] The development of a transcription / translation system that reconstructs the molecules (factors) required for protein synthesis has also been reported (Shimizu, Y. et al.: Nature Biotech., 19, 751-755, 2001). In this synthesis system, genes for 31 factors, including 3 initiation factors, 3 elongation factors, 4 factors related to termination, 20 aminoacyl tRNA synthetases that bind each amino acid to tRNA, and methionyl tRNA formyltransferase, that constitute the protein synthesis system of bacteria are amplified from the Escherichia coli genome, and the protein synthesis system is reconstructed in vitro using these. The synthesis system reconstructed in this way can be used in the present invention.

[0179] The term "cell-free transcription / translation system" can be used interchangeably with a cell-free protein synthesis system, an in vitro translation system, or an in vitro transcription / translation system. In an in vitro translation system, RNA is used as a template to synthesize protein. As template RNA, total RNA, mRNA, in vitro transcription products, etc. can be used. In other in vitro transcription / translation systems, DNA is used as a template. The template DNA should contain a ribosome binding region and, in addition, preferably contains an appropriate terminator sequence. It should be noted that in an in vitro transcription / translation system, the conditions for adding the factors required for each reaction are set in a manner that the transcription reaction and the translation reaction are carried out continuously.

[0180] 5. Uses of modified esterase

[0181] Another aspect of the present invention relates to the use of a 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)-chrysanthemic acid ethyl ester to hydrolyze it to produce (1R, 3S)-chrysanthemic acid. As long as the desired hydrolysis reaction can be achieved, the reaction conditions are not particularly limited. For example, a buffer such as glycine-NaOH, PIPES, etc. can be used to react at pH 6 to 11 and a temperature of 40°C to 60°C. As a substrate, a high-purity (1R, 3S)-chrysanthemic acid ethyl ester (for example, a purity of 98% or more) can be used, or a substrate in which (1R, 3R)-chrysanthemic acid ethyl ester, (1S, 3S)-chrysanthemic acid ethyl ester and (1S, 3R)-chrysanthemic acid ethyl ester coexist (for example, a mixed chrysanthemic acid ethyl ester of (1R, 3R): (1S, 3S): (1R, 3S): (1S, 3R) = 3:3:2:2, etc.) can be used. The reaction product is optically resolved as needed and used for various purposes. Column chromatography can be used for optical resolution. An immobilized modified enzyme can be used as a modified enzyme.

[0182] Example

[0183] The wild-type esterase from Arthrobacter sphaeroides (the amino acid sequence is shown in SEQ ID NO: 1, and the gene sequence is shown in SEQ ID NO: 12) has high selectivity for (1R,3R)-ethyl chrysanthemate. We attempted to modify the stereoselectivity of this esterase by protein engineering.

[0184] 1. Preparation of variant library

[0185] The variant library (CASTing Library and 2-point combinatorial variants) was prepared by the following method.

[0186] (1) Introduction and transformation of mutations

[0187] PCR primers for introducing mutations were designed, and PCR was performed under the following conditions to introduce mutations.

[0188] <Template plasmid>

[0189] pET21b (introducing wild-type enzyme gene) about 30ng / μL

[0190] <PCR reaction composition> (total volume 20 μL)

[0191] 5×Prime STAR GXLBuffer (Takara Bio): 4μL

[0192] dNTP Mixture(2.5mM each):1.6μL

[0193] Template (about 30ng / μL): 0.25μL

[0194] F-primer: 0.2 μL

[0195] R-primer: 0.2 μL

[0196] Prime STAR GXLDNA Polymerase(Takara Bio):0.8μL

[0197] Mix the above ingredients and adjust to 20 μL with ultrapure water (Milli Q water)

[0198] <PCR conditions>

[0199] React at 98°C for 1 minute

[0200] 20 cycles of 98°C for 10 seconds, 60°C for 15 seconds, and 68°C for 2 minutes were performed.

[0201] React at 68°C for 5 minutes

[0202] Place at 4°C

[0203] 1.5 μL of DpnI was added to 15 μL of the PCR reaction solution and treated (37°C, 3 hours or more). Next, 2 μL of the DpnI treatment solution was used to perform ligation treatment (16°C, O / N) according to the following ligation reaction composition.

[0204] <Ligation reaction composition> (total volume 5.5 μL)

[0205] DpnI treated PCR product: 2 μL

[0206] T4 polynucleotide kinase: 1 μL

[0207] LigationHigh(TOYOBO):2.5μL

[0208] 5.5 μL of the ligation reaction solution was used to transform (treated at 42°C for 45 seconds and then chilled on ice) 25 μL of E. coli BL21 (DE3). About 30 μL of the transformation solution was spread on an LB agar plate supplemented with ampicillin (final concentration 0.1 mg / mL) and cultured at 37°C, O / N (pre-culture).

[0209] (2) Obtaining enzyme extract

[0210] Each mutant after pre-culture was inoculated into 1 mL of TB medium (invitrogen) supplemented with ampicillin (final concentration 0.1 mg / mL). Cultured at 33°C for 48 hours (IPTG (final concentration 0.1 mM) was added 24 hours after the start of culture), and centrifuged (3000g×10 minutes). After removing the supernatant and recovering the bacteria, the bacteria were lysed with a lysing agent (25°C, 2 hours or more) to extract the enzyme. After centrifuging the lysate (3000g×10 minutes), the supernatant was recovered as an enzyme extract.

[0211] (3) Preparation of combined variants

[0212] Using the A328G variant as a template, PCR was performed in the same manner as in (1) above. The PCR product was recovered and an enzyme extract was obtained in the same manner as in (2) above. It should be noted that the candidate mutation point combined with A328G was selected based on the evaluation results of CASTing Library and the results of docking simulation using MOE (MOLSIS).

[0213] (4) Preparation of multiple variants

[0214] Using the A328G / A221F variant as a template, random mutations were introduced by Error-prone PCR. Using trans-pNP chrysanthemic acid, screening was performed using the amount of free pNP as an indicator. The same mutation introduction and screening were repeated multiple times using the obtained variant as a template, thereby producing multiple variants with increased reactivity to (1R,3S)-chrysanthemic acid ethyl ester.

[0215] 2. Evaluation of variants

[0216] 2-1. Evaluation of CASTing Library

[0217] CASTing Library was evaluated by the following method: To evaluate the change in reactivity to (1R,3S)-chrysanthemic acid ethyl ester, the following two substrates were used.

[0218] Substrate 1 (mixed substrate): chrysanthemic acid ethyl ester (purity 95% or more, a mixture of (1R, 3R) isoform, (1R, 3S) isoform, (1S, 3R) isoform, and (1S, 3S) isoform ((1R, 3R): (1S, 3S): (1R, 3S): (1S, 3R) = 3:3:2:2), Tokyo Chemical Industry)

[0219] Substrate 2 (single substrate): (1R,3S)-chrysanthemic acid ethyl ester

[0220] 1 μL of substrate (substrate 1 or substrate 2) is dispensed into a reaction container, and 99 μL of the test sample (the enzyme extract prepared in 1. (2) above) is added to react (40°C, 24 hours). After the reaction, 10 μL of 6N HCl and 150 μL of hexane are added. After sufficient stirring, centrifugation is performed and 100 μL of the organic layer is collected. The extract is dried, redissolved in an analytical solvent, and analyzed by supercritical fluid chromatography (SFC). The analytical conditions are shown below.

[0221] <Analysis conditions of supercritical fluid chromatography (SFC)>

[0222] Apparatus: ACQUITY UPC2 PDA system (Waters)

[0223] Column: ACQUITY UPC2 Trefoil AMY1 (Waters) 2.5 μm, 3.0×150 mm

[0224] Mobile phase: CO2 / 10mM 2-propanol containing ammonium acetate (0.5% acetic acid) = 92 / 8

[0225] Flow rate: 2.0mL / min

[0226] Injection volume: 1μL

[0227] Analysis time: 1.5 minutes

[0228] PDA detector: 205-400nm

[0229] Resolution: 1.2nm

[0230] The change in reactivity to (1R,3S)-chrysanthemic acid ethyl ester 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)-chrysanthemic acid ethyl ester (single substrate) to the wild-type enzyme" / "the ratio (%) of the amount of (1R,3R)-chrysanthemic acid produced in the reaction using the mixed substrate to the wild-type enzyme" was calculated from the area of ​​the SFC region. If the value exceeded 1, it was judged that the reactivity to (1R,3S)-chrysanthemic acid ethyl ester increased.

[0231] The evaluation results are shown in Figures 1 to 5 The following mutations were identified as mutations that increase the reactivity to (1R,3S)-chrysanthemic acid ethyl ester.

[0232] F58E, F58I, F58C, F58A, F58M, F58Y

[0233] D227I, D227S, D227A, D227W, D227H, D227G, D227F, D227VI228Y, I228S, I228T, I228W

[0234] L229Y, L229F, L229V

[0235] L231M, L231A, L231G

[0236] S244F

[0237] A245N, A245G

[0238] V297R, V297W, V297Q, V297A, V297I

[0239] F298S

[0240] A328N, A328G

[0241] The following variations show values ​​of 2.0 or more and are more preferred for improving the reactivity to (1R,3S)-ethyl chrysanthemate.

[0242] F58E, F58I, F58C, F58A, F58M

[0243] D227I, D227S, D227A, D227W, D227H, D227G

[0244] I228Y、I228S

[0245] L229Y、L229F

[0246] L231M, L231A

[0247] V297R, V297W, V297Q

[0248] A328G

[0249] The following mutations have values ​​of 3.0 or more, which can be said to be more preferable. In addition, A328G was also confirmed to produce (1R, 3S)-chrysanthemic acid in the reaction using substrate 1 (mixed substrate), and is a particularly effective mutation.

[0250] F58E, F58I, F58C, F58A, F58M

[0251] D227I, D227S, D227A, D227W, D227H

[0252] I228Y、I228S

[0253] L229Y

[0254] L231M

[0255] V297R, V297W

[0256] A328G

[0257] 2-2. Evaluation of Combinatorial Variants

[0258] Pour 1 μL of substrate 1 into a reaction container, add 99 μL of the test sample (the enzyme extract prepared in 1. (3) above), and react (40°C, 24 hours). After the reaction, add 10 μL of 6N HCl and 150 μL of hexane. After sufficient stirring, centrifuge and collect 100 μL of the organic layer. Dry the extract, dissolve it in an analytical solvent, and analyze it by supercritical fluid chromatography (SFC). The analysis conditions are the same as those evaluated by CASTingLibrary.

[0259] The change in selectivity for (1R,3S)-chrysanthemic acid ethyl ester was evaluated based on the reactivity / selectivity of the mutant enzyme (A328G variant) having a mutation (A328G) that improves the selectivity for (1R,3S)-chrysanthemic acid ethyl ester. Specifically, the value of "the ratio (%) of the amount of (1R,3S)-chrysanthemic acid produced in the reaction using a mixed substrate to the A328G variant" / "the ratio (%) of the amount of (1R,3R)-chrysanthemic acid produced in the reaction using a mixed substrate to the A328G variant" was calculated from the area of ​​the SFC region. If the value was 1 or more, it was determined that the mutation was effective in improving the selectivity for (1R,3S)-chrysanthemic acid ethyl ester.

[0260] The evaluation results are shown in Figures 6 to 8 The following combinations of mutations were identified as mutations effective in improving the selectivity for (1R, 3S)-chrysanthemic acid ethyl ester. It should be noted that surprisingly, A328G / F217E, A328G / F217V, A328G / F217Y, A328G / S220Q, A328G / S220L, A328G / S220N, and A328G / S220G did not improve the reactivity when the mutation points F217 and S220 were alone, but when they were combined with the A328G mutation, the selectivity for (1R, 3S)-chrysanthemic acid ethyl ester was improved.

[0261] A328G / F58L, A328G / F58E

[0262] A328G / L151Q, A328G / L151R, A328G / L151H, A328G / L151W, A328G / L151A, A328G / L151V, A328G / L151M, A328G / L151N, A328G / L151C, A328G / L151T, A328G / L151I

[0263] A328G / F217W, A328G / F217G, A328G / F217A, A328G / F217D, A328G / F217T, A328G / F217R, A328G / F217E、A328G / F217V、A328G / F217YA328G / S220Q、A328G / S220L、A328G / S220N、A328G / S220G

[0264] A328G / A221F, A328G / A221M, A328G / A221L, A328G / A221V, A328G / A221E, A328G / A221R, A328G / A221G, A328G / A221S

[0265] A328G / D227E, A328G / D227C

[0266] A328G / I228L, A328G / I228G

[0267] A328G / L229R, A328G / L229V, A328G / L229I, A328G / L229H

[0268] A328G / V297C, A328G / V297L

[0269] The following combinations of variations show values ​​of 1.5 or more, and are more preferred for improving the selectivity for ethyl (1R, 3S)-chrysanthemate.

[0270] A328G / F58L

[0271] A328G / L151Q, A328G / L151R

[0272] A328G / F217W, A328G / F217G, A328G / F217A, A328G / F217D, A328G / F217T, A328G / F217R, A328G / F217E

[0273] A328G / S220Q, A328G / S220L

[0274] A328G / A221F, A328G / A221M, A328G / A221L, A328G / A221V, A328G / A221E, A328G / A221R, A328G / A221G

[0275] A328G / I228L, A328G / I228G

[0276] A328G / L229R, A328G / L229V, A328G / L229I

[0277] The value of the combination of the following mutations is 2.0 or more, which is more preferable. It should be noted that the mutation A328G / A221F shows specific reactivity to (1R, 3S)-chrysanthemic acid ethyl ester.

[0278] A328G / L151Q

[0279] A328G / F217W, A328G / F217G, A328G / F217A, A328G / F217D, A328G / F217T

[0280] A328G / A221F, A328G / A221M, A328G / A221L, A328G / A221V, A328G / A221E, A328G / A221R

[0281] A328G / I228L, A328G / I228G

[0282] 2-3. Evaluation of multiple variants

[0283] 2 μL of substrate 1 was injected into a reaction container, and 98 μL of the test sample (the enzyme extract prepared in 1. (3) above) was added to react (40°C, 24 hours). After the reaction, 10 μL of 6N HCl and 150 μL of hexane were added. After sufficient stirring, centrifugation was performed and 100 μL of the organic layer was collected. The extract was dried, dissolved in an analytical solvent, and analyzed by supercritical fluid chromatography (SFC). The analysis conditions were the same as those in the evaluation of CASTingLibrary.

[0284] The change in selectivity for (1R,3S)-chrysanthemic acid ethyl ester was evaluated based on the reactivity / selectivity of the mutant enzyme (A328G mutant) having a mutation (A328G) that improves the selectivity for (1R,3S)-chrysanthemic acid ethyl ester. Specifically, the value of "the ratio of the amount of (1R,3S)-chrysanthemic acid produced in the reaction using the mixed substrate to the A328G mutant (%)" / "the ratio of the amount of (1R,3R)-chrysanthemic acid produced in the reaction using the mixed substrate to the A328G mutant (%)" was calculated from the area of ​​the SFC region. If the value was 1 or more, it was determined that the mutant was effective in improving the selectivity for (1R,3S)-chrysanthemic acid ethyl ester. The higher the ratio of the amount of (1R,3R)-chrysanthemic acid ethyl ester produced to the A328G mutant, the more effective the mutant was in improving the productivity of (1R,3S)-chrysanthemic acid ethyl ester.

[0285] The evaluation results are shown in Fig. 9 The following combinations of mutations were identified as mutations that showed very high selectivity and reactivity to (1R,3S)-chrysanthemic acid ethyl ester.

[0286] A328G / A221F / D326V

[0287] A328G / A221F / D326V / F298L

[0288] A328G / A221F / D326V / F298L / N222D

[0289] 3. Summary

[0290] Multiple variants that are effective for improving reactivity / selectivity for (1R,3S)-chrysanthemic acid ethyl ester have been successfully identified. The A328G / A221F variant shows specific reactivity for (1R,3S)-chrysanthemic acid ethyl ester, which is very characteristic. The A328G / A221F / D326V / F298L / N222D variant has greatly improved reactivity in addition to greatly improved selectivity for (1R,3S)-chrysanthemic acid ethyl ester, and is extremely useful. The amino acid sequences of variants containing particularly effective variants including these variants and the genes encoding them (an example) are shown below.

[0291] A328G variant: SEQ ID NO: 2 (amino acid sequence), SEQ ID NO: 13 (gene sequence)

[0292] A328G / F217W variant: SEQ ID NO: 3 (amino acid sequence), SEQ ID NO: 14 (gene sequence)

[0293] A328G / A221F variant: SEQ ID NO: 4 (amino acid sequence), SEQ ID NO: 15 (gene sequence)

[0294] A328G / A221M variant: SEQ ID NO: 5 (amino acid sequence), SEQ ID NO: 16 (gene sequence)

[0295] A328G / A221L variant: SEQ ID NO: 6 (amino acid sequence), SEQ ID NO: 17 (gene sequence)

[0296] A328G / A221V variants: SEQ ID NO: 7 (amino acid sequence), SEQ ID NO: 18 (gene sequence)

[0297] A328G / I228L variant: SEQ ID NO: 8 (amino acid sequence), SEQ ID NO: 19 (gene sequence)

[0298] A328G / A221F / D326V variants: SEQ ID NO: 9 (amino acid sequence), SEQ ID NO: 20 (gene sequence)

[0299] A328G / A221F / D326V / F298L variants: SEQ ID NO: 10 (amino acid sequence), SEQ ID NO: 21 (gene sequence)

[0300] A328G / A221F / D326V / F298L / N222D variants: SEQ ID NO: 11 (amino acid sequence), SEQ ID NO: 22 (gene sequence)

[0301] Industrial Applicability

[0302] The modified esterase of the present invention has high reactivity to (1R, 3S)-chrysanthemic acid ethyl ester and is useful for the production of (1R, 3S)-chrysanthemic acid. By using the modified esterase of the present invention, (1R, 3S)-chrysanthemic acid can be efficiently produced by an enzymatic method, which can solve the problems of the previous production method (chemical hydrolysis).

[0303] The present invention is not limited by the embodiments and examples of the invention described above. Various modifications that do not deviate from the scope of the claims and that can be easily thought of by those skilled in the art are also included in the present invention. The contents of the papers, published patent publications, patent publications, etc. shown in this specification are cited by citing their entire contents.

[0304] Sequence Listing Free Text

[0305] Sequence number 2: Description of artificial sequence: A328G variant

[0306] Sequence number 3: Description of artificial sequence: A328G / F217W variant

[0307] Sequence number 4: Description of artificial sequence: A328G / A221F variant

[0308] Sequence number 5: Description of artificial sequence: A328G / A221M variant

[0309] SEQ ID NO. 6: Description of artificial sequence: A328G / A221L variant

[0310] Sequence number 7: Description of artificial sequence: A328G / A221V variant

[0311] SEQ ID NO. 8: Description of artificial sequence: A328G / I228L variant

[0312] Sequence number 9: Description of artificial sequence: A328G / A221F / D326V variant

[0313] SEQ ID NO. 10: Description of artificial sequence: A328G / A221F / D326V / F298L variant

[0314] SEQ ID NO. 11: Description of artificial sequence: A328G / A221F / D326V / F298L / N222D variant

[0315] SEQ ID NO. 13: Description of artificial sequence: A328G variant

[0316] SEQ ID NO. 14: Description of artificial sequence: A328G / F217W variant

[0317] SEQ ID NO. 15: Description of artificial sequence: A328G / A221F variant

[0318] SEQ ID NO. 16: Description of artificial sequence: A328G / A221M variant

[0319] SEQ ID NO. 17: Description of artificial sequence: A328G / A221L variant

[0320] SEQ ID NO. 18: Description of artificial sequence: A328G / A221V variant

[0321] SEQ ID NO. 19: Description of artificial sequence: A328G / I228L variant

[0322] SEQ ID NO. 20: Description of artificial sequence: A328G / A221F / D326V variant

[0323] SEQ ID NO. 21: Description of artificial sequence: A328G / A221F / D326V / F298L variant

[0324] SEQ ID NO. 22: Description of artificial sequence: A328G / A221F / D326V / F298L / N222D variant

Claims

1. A modified esterase comprising an amino acid sequence containing any one of the following (1) to (10) as substitution in the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence showing 90% or more homology with the amino acid sequence, wherein the reactivity to (1R, 3S)-chrysanthemic acid ethyl ester is improved compared to the esterase consisting of the amino acid sequence of SEQ ID NO: 1, wherein: The amino acid sequence showing 90% or more homology with the amino acid sequence has an amino acid sequence difference in the part other than the position where the amino acid is substituted, (1) The mutation point is F58, and the substituted amino acid is E, I, C, A, M or Y, (2) The mutation point is D227, and the substituted amino acid is I, S, A, W, H, G, F or V, (3) The mutation point is I228, and the substituted amino acid is Y, S, T or W, (4) The mutation point is L229, and the substituted amino acid is Y, F or V, (5) The mutation point is L231, and the substituted amino acid is M, A or G. (6) The mutation point is S244, and the substituted amino acid is F. (7) The mutation point is A245, and the substituted amino acid is N or G. (8) The mutation point is V297, and the substituted amino acid is R, W, Q, A or I, (9) The mutation point is F298, and the substituted amino acid is S. (10) The mutation point is A328, and the substituted amino acid is N.

2. The modified esterase according to claim 1, wherein (1) wherein the substituted amino acid is E, I, C, A or M, (2) The substituted amino acid is I, S, A, W, H or G, (3) wherein the substituted amino acid is Y or S, (4) wherein the substituted amino acid is Y or F, (5) wherein the substituted amino acid is M or A, The amino acid after substitution in (8) is R, W or Q.

3. The modified esterase according to claim 1, wherein The amino acid after substitution of (4) is Y, The amino acid after substitution in (5) is M, The amino acid after substitution in (8) is R or W. 4 . A gene encoding the modified esterase according to claim 1 . A recombinant DNA comprising the gene according to claim 4.

6. A microorganism comprising the recombinant DNA according to claim 5. 7 . An enzyme agent comprising the modified esterase according to claim 1 .

8. 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 claims 1 to 3, (II) a step of expressing the nucleic acid, and (III) A step of recovering the expression product.

9. A method for producing (1R, 3S)-chrysanthemic acid, characterized in that: The modified esterase according to any one of claims 1 to 3 is allowed to act on (1R, 3S)-ethyl chrysanthemate.

10. The manufacturing method according to claim 9, wherein: (1R,3S)-Chrysanthemic acid ethyl ester is in a state of coexistence with (1R,3R)-chrysanthemic acid ethyl ester, (1S,3S)-chrysanthemic acid ethyl ester and (1S,3R)-chrysanthemic acid ethyl ester.

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