Modified esterase

Abstract

The present invention provides any one type of modified esterase selected from (i) to (iii) below: (i) a modified esterase containing an amino acid sequence in which at least one amino acid selected from the group consisting of (1) to (5) below is substituted with another amino acid in the amino acid sequence represented by SEQ ID NO: 1, 7 or 35: (1) S12 (2) R25 (3) S220 (4) A313 (5) S315; and (ii) a modified esterase which is a modified esterase in the modified esterase (i), one or more amino acids are further substituted (excluding the amino acid site substituted in (i)), added, inserted or deleted (excluding the amino acid site substituted in (i)), and the temperature stability and / or the tolerance to organic solvents of the esterase are further improved compared with those of the esterase composed of the amino acid sequence of SEQ ID NO: 1, 7 or 35; and (iii) a modified esterase in which amino acids other than the amino acid substituted in (i) are further substituted with another amino acid in the modified esterase in (i), the modified esterase has 70% or more identity with the modified esterase of (i), and the temperature stability and / or the tolerance to an organic solvent of the modified esterase are further improved compared with those of an esterase comprising the amino acid sequence of SEQ ID NO: 1, 7 or 35.

Description

Technical Field

[0001] The present invention relates to a novel modified esterase. More specifically, the present invention relates to a modified esterase having improved high temperature stability and / or tolerance to organic solvents compared to existing esterases. Background Art

[0002] Chrysanthemic acid is widely used as a raw material for the synthesis of 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 or by an enzyme reaction using chrysanthemic acid esterase.

[0003] For example, Patent Document 1 discloses that esterase 1R, 3R ((+)-trans) derived from Arthrobacter globiformis specifically decomposes chrysanthemic acid ethyl ester. Patent Document 2 discloses an example of modifying esterase derived from Arthrobacter globiformis to an enzyme 1R, 3S ((+)-cis) that specifically decomposes chrysanthemic acid ethyl ester. In addition, Non-Patent Document 1 discloses that esterase derived from Arthrobacter globiformis or esterase derived from other bacteria 1R, 3R ((+)-trans) specific or 1S, 3R ((-)-trans) specific decomposes chrysanthemic acid ethyl ester. Prior art literature Patent Literature

[0004] Patent Document 1: Japanese Patent Application Laid-Open No. 5-56787 Patent Document 2: International Publication No. 2020 / 116331 Non-patent literature

[0005] Non-patent document 1: Satoshi Mitsuda et al., Agric. Biol. Chem., 55(11), 2865-2870, 1991. Summary of the invention Problem that the invention aims to solve

[0006] The industrial usefulness of the esterase derived from Arthrobacter spherical is higher. In the manufacture of chrysanthemic acid utilizing the enzyme reaction of the esterase, a stable esterase with high tolerance to heat, organic solvents, etc. is expected. However, the temperature stability and tolerance to organic solvents of the wild-type esterase derived from Arthrobacter spherical are subject to improvement. Therefore, the present invention provides a novel esterase with a more preferred high temperature stability and / or tolerance to organic solvents as its subject. Means of solving the problem

[0007] The present inventors modified the amino acid sequence of an esterase derived from Arthrobacter sphericalus by using protein engineering techniques, attempting to create a modified esterase that is more stable at high temperatures. Through trial and error, the present inventors successfully identified a mutation point (amino acid residue) in the esterase that is effective in improving temperature stability, and further found that the mutation point is also effective in improving tolerance to organic solvents. By further advancing research based on the relevant findings, the present invention was completed. That is, the present invention is as described below.

[0008] [1] A modified esterase, which is any one of the following modified esterases (i) to (iii): (i) a modified esterase comprising an amino acid sequence in which at least one amino acid at a position selected from the group consisting of the following (1) to (5) in the amino acid sequence of SEQ ID NO: 1, 7 or 35 is substituted with another amino acid, (1)S12 (2)R25 (3)S220 (4)A313 (5) S315; (ii) a modified esterase, wherein in the modified esterase of (i), one or more amino acids are further substituted (excluding the amino acid site substituted in (i)), added, inserted or deleted (excluding the amino acid site substituted in (i)), and its temperature stability and / or tolerance to organic solvents are further improved compared to these properties of the esterase consisting of the amino acid sequence of SEQ ID NOs: 1, 7 or 35; (iii) a modified esterase, wherein in the modified esterase of (i), amino acids other than the amino acids substituted in (i) are further substituted by other amino acids, wherein the modified esterase has more than 70% identity with the modified esterase of (i) and its temperature stability and / or tolerance to organic solvents are further improved compared to these properties of the esterase composed of the amino acid sequence of sequence number 1, 7 or 35. [2] The modified esterase according to [1], wherein in (i), the amino acid substitution is at least one selected from the group consisting of: (1)S12P (2)R25P (3)S220A (4) A313S (5)S315M. [3] A nucleic acid encoding the modified esterase described in [1] or [2]. [4] An expression cassette or recombinant vector comprising the nucleic acid described in [3]. [5] A transformant obtained by transformation using the expression cassette or recombinant vector described in [4]. [6] A method for producing a modified esterase, comprising the step of culturing the transformant described in [5]. [7] An enzyme agent comprising the modified esterase described in [1] or [2]. [8] The enzyme agent according to [7], which is used for producing chrysanthemic acid. [9] A method for producing chrysanthemic acid, comprising the step of allowing the modified esterase described in [1] or [2] to act on ethyl chrysanthemate. Effects of the Invention

[0009] The temperature stability and tolerance to organic solvents of the modified esterase of the present invention are further improved compared to the wild-type esterase. Therefore, the modified esterase of the present invention can be used to produce chrysanthemic acid at a higher temperature band than ever before. In addition, the modified esterase of the present invention can also efficiently produce chrysanthemic acid in the presence of an organic solvent. DETAILED DESCRIPTION

[0010] Some of the terms used to describe the present invention will be described below.

[0011] <Amino Acids> In this specification, according to convention, there are cases where amino acids are represented by one letter. The details are as follows: Glycine: G Alanine: A Valine: V Leucine: L Isoleucine: I Phenylalanine: F Tyrosine: Y Tryptophan: W Serine: S Threonine: T Cysteine: C Methionine: M Aspartic acid: D Glutamate: E Asparagine: N Glutamine: Q Lysine: K Arginine: R Histidine: H Proline: P

[0012] <Amino acid sequence> In the present specification, the left end of the amino acid sequence is the N-terminus, and the right end is the C-terminus.

[0013] <Amino Acid Types> In the present specification, "non-polar amino acids" include alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan. "Uncharged amino acids" include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. “Acidic amino acids” include aspartic acid and glutamic acid, and “basic amino acids” include lysine, arginine, and histidine.

[0014] <Replace> In this specification, "substitution" refers not only to the situation of artificially introducing substitution of amino acid residues, but also includes the situation of naturally introducing substitution of amino acid residues, that is, it also includes the situation that the original amino acid residues are different. In this specification, the substitution of amino acid residues can be artificial substitution or natural substitution, preferably artificial substitution.

[0015] In this specification, the mutation caused by amino acid substitution is represented by a combination of one letter representing the amino acid residue before substitution, a number representing the amino acid residue position where the amino acid substitution occurs, and one letter representing the amino acid residue after substitution. For example, when the 328th alanine is substituted with glycine, it is represented as "A328G". In addition, when representing the combination (shared) of two mutations, the symbol " / " is used. For example, if it is a combination of a mutation in which the 328th alanine is substituted with glycine and a mutation in which the 221st alanine is substituted with phenylalanine, it is represented as "A328G / A221F".

[0016] <Modified esterase> In this specification, "modified esterase" is an esterase obtained by modifying an esterase as a benchmark (hereinafter referred to as "benchmark esterase"). In this specification, the benchmark esterase is typically an esterase with an amino acid sequence of sequence number 1, an esterase with an amino acid sequence of sequence number 7, or an esterase with an amino acid sequence of sequence number 35. In addition, the esterase with the amino acid sequence of sequence number 1 is a wild-type esterase derived from Arthrobacter sphericalus. In addition, the esterase with the amino acid sequence of sequence number 7 is a double mutant esterase in which two amino acids (A221F / A328G) are substituted in the wild-type esterase derived from Arthrobacter sphericalus. In addition, the esterase with the amino acid sequence of sequence number 35 is a quintuple mutant esterase in which five amino acids (A221F / N222D / F298L / D326V / A328G) are substituted in the wild-type esterase derived from Arthrobacter sphericalus.

[0017] <Nucleic Acids> In this specification, "nucleic acid" includes both deoxyribonucleic acid and ribonucleic acid. In one embodiment, the "nucleic acid" can be deoxyribonucleic acid. In addition, in another embodiment, the "nucleic acid" can be ribonucleic acid. In addition, in another embodiment, the "nucleic acid" can be a chimera of deoxyribonucleic acid and ribonucleic acid.

[0018] 1. Modified esterase The modified esterase of the present invention is an enzyme reactive with chrysanthemic acid ethyl ester and is stable even at high temperatures or in organic solvents. As a result, it is a novel esterase that can efficiently synthesize chrysanthemic acid.

[0019] The modified esterase of the present invention is a substance obtained by substituting specific amino acids in a standard esterase, and specifically contains the following specific amino acid sequences (i) to (iii) or a sequence consisting of the amino acid sequences:

[0020] (i) a modified esterase comprising an amino acid sequence in which at least one amino acid at a position selected from the group consisting of the following (1) to (5) in the amino acid sequence of SEQ ID NO: 1, 7 or 35 is substituted with another amino acid; (1)S12 (2)R25 (3)S220 (4)A313 (5)S315

[0021] (ii) a modified esterase, wherein in the modified esterase of (i), one or more amino acids are further substituted (excluding the amino acid site substituted in (i)), added, inserted or deleted (excluding the amino acid site substituted in (i)), and its temperature stability and / or tolerance to organic solvents are further improved compared to these properties of the esterase consisting of the amino acid sequence of SEQ ID NOs: 1, 7 or 35;

[0022] (iii) a modified esterase, wherein in the modified esterase of (i), amino acids other than the amino acids substituted in (i) are further substituted by other amino acids, wherein the modified esterase has more than 70% identity with the modified esterase of (i) and its temperature stability and / or tolerance to organic solvents are further improved compared to these properties of the esterase composed of the amino acid sequence of sequence number 1, 7 or 35.

[0023] First, the modified esterase (i) will be described. The modified esterase of (i) comprises an amino acid sequence in which at least one of the original amino acids in (1) to (5) above in the reference esterase is substituted with another amino acid. In the esterase of the present invention, the number of substituted amino acids may generally be 1 or more, preferably 2 or more, and more preferably 3 or more (e.g. 3, 4, 5 or more).

[0024] Regarding the original amino acid substitution with other amino acids, the type of the substituted amino acid is not particularly limited as long as it can exert the desired effect of the present invention, but there is a situation where conservative substitution is preferred. Conservative substitution of amino acids is a well-known technique for those skilled in the art. As an example, the type of amino acids after substitution can be selected with reference to the above-mentioned <type of amino acids>, but is not limited thereto.

[0025] In one embodiment, the number of substituted amino acids is 1 to 5, and the substitution sites may be 1 to 5 sites selected from the group consisting of: (1) S12, (2) R25, (3) S220, (4) A313 and (5) S315.

[0026] In another embodiment, the number of substituted amino acids is 1, and the substitution sites may be the following sites. (1)S12 (2)R25 (3)S220 (4)A313 (5)S315

[0027] In another embodiment, the number of substituted amino acids is 2, and the substitution sites may be the following sites. (4) A313 and (5) S315

[0028] In another embodiment, the number of substituted amino acids is 4, and the substitution sites may be the following sites. (2) R25, (3) S220, (4) A313 and (5) S315 (1) S12, (3) S220, (4) A313 and (5) S315

[0029] In another embodiment, the number of substituted amino acids is 5, and the substitution sites may be the following sites. (1) S12, (2) R25, (3) S220, (4) A313 and (5) S315

[0030] In a preferred embodiment, the modified esterase of the present invention comprises an amino acid sequence having the following amino acid substitutions in SEQ ID NO: 1, 7 or 35, or consists of the amino acid sequence.

[0031] <Modified esterase having one amino acid substitution in the reference esterase> (E-1)S12P (E-2)R25P (E-3)S220A (E-4)A313S (E-5)S315M

[0032] <Modified esterase having two amino acid substitutions in the reference esterase> (E-6)A313S / S315M

[0033] <Modified esterase having 4 amino acid substitutions in the benchmark esterase> (E-7)R25P / S220A / A313S / S315M (E-8)S12P / S220A / A313S / S315M

[0034] <Modified esterase having 5 amino acid substitutions in the benchmark esterase> (E-9)S12P / R25P / S220A / A313S / S315M

[0035] In one embodiment, the modified esterase of the present invention may be a substance in which an esterase part is fused with other polypeptides or proteins. In the modified esterase of the present invention, the fused polypeptide or protein is not particularly limited, but examples thereof include sequences that play a role in purification, such as multiple amino acid residues, and polypeptides that can improve the stability of the esterase during recombinant production.

[0036] In addition, in this specification, "organic solvent" refers to an organic compound that is liquid at room temperature and pressure and can dissolve other substances. Examples of organic solvents include acetaldehyde, acetic acid, acetic anhydride, acetone, acetonitrile, acetophenone, acetylacetone, allyl alcohol, ethanolamine, aniline, benzaldehyde, benzene, benzyl alcohol, benzyl benzoate, 1-butanol, 2-butanol, methyl ethyl ketone, butyl acetate, 2-methyl-2-propanol, dibutyl ether, carbon disulfide, etc. Disulfide、Chloroform、Epichlorohydrin、o-Cresol、m-Cresol、p-Cresol、Cyclohexane、Cyclohexanol、1,2-Dichloroethane、Dichloromethane、Diethyl Carbonate、Diethylene Glycol、Diethylene Glycol Monobutyl Ether、Butylcarbitol acetate、Diethylene Glycol Monoethyl Ether、Diethylene Glycol Monoethyl Ether Acetate、Diethylene Glycol Monomethyl Ether、Diethyl Ether Ether), Dimethylacetamide, N,N-dimethylformamide, Dimethyl Sulfoxide, 1,4-dioxane (1,4-Dioxane、Ethanol、Absolute Ethanol、Ethyl Acetate、Ethyl Benzoate、2-Chloroethanol、Ethylene Glycol、1,2-Dimethoxyethane、2-Butoxyethanol、Ethylene Glycol Monobutyl Ether Acetate、2-Ethoxyethanol、Ethylene Glycol Monoethyl Ether Acetate、Ethylene Glycol Monomethyl Ether Acetate、Ethyl Formate、2-Ethylhexanol、Ethyl Ethyl Acetate Acetoacetate、Ethyl-3-Oxobutanate、Ethyl Propionate、Formamide、Formic Acid、Furfuryl Alcohol、Glycerol、Heptane、Hexane、1-Hexanol、Ligroin、2,6-Dimethylpyridine6-Lutidine), Methanol, 2-Methoxyethanol, Methyl Acetate, 2-Methyl-2-butanol, 3-Methyl-1-butanol, Isoamyl Acetate, Methyl Propionate, Triethanolamine, Nitrobenzene, Nitromethane, n-Octane, 1-Octanol, 2-Octanol, Pentane, 1-Pentanol, 3-Pentanol, n-Pentyl Acetate, Petroleum Ether Benzine), Phenol, 1-Propanol, n-Propyl Acetate, Propylene Glycol, Propylene Oxide, Methyloxirane, 1,2-Epoxypropane, n-Propyl Ether, Pyridine, 1,1,2,2-Tetrachloroethane, Tetrachloroethylene, Tetrahydrofuran, Tetralin, Toluene, 1,1,2-Trichloroethane, 1,1,2-TCA, Trichloroethylene, Triethylamine, TEA, Trifluoroacetic acid Acid), TFA, xylene, o-xylene, m-xylene or p-xylene, but not limited to these. In a preferred embodiment, the organic solvent may be acetonitrile and methanol.

[0037] Next, the modified esterase (ii) will be described. The modified esterase of (ii) is an esterase obtained by further introducing amino acid modifications to the modified esterase of (i) above. However, the additionally introduced modification site is introduced into a site other than a specific amino acid substitution site in the modified esterase of (i). The additionally introduced amino acid modification can be any one (e.g., only substitution) or two or more (e.g., substitution and insertion) of amino acid substitution, addition, insertion, and deletion. In the modified esterase of (ii), as long as the desired effect of the present invention can be exerted, the number of additionally introduced amino acid modifications is not particularly limited, but is usually more than 1, for example, 1 to 80, preferably 1 to 70, 1 to 60, 1 to 50, 1 to 40 or 1 to 30, more preferably 1 to 20, 1 to 10, 1 to 8, 1 to 7, 1 to 6, 1 to 5 or 1 to 4, further preferably 1 to 3, and particularly preferably 1 or 2.

[0038] In addition, although not wishing to be bound by theory, positions 59 (serine residue), 62 (lysine residue) and 148 (tyrosine residue) of the esterase of sequence number 1, 7 or 35 are considered to be active catalyst residues. Therefore, there may be a case where it is preferred not to replace or delete these sites. Considering the amino acid residues that are important in the optical selectivity for chrysanthemic acid ethyl ester, it is more preferred that, for the amino acids at positions (221) (alanine residue or phenylalanine residue), (222) (asparagine residue), (298) (phenylalanine residue), (326) (aspartic acid residue) and (328) (alanine or glycine residue) in sequence number 1, 7 or 35, when it is not desired to change the optical selectivity, no substitution or deletion is introduced at these sites.

[0039] In addition, the modified esterase of (ii) is characterized in that, while having the above-mentioned amino acid sequence, the temperature stability and / or tolerance to organic solvents are further improved compared to these properties of the benchmark esterase (i.e., the esterase composed of the amino acid sequence of sequence number 1, 7 or 35). More specifically, relative to the benchmark esterase (esterase having the amino acid sequence of sequence number 1, 7 or 35), the T50 value (°C) of the modified esterase of (ii) is increased by 1.0°C or more, preferably by 1.2°C or more, and more preferably by 1.5°C or more. Alternatively, relative to the benchmark esterase (esterase having the amino acid sequence of sequence number 1, 7 or 35), the stability of the modified esterase of (ii) in an organic solvent (such as methanol or acetonitrile, etc.) is increased by 1.1 times or more, preferably by 1.2 times or more. Or the modified esterase of (ii) has the same temperature stability and / or tolerance to organic solvents as the modified esterase of (i).

[0040] Next, the modified esterase (iii) will be described. The modified esterase (iii) is an esterase obtained by further introducing an amino acid substitution into the modified esterase (i) above. However, the site of the additionally introduced amino acid substitution is introduced into a site other than the specific amino acid substitution site in the modified esterase (i).

[0041] The sequence identity between the modified esterase of (iii) and the modified esterase of (i) is not particularly limited as long as the desired effect can be exerted, but is usually 70% or more, preferably 75% or more, 80% or more, or 85% or more, more preferably 90% or more, 95% or more, 96% or more, 97% or more, or 98% or more, and particularly preferably 99% or more.

[0042] In addition, sequence identity can be determined by a known method. For example, it can be obtained by bl2seq program (Tatiana A. Tatsusova, Thomas L. Madden, FEMS Microbiol. Lett., Vol. 174, p247-250, 1999) of BLASTPACKAGE [sgi 32 bit edition, Version 2.0.12; available from National Center for Biotechnology Information (NCBI)]. When this method is used, the parameters can be set to Gap insertion Cost value: 11 and Gap extension Cost value: 1.

[0043] The modified esterase of (iii) is also the same as the modified esterase of (ii), characterized in that the temperature stability and / or tolerance to organic solvents are further improved compared to these properties of the benchmark esterase. That is, relative to the benchmark esterase, the T50 value (°C) of the modified esterase of (iii) is increased by more than 1.0°C, preferably by more than 1.2°C, and more preferably by more than 1.5°C. Alternatively, relative to the benchmark esterase (esterase having the amino acid sequence of sequence number 1, 7 or 35), the stability of the modified esterase of (ii) in an organic solvent (such as methanol or acetonitrile, etc.) is increased by more than 1.1 times, preferably by more than 1.2 times. Or the modified esterase of (iii) has the same temperature stability and / or tolerance to organic solvents as the modified esterase of (i).

[0044] 2. Nucleic acid encoding modified esterase The present invention also provides a nucleic acid encoding the modified esterase of the present invention (hereinafter sometimes referred to as "nucleic acid of the present invention").

[0045] The nucleic acid of the present invention is a nucleic acid encoding the modified esterase of the present invention. The nucleic acid of the present invention is not particularly limited as long as it is a nucleic acid encoding the modified esterase of (i) to (iii) described above.

[0046] Nucleic acid of the present invention can be prepared by a method known to itself. As an example, the base sequence shown in sequence number 13 is a base sequence encoding a benchmark esterase consisting of the amino acid sequence shown in sequence number 1, the base sequence shown in sequence number 19 is a base sequence encoding a benchmark esterase consisting of the amino acid sequence shown in sequence number 7, and the base sequence shown in sequence number 41 is a base sequence encoding a benchmark esterase consisting of the amino acid sequence shown in sequence number 35. Therefore, nucleic acid of the present invention can be appropriately designed using sequence numbers 13, 19 or 41 as a benchmark sequence by a method known to itself. In addition, codon degeneracy can be considered according to the host expressing nucleic acid of the present invention.

[0047] In one embodiment, an example of the nucleic acid of the present invention is shown in SEQ ID NOs: 14 to 18, 20 to 24, and 42 to 46. In addition, these are nucleic acid sequences encoding modified esterases prepared based on the reference esterase of SEQ ID NOs: 1, 7, or 35.

[0048] SEQ ID NO: 14: S220A mutant of SEQ ID NO: 1 SEQ ID NO: 15: A313S / S315M mutant of SEQ ID NO: 1 SEQ ID NO: 16: R25P / S220A / A313S / S315M mutant of SEQ ID NO: 1 SEQ ID NO. 17: S12P / S220A / A313S / S315M mutant of SEQ ID NO. 1 SEQ ID NO: 18: S12P / R25P / S220A / A313S / S315M mutant of SEQ ID NO: 1 SEQ ID NO: 20: S220A mutant of SEQ ID NO: 7 SEQ ID NO: 21: A313S / S315M mutant of SEQ ID NO: 7 SEQ ID NO: 22: R25P / S220A / A313S / S315M mutant of SEQ ID NO: 7 SEQ ID NO: 23: S12P / S220A / A313S / S315M mutant of SEQ ID NO: 7 SEQ ID NO: 24: S12P / R25P / S220A / A313S / S315M mutant of SEQ ID NO: 7 SEQ ID NO: 42: S220A mutant of SEQ ID NO: 35 SEQ ID NO: 43: A313S / S315M mutant of SEQ ID NO: 35 SEQ ID NO. 44: R25P / S220A / A313S / S315M mutant of SEQ ID NO. 35 SEQ ID NO. 45: S12P / S220A / A313S / S315M mutant of SEQ ID NO. 35 SEQ ID NO: 46: S12P / R25P / S220A / A313S / S315M mutant of SEQ ID NO: 35

[0049] In addition, the nucleic acid of the present invention is not limited to the nucleic acid sequence encoding the modified esterase of the present invention itself. As long as it encodes a polypeptide having esterase activity and temperature stability and / or tolerance to organic solvents equivalent to the modified esterase of the present invention, a nucleic acid sequence having a homology of 75% or more, preferably 80% or more, 85% or more, 90% or more, more preferably 95% or more, 96%, 97% or more, 98% or more, and particularly preferably 99% or more with the nucleic acid sequence is also included in the nucleic acid of the present invention.

[0050] Among them, the "homology" of the nucleic acid sequence is calculated using public or commercially available software equipped with an algorithm for comparing the reference sequence as the query sequence. Specifically, BLAST, FASTA or GENETYX (manufactured by Software Development Co., Ltd.) can be used. When determining the homology of sequences using these, the homology score can be determined using the default parameters, or the homology score can be determined using appropriately changed parameters.

[0051] In addition, the same applies to nucleic acids composed of nucleic acid sequences complementary to the nucleic acid of the present invention and nucleic acids that hybridize under stringent conditions, as long as they encode a polypeptide having an activity equivalent to the esterase activity possessed by the modified esterase of the present invention, they are included in the nucleic acid of the present invention. In this case, "stringent conditions" refers to conditions of incubating at 50°C to 65°C for 4 hours to overnight in 6×SSC (1×SSC, 0.15M NaCl, 0.015M sodium citrate, pH 7.0) containing 0.5% SDS, 5×Denhartz〔Denhartz's, 0.1% bovine serum albumin (BSA), 0.1% polyvinyl pyrrolidone, 0.1% Ficoll 400〕and 100μg / ml salmon sperm DNA. Hybridization under stringent conditions can be specifically performed by the following method. That is, a nylon membrane on which a DNA library or cDNA library is immobilized is prepared, and the nylon membrane is blocked at 65°C in a prehybridization solution containing 6×SSC, 0.5% SDS, 5×Denhartz, and 100μg / ml salmon sperm DNA. Then, each probe labeled with 32P is added and incubated at 65°C overnight. The nylon membrane is washed in 6×SSC at room temperature for 10 minutes; in 2×SSC containing 0.1% SDS at room temperature for 10 minutes; and in 0.2×SSC containing 0.1% SDS at 45°C for 30 minutes, and then the DNA specifically hybridized with the probe can be detected by radioautography.

[0052] In one embodiment, as described above, the modified esterase of the present invention may be a substance formed by fusion of an esterase part with other polypeptides or proteins. Therefore, the nucleic acid of the present invention may be a base sequence encoding an esterase part and other polypeptides or proteins.

[0053] The method for expressing the nucleic acid of the present invention in a host is not particularly limited, and a known method per se can be used.

[0054] 3. Expression cassette or recombinant vector The present invention also provides an expression cassette or recombinant vector containing the nucleic acid of the present invention (hereinafter referred to as "the expression cassette of the present invention", "the recombinant vector of the present invention", or "the expression cassette or recombinant vector of the present invention").

[0055] The expression cassette or recombinant vector of the present invention can be prepared by linking a promoter and a terminator to the nucleic acid of the present invention, or by inserting the expression cassette of the present invention or the nucleic acid of the present invention into an expression vector.

[0056] In the expression cassette of the present invention or the recombinant vector of the present invention, as a control factor, in addition to a promoter and a terminator, transcription factors such as an enhancer, a CCAAT box, a TATA box, and an SPI site may also be contained as needed. These control factors only need to be connected to the nucleic acid of the present invention in a manner that can act. The connection that can act means that various control factors that regulate the nucleic acid of the present invention and the nucleic acid of the present invention are connected in a state that can act in the host cell.

[0057] In the recombinant vector of the present invention, the vector is preferably an expression vector. As an expression vector, a vector constructed from a phage, plasmid or virus that can proliferate autonomously in a host for gene recombination can be preferably used. Related expression vectors are well known, and for example, commercially available expression vectors include: pQE vectors (QIAGEN Co., Ltd.), pDR540, pRIT2T (GE Healthcare Bioscience Co., Ltd.), pET vectors (Merck Co., Ltd.), etc. The expression vector can be used in combination with a host cell as long as it is selected. For example, when Escherichia coli is used as the host cell, a combination of a pQE vector and a DH5α Escherichia coli strain, a combination of a pET vector and a BL21 (DE3) Escherichia coli strain, or a combination of a pDR540 vector and a JM109 Escherichia coli strain can be mentioned.

[0058] 4. Transformants The present invention also provides a transformant transformed using the expression cassette or recombinant vector of the present invention (hereinafter sometimes referred to as "the transformant of the present invention").

[0059] The host used for producing the transformant of the present invention is not particularly limited as long as it has the following characteristics (1) to (4): (1) can be introduced into expression cassettes or recombinant vectors, (2) The expression cassette or recombinant vector is stable, (3) It can proliferate autonomously and (4) The ability to express the gene in the introduced expression cassette or recombinant vector. Examples of the host include bacteria belonging to the genus Escherichia such as Escherichia coli, the genus Bacillus such as Bacillus subtilis, the genus Pseudomonas such as Pseudomonas putida, filamentous fungi, yeast, etc. In addition, animal cells, insect cells, plants, etc. may be used.

[0060] The transformant of the present invention can be prepared by introducing the nucleic acid of the present invention, the expression cassette of the present invention or the recombinant vector of the present invention into the host. As for the site where the nucleic acid of the present invention is introduced, there is no particular limitation as long as the target gene can be expressed. It can be on a plasmid or on a genome. As a specific method for introducing the expression cassette of the present invention or the recombinant vector of the present invention, for example, a recombinant vector method and a genome editing method can be cited. The conditions for introducing the expression cassette or the recombinant vector into the host can be appropriately set according to the type of the host. When the host is a bacterium, for example, a method using competent cells treated with calcium ions and an electrofection method can be cited. When the host is yeast, for example, electroporation (electroporation), spheroplast method and lithium acetate method can be cited. When the host is an animal cell, for example, electrofection, calcium phosphate method and lipid transfection method can be cited. When the host is an insect cell, for example, calcium phosphate method, lipid transfection method and electrofection method can be cited. When the host is a plant cell, for example, electrofection, Agrobacterium method, gene gun method and PEG method can be cited.

[0061] Whether the expression cassette of the present invention or the recombinant vector of the present invention has been introduced into the host can be confirmed by a known method such as PCR, Southern hybridization or Northern hybridization.

[0062] When confirming by PCR whether the expression cassette of the present invention or the recombinant vector of the present invention has been introduced into the host, for example, genomic DNA or the expression cassette or the recombinant vector can be separated / purified from the transformant. For the separation / purification of the expression cassette or the recombinant vector, for example, when the host is a bacterium, the lysate obtained by dissolving the bacteria is carried out. As a method of lysing bacteria, for example, lysozymes such as lysozyme can be used for treatment, and surfactants such as protease and other enzymes and sodium lauryl sulfate (SDS) can be used as needed.

[0063] Furthermore, physical disruption methods such as freeze-thaw and French press treatment may be combined. Isolation / purification of nucleic acids from lysates can be performed by, for example, appropriately combining protein removal treatment using phenol treatment and protease treatment, ribonuclease treatment, alcohol precipitation treatment, and commercially available kits.

[0064] The cleavage of the nucleic acid can be carried out according to conventional methods (e.g., restriction enzyme treatment). As the restriction enzyme, for example, a type II restriction enzyme that acts on a specific nucleotide sequence is used. The nucleic acid is bound to the expression cassette or expression vector using, for example, DNA ligase.

[0065] Afterwards, using the separated / purified DNA as a template, PCR is performed with primers designed to be specific to the DNA of the present invention. The amplified product obtained by PCR is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., stained with ethidium bromide and SYBR Green solution, etc., and then detected as a band using the amplified product to confirm transformation.

[0066] In addition, the amplification product can also be detected by performing PCR using primers previously labeled with a fluorescent dye, etc. Furthermore, a method can also be adopted in which the amplification product is bound to a solid phase such as a microplate and the amplification product is confirmed by fluorescence and enzyme reaction, etc.

[0067] 5. Method for producing modified esterase The present invention also provides a method for producing a modified esterase comprising the step of culturing the transformant of the present invention (hereinafter sometimes referred to as "the production method of the present invention").

[0068] The culture conditions used in the production method of the present invention may be appropriately set in consideration of the nutritional and physiological properties of the transformant to be used.

[0069] The culture of the transformant can use a solid culture medium or a liquid culture medium, and can be preferably a liquid culture medium. In addition, when industrial production is carried out, it is preferably a ventilation stirring culture. As a nutrient source for the culture medium, it is sufficient to use the substances required for the growth and development of the transformant appropriately. As a carbon source, it is sufficient to use a carbon compound that can be recycled, for example, glucose, sucrose, lactose, maltose, syrup, pyruvic acid, etc. As a nitrogen source, it is sufficient to use an internalizable nitrogen compound, for example, peptone, meat extract, yeast extract, casein hydrolyzate, soybean meal alkaline extract. In addition to carbon sources and nitrogen sources, for example, salts of phosphates, carbonates, sulfates, magnesium, calcium, potassium, iron, manganese and zinc, specific amino acids and specific vitamins and biotin, etc. can also be used as needed.

[0070] The culture temperature only needs to be appropriately set within the range in which the transformant of the present invention can grow and develop and the transformant produces the modified esterase. In one embodiment, the culture temperature is about 15 to 37° C., but is not limited to these. As the timing of terminating the culture, it is sufficient to terminate the culture when the modified esterase reaches the maximum yield. The culture time can be, for example, about 12 to 48 hours, but is not limited to these.

[0071] The modified esterase expressed by the transformant is present inside the transformant or in the culture medium. The modified esterase can be recovered by a known method per se, but an example of the recovery method is briefly described below.

[0072] In one embodiment, when the expressed modified esterase is present inside the transformant, it is separated from the culture supernatant by a method known per se, such as centrifuging the transformant. The culture supernatant is removed and the separated transformant is recovered. The separated transformant is treated with a mechanical method such as ultrasound or French pressing or an enzymatic method such as lysozyme, and is solubilized as needed using an enzyme such as a protease or a surfactant such as sodium lauryl sulfate (SDS), thereby obtaining a water-soluble fraction containing the modified esterase.

[0073] In addition, in other embodiments, when the expressed modified esterase is present in the culture medium, it can be directly provided for purification treatment, or it can be provided for purification treatment after the modified esterase in the water-soluble component is concentrated. Concentration can be performed, for example, by using reduced pressure concentration, membrane concentration, salting-out treatment, step-by-step precipitation method of hydrophilic organic solvents (e.g., methanol, ethanol and acetone). In addition, the purification treatment of the modified esterase can be performed, for example, by appropriately combining methods such as gel filtration, adsorption chromatography, ion exchange chromatography, affinity chromatography, etc. The modified esterase purified in this way can be circulated to the market after being powdered by freeze drying, vacuum drying, spray drying, etc. as needed.

[0074] 6. Enzyme containing modified esterase The present invention also provides an enzyme agent comprising the modified esterase of the present invention (hereinafter sometimes referred to as "the enzyme agent of the present invention").

[0075] The amount of the modified esterase of the present invention contained in the enzyme agent of the present invention can generally be 0.001 to 100% by weight, preferably 0.01 to 100% by weight, 0.1 to 100% by weight or 1 to 100% by weight, more preferably 5 to 100% by weight, 10 to 100% by weight, 20 to 100% by weight, 30 to 100% by weight, 40 to 100% by weight or 50 to 100% by weight, but is not limited to these.

[0076] The enzyme agent of the present invention may contain only the modified esterase of the present invention, but may contain other components as long as the desired effect is exerted. Examples of other components include: other enzymes other than the modified esterase of the present invention, additives, and culture residues produced in the production method of the present invention.

[0077] As other enzymes, for example, amylase (α-amylase, β-amylase, glucoamylase), glucosidase (α-glucosidase, β-glucosidase), β-galactosidase (α-galactosidase, β-galactosidase), protease (acid protease, neutral protease, alkaline protease), peptidase (leucine peptidase, aminopeptidase), lipase, esterase, cellulase, phosphatase (acid phosphatase, alkaline phosphatase), nuclease, deaminase, oxidase, dehydrogenase, glutaminase, pectinase, catalase, glucanase, transglutaminase, protein deamidase, pullulanase, etc. These other enzymes may contain one alone or in combination of a plurality of.

[0078] As additives, there can be mentioned: excipients, buffers, suspending agents, stabilizers, preservatives, preservatives, physiological saline, etc. As excipients, there can be mentioned: starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, white sugar, glycerol, etc. As buffers, there can be mentioned: phosphates, citrates, acetates, etc. As stabilizers, there can be mentioned: propylene glycol, ascorbic acid, etc. As preservatives, there can be mentioned: phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methyl parahydroxybenzoate, etc. As preservatives, there can be mentioned: ethanol, benzalkonium chloride, p-hydroxybenzoic acid, chlorobutanol, etc. These additives can be contained alone or in combination of multiple types.

[0079] Examples of the culture residues include components derived from the culture medium, impurity proteins, and bacterial components.

[0080] The form of the enzyme agent of the present invention is not particularly limited, and examples thereof include liquid, solid (powder, granules, etc.), etc. The shape of the enzyme agent may be formed by a method known per se.

[0081] The modified esterase of the present invention, which is an active ingredient of the enzyme agent of the present invention, has high enzyme activity in the process of converting chrysanthemic acid ethyl ester to chrysanthemic acid (preferably converting (1R, 3S)-chrysanthemic acid ethyl ester to (1R, 3S)-chrysanthemic acid). Therefore, in one embodiment of the enzyme agent of the present invention, the enzyme agent of the present invention is an enzyme agent for producing chrysanthemic acid (preferably (1R, 3S)-chrysanthemic acid).

[0082] By allowing the enzyme of the present invention to act on a composition containing chrysanthemic acid ethyl under appropriate conditions, a composition containing chrysanthemic acid can be efficiently produced. In addition, the substrate of the enzyme of the present invention is described in detail in the "method for producing chrysanthemic acid" described later.

[0083] 7. Method for producing chrysanthemic acid The present invention also provides a method for producing chrysanthemic acid comprising the step of allowing ethyl chrysanthemate to act on the modified esterase of the present invention (hereinafter sometimes referred to as "the method for producing chrysanthemic acid of the present invention").

[0084] In the method for producing chrysanthemic acid of the present invention, chrysanthemic acid ethyl ester serving as a matrix may be a purified substance, or may be a composition containing other components (hereinafter referred to as "chrysanthemic acid ethyl ester-containing composition"). The chrysanthemic acid ethyl ester-containing composition may use high-purity (1R, 3S)-chrysanthemic acid ethyl ester (for example, purity of 98% or more), or may use a matrix 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.).

[0085] The content of chrysanthemic acid ethyl contained in the composition containing chrysanthemic acid ethyl is not particularly limited, and for example, 20% or more can be cited. From the viewpoint of further improving the content of chrysanthemic acid ethyl in the product, the content of chrysanthemic acid ethyl in the matrix can be preferably 30% or more, more preferably 40% or more, and further preferably 45% or more. The upper limit of the content range is not particularly limited, but for example, 100% or less, preferably 90% or less, and more preferably 70% or less can be cited. There is no particular limitation on the source of the composition containing chrysanthemic acid ethyl, and for example, chemically synthesized substances can be cited.

[0086] As long as chrysanthemic acid can be produced, the reaction time, reaction temperature, pH of the reaction solution, solvent, etc. when the modified esterase is allowed to act on the substrate (e.g., a composition containing ethyl chrysanthemic acid) are not particularly limited. The reaction time is, for example, 10 minutes to 96 hours, preferably 1 hour to 72 hours, and more preferably 12 hours to 48 hours. The reaction temperature can be, for example, 10 to 90°C, preferably 20 to 80°C, more preferably 30 to 75°C, further preferably 40 to 70°C, and further preferably 50 to 65°C. The pH of the reaction solution can be, for example, 3 to 12, preferably 6 to 11, and more preferably 8 to 11. As a solvent in the reaction, an organic solvent may be contained or not. As an organic solvent, methanol and acetonitrile can be used. The amount of the organic solvent is not particularly limited, and for example, as the lower limit, 10%, 20%, 30% or more, 40% or more can be cited, and as the upper limit, 100% or less, 80% or less, 70% or less, 50% or less can be cited. These reaction conditions can be appropriately selected according to the raw materials used and the target product. In addition, the optimal reaction conditions can be determined by preliminary experiments.

[0087] By using the method for producing chrysanthemic acid of the present invention, a composition containing chrysanthemic acid can be produced very efficiently. One embodiment of the method for producing a composition containing chrysanthemic acid of the present invention comprises the following steps (1) and (2). In addition, after step (2), a step of recovering chrysanthemic acid from the obtained composition containing chrysanthemic acid can be added. (1) Step of providing a composition containing ethyl chrysanthemate (2) A step of treating the provided composition containing ethyl chrysanthemate with the modified esterase of the present invention

[0088] The content of chrysanthemic acid in the chrysanthemic acid-containing composition obtained by the method for producing chrysanthemic acid of the present invention is not particularly limited, and may generally be 1% by weight or more, preferably 2% by weight or more, 3% by weight or more, 4% by weight or more, or 5% by weight or more, more preferably 10% by weight or more, 12% by weight or more, or 14% by weight or more, and particularly preferably 16% by weight or more.

[0089] The reaction product may be subjected to an additional optical resolution step as required. Column chromatography may be used for optical resolution. An immobilized modified esterase may be used as the modified esterase.

[0090] In addition, the composition containing chrysanthemic acid is preferably used as a pesticide intermediate.

[0091] The present invention will be further specifically described in the following examples, but the present invention is not limited to these examples. Example

[0092] A double mutant of a wild-type esterase (SEQ ID NO: 1) derived from Arthrobacter globiformis, the esterase of SEQ ID NO: 7 with high reactivity to ethyl chrysanthemate is a (1R,3S)-specific esterase with high selectivity for (1R,3S)-ethyl chrysanthemate. Therefore, using this (1R,3S)-specific esterase and the wild-type esterase as reference esterases, attempts were made to produce modified esterases with improved heat resistance and stability in organic solvents through protein engineering techniques. The detailed steps are as follows.

[0093] 1. Preparation of mutant library A mutant library was prepared according to the following method. (1) Introduction / transformation of mutations PCR primers for introducing mutations (SEQ ID NOs: 25 - 34) were designed, and PCR was performed under the following conditions to introduce mutations.

[0094] <Template plasmid> pET21b (into which the gene sequence of the reference esterase was introduced) approximately 30 ng / μL

[0095] <PCR reaction composition> (total volume 20 μL) 5×Prime STAR GXL Buffer (TakaraBio): 4 μL dNTP Mixture (2.5 mM each): 1.6 μL Template (approximately 30 ng / μL): 0.25 μL F - primer: 0.2 μL R - primer: 0.2 μL Prime STAR GXL DNA Polymerase (Takara Bio): 0.8 μL These were mixed and adjusted to 20 μL using ultrapure water (Milli Q water).

[0096] <PCR conditions> (1) 98°C, 1 minute (2) <98°C, 10 seconds; 60°C, 15 seconds; 68°C, 2 minutes> × 20 cycles (3) 68°C, 5 minutes (4) Incubate at 4°C

[0097] 0.5 μL of DpnI was added to the 20 μL PCR reaction solution and incubated (37°C, for 3 hours or more). The DNA was purified using the DNA purification kit NucleoSpin Gel and PCR Clean-up (manufactured by Takara Bio Inc.).

[0098] <Seamless (In-fusion) Response> A reaction solution having the following composition was prepared (total amount 10 μL), and heated at 50° C. for 15 minutes using a thermal cycler. Purified PCR solution: 2-5 μL (50-200 ng equivalent) Milli Q water: 3-6 μL 5× seamless reagent: 2μL

[0099] 2 μL of the seamless reaction solution was mixed with 18 μL of E. coli BL21 (DE3) (NIPPON GENE) for transformation. After recovery culture at 37°C for 20 minutes, the transformation solution was spread on an LB agar plate (manufactured by Invitrogen) supplemented with ampicillin (final concentration 100 μg / mL) and cultured at 37°C for 18 hours. (Pre-culture).

[0100] (2) Sequence confirmation Each mutant strain after pre-culture was inoculated into LB medium (manufactured by Invitrogen Co., Ltd.) supplemented with ampicillin (final concentration 100 μg / mL). Cultured at 37°C for 18 hours as bacterial culture solution. The bacterial cells were recovered by centrifugation, and the respective plasmids were purified using a plasmid miniprep kit (Miniprep Kit) NucleoSpin Plasmid EasyPure (TakaraBio). The DNA sequence of the gene was confirmed by Sanger sequencing.

[0101] (3) Preparation of enzyme extract Each mutant strain after pre-culture was inoculated into 1 mL of TB medium (manufactured by Invitrogen) supplemented with ampicillin (final concentration 100 μg / mL). Culture was carried out at 33°C for 48 hours (IPTG (final concentration 0.1 mM) was added 24 hours after the start of culture), and centrifugation was performed (3,000 g × 10 minutes). The supernatant was removed, and after the bacterial cells were recovered, the bacterial cells were lysed using a lytic agent (25°C, 2 hours or more), and the enzyme was extracted. After the lysate was centrifuged (3,000 g × 10 minutes), the supernatant was recovered as the enzyme extract.

[0102] (4) Activity assay The activity of each enzyme was evaluated by measuring the activity according to the following activity measuring method.

[0103] Activity assay Measure the matrix (p-nitrophenyl-chrysanthemic acid ethyl ester: 6 mg) and suspend it in 16 mL of 4% Triton X-100. Heat to 50°C to dissolve the matrix. After cooling at room temperature, add 4 mL of 0.5 M PIPES pH 7.0 and mix them. Mix 190 μL of 0.1 M PIPES pH 7.0 and 10 μL of lysate. Add 3 μL of sample + 197 μL of each matrix solution to each well. React at 37°C for 2 hours. Use a plate reader to measure the absorbance at 416 nm.

[0104] (5) Preparation of combined mutants Using the obtained mutant as a template, PCR was carried out in the same manner as in the above (1). The PCR product was recovered and an enzyme extract was prepared in the same manner as in the above (3).

[0105] 2. Test Example 1 (1) Modification of wild-type esterase The following mutations were introduced into the wild-type esterase. The closely related esterases were searched for homology, and amino acids with no homology were selected. As a result, mutations were introduced into the following amino acid residues (one letter representing the amino acid residue, a number representing the position of the amino acid residue).

[0106] Q4, S12, R25, V64, F77, A93, H107, Y123, L128, C161, H184, Q208, H216, S220, L242, A245, M254, V286, F292, C303, A313, S315, S363

[0107] The enzyme activity of the prepared mutant enzymes was measured, and samples showing enzyme activity were collected. Further, by repeatedly using PCR mutation introduction, combined mutations of multiple mutation sites were prepared to simply evaluate the improvement of heat resistance. The result was that the following 5 mutant enzymes had changes in heat resistance, etc. compared to the wild-type enzyme.

[0108] [Table 1] Mutant enzyme (reference sequence: SEQ ID NO: 1) Introducing mutations Amino acid sequence Base sequence S220A Sequence number 2 Sequence Number 14 A313S / S315M Sequence number 3 Sequence Number 15 R25P / S220A / A313S / S315M Sequence Number 4 Sequence Number 16 S12P / S220A / A313S / S315M Sequence number 5 Sequence Number 17 S12P / R25P / S220A / A313S / S315M Sequence Number 6 Sequence Number 18

[0109] Therefore, detailed evaluation was performed on each mutant enzyme.

[0110] (2) Evaluation of heat resistance (temperature stability) Prepare an enzyme extract using the method of 1.(3) above. Use a thermal cycler to heat the enzyme extract at 30, 40, 50, 60, 70, 80 or 90°C for 30 minutes. After heating, remove the solid component by centrifugation as an enzyme sample solution. According to the enzyme activity determination method, the enzyme activity is determined by measuring the absorbance of each sample. Assuming the maximum value of each sample is 100%, the change in enzyme activity caused by temperature is determined using a relative value, and the 50% inactivation temperature (T50 value) is measured (Table 2). The result shows an improvement in heat resistance of up to 3.41°C.

[0111] [Table 2] T50 value (℃) T50 value (difference from wild type) (℃) wild type 69.59 - S220A 72.05 2.46 A313S / S315M 70.74 1.15 R25P / S220A / A313S / S315M 72.67 3.08 S12P / S220A / A313S / S315M 73 3.41 S12P / R25P / S220A / A313S / S315M 72.52 2.93

[0112] (3) Acetonitrile tolerance evaluation Enzyme extracts were prepared for wild-type and mutant enzymes. Acetonitrile was added to the enzyme extract to a final concentration of 30% and incubated at 30°C for 3 hours. The relative activity value when acetonitrile was added was calculated based on the sample to which an equal amount of ultrapure water was added instead of acetonitrile. The experiments were performed 3 times and the average value was calculated (Table 3). The results confirmed that the tolerance to acetonitrile was significantly improved, especially in the mutant enzyme of S220A and the quadruple mutant enzyme (S12P / S220A / A313S / S315M).

[0113] (4) Methanol tolerance evaluation Enzyme extracts were prepared for wild-type esterases and mutant enzymes. Methanol was added to the enzyme extract to a final concentration of 40% and incubated at 30°C for 3 hours. The relative activity value when methanol was added was calculated based on the sample to which an equal amount of ultrapure water was added instead of methanol. The relative activity value of each mutant enzyme was calculated based on the activity value of the wild type. The experiments were performed 3 times and the average value was calculated (Table 3). The results confirmed that the tolerance to methanol was significantly improved, especially in the quintuple mutant enzyme (S12P / R25P / S220A / A313S / S315M).

[0114] [Table 3]

[0115] 3. Test Example 2 (1) Modification of (1R,3S)-specific esterase As an esterase with high selectivity for (1R, 3S)-chrysanthemic acid ethyl ester, an esterase with double mutation of A221F / A328G is an esterase with high selectivity for (1R, 3S)-chrysanthemic acid ethyl ester that cannot be hydrolyzed by wild-type esterase ((1R, 3S)-specific esterase). The mutation of Test Example 1 was further introduced into the above-mentioned (1R, 3S)-specific esterase to study whether it has an effect of improving heat resistance (temperature stability). Using the base sequence (sequence number 19) with the mutation of A221F / A328G introduced as a template, mutation introduction and sequence confirmation were performed using PCR. For multiple mutation points, PCR was repeatedly performed to introduce the mutation points. Five mutant enzymes were prepared (Table 4).

[0116] [Table 4] Mutant enzyme (reference sequence: SEQ ID NO: 7) Introducing mutations Amino acid sequence Base sequence S220A Sequence Number 8 Sequence number 20 A313S / S315M Sequence Number 9 Sequence Number 21 R25P / S220A / A313S / S315M Sequence number 10 Sequence Number 22 S12P / S220A / A313S / S315M Sequence Number 11 Sequence Number 23 S12P / R25P / S220A / A313S / S315M Sequence Number 12 Sequence Number 24

[0117] (2) Heat resistance (temperature stability) evaluation Prepare enzyme extracts for (1R, 3S)-specific esterases and mutant enzymes. Use a thermal cycler to heat the enzyme extracts at 30, 40, 50, 60, 70, 80 or 90°C for 30 minutes. After heating, remove the solid components by centrifugation as enzyme sample solutions. According to the enzyme activity determination method, the enzyme activity is determined by measuring the absorbance of each sample. Assuming the maximum value of each sample is 100%, the change in enzyme activity caused by temperature is determined using relative values, and the 50% inactivation temperature (T50 value) is measured (Table 5). As a result, it can be seen that the maximum heat resistance is improved by 1.72°C.

[0118] [Table 5] T50 value (℃) T50 value (difference from wild type) (℃) (1R,3S) specific esterase 73.16 - S220A 74.34 1.18 A313S / S315M 74.39 1.23 R25P / S220A / A313S / S315M 74.69 1.53 S12P / S220A / A313S / S315M 74.88 1.72 S12P / R25P / S220A / A313S / S315M 73.86 0.7

[0119] (3) Acetonitrile tolerance evaluation Enzyme extracts were prepared for (1R, 3S)-specific esterases and mutant enzymes. Acetonitrile was added to the enzyme extract to a final concentration of 40% and incubated at 30°C for 3 hours. The relative activity value when acetonitrile was added was calculated based on the sample to which ultrapure water was added. The relative activity value of each mutant enzyme was calculated based on the activity value of the (1R, 3S)-specific esterase. The experiments were performed 3 times and the average value was calculated (Table 6). The results confirmed that the tolerance to acetonitrile was significantly improved, especially in the S220A mutant enzyme, the quadruple mutant enzyme, and the quintuple mutant enzyme.

[0120] (4) Methanol tolerance evaluation Enzyme extracts were prepared for (1R, 3S)-specific esterases and mutant enzymes. Methanol was added to the enzyme extract to a final concentration of 40% and incubated at 30°C for 3 hours. The relative activity value when methanol was added was calculated based on the sample to which ultrapure water was added. The relative activity value of each mutant enzyme was calculated based on the activity value of the (1R, 3S)-specific esterase. The experiments were performed three times and the average value was calculated (Table 6). The results confirmed that the tolerance to methanol was significantly improved, especially in the quadruple mutant enzyme (R25P / S220A / A313S / S315M).

[0121] [Table 6]

[0122] 4. Test Example 3 (1) Improved heat resistance of chrysanthemoyl esterase with five mutations It was further investigated whether the five amino acid substitutions that were identified in the above test examples as being capable of imparting heat resistance to esterases could impart heat resistance to other esterases. The amino acid substitution identified this time was introduced into the modified chrysanthemic esterase (having a five-fold mutation (A221F / N222D / F298L / D326V / A328G) relative to the wild-type esterase) prepared according to the existing report (WO2020 / 116331) to confirm whether the heat resistance is improved. The prepared modified enzymes are shown in Table 7.

[0123] [Table 7]

[0124] Regarding the quintuple mutant chrysanthemoyl esterase and mutant enzyme, they were expressed in Escherichia coli, and the enzyme extract was prepared by the method of 1. (3) above. The enzyme extract was heated at 30, 40, 50, 60, 70, 80 or 90°C for 30 minutes using a thermal cycler. After heating, the solid component was removed by centrifugation as an enzyme sample solution. According to the enzyme activity determination method, the absorbance of each sample was measured as the enzyme activity. Assuming the maximum value of each sample is 100%, the change in enzyme activity caused by temperature is measured using a relative value, and the 50% inactivation temperature (T50 value) is calculated. The results are shown in Table 8. As shown in Table 8, a significant improvement in heat resistance can be seen in all mutant enzymes.

[0125] [Table 8]

[0126] (2) Improved tolerance to acetonitrile Regarding the quintuple mutant chrysanthemoyl esterase and mutant enzyme, they were expressed in Escherichia coli, and the enzyme extract was prepared by the method of 1. (3) above. 30% ultrapure water or acetonitrile was added to the enzyme extract and incubated at 30°C for 3 hours. The relative activity value when acetonitrile was added was calculated based on the sample to which ultrapure water was added. The relative activity value of each mutant enzyme was calculated based on the activity value of the quintuple mutant chrysanthemoyl esterase. The experiments were performed 3 times and the average value was calculated. The results are shown in Table 9. As shown in Table 9, a significant improvement in tolerance to acetonitrile was confirmed in all mutant enzymes.

[0127] [Table 9]

[0128] (3) Improved methanol tolerance Regarding the quintuple mutant chrysanthemoyl esterase and mutant enzyme, they were expressed in Escherichia coli, and the enzyme extract was prepared by the method of 1. (3) above. 30% ultrapure water or methanol was added to the enzyme extract and incubated at 30°C for 3 hours. The relative activity value when methanol was added was calculated based on the sample to which ultrapure water was added. The relative activity value of each mutant enzyme was calculated based on the activity value of the quintuple mutant chrysanthemoyl esterase. The experiments were performed 3 times and the average value was calculated. The results are shown in Table 10. As shown in Table 10, a significant improvement in methanol tolerance was confirmed in some mutant enzymes.

[0129] [Table 10] Industrial Applicability

[0130] By using the modified esterase of the present invention, chrysanthemic acid, which is an important intermediate of pesticides, can be efficiently produced. Therefore, the present invention is extremely useful in the field of pesticide production, for example.

[0131] This application is based on Japanese Patent Application No. 2022-132082 (filing date: August 22, 2022), the contents of which are incorporated herein in their entirety.

Claims

1. A modified esterase, which is any one of the following modified esterases (i) to (iii): (i) a modified esterase comprising an amino acid sequence in which at least one amino acid at a position selected from the group consisting of the following (1) to (5) in the amino acid sequence of SEQ ID NO: 1, 7 or 35 is substituted with another amino acid, (1)S12 (2)R25 (3)S220 (4)A313 (5)S315； (ii) a modified esterase, wherein one or more amino acids are further substituted, added, inserted or deleted in the modified esterase of (i), and the temperature stability and / or tolerance to organic solvents thereof are further improved compared to these properties of the esterase consisting of the amino acid sequence of SEQ ID NOs: 1, 7 or 35, wherein the substitution does not include the amino acid site substituted in (i), and the deletion does not include the amino acid site substituted in (i); (iii) a modified esterase, wherein in the modified esterase of (i), amino acids other than the amino acids substituted in (i) are further substituted by other amino acids, the modified esterase having more than 70% identity with the modified esterase of (i), and having further improved temperature stability and / or tolerance to organic solvents compared to these properties of the esterase composed of the amino acid sequence of sequence number 1, 7 or 35.

2. The modified esterase according to claim 1, wherein in (i), the amino acid substitution is at least one selected from the group consisting of: (1)S12P (2)R25P (3)S220A (4) A313S (5)S315M.

3. A nucleic acid encoding the modified esterase according to claim 1 or 2. An expression cassette or a recombinant vector comprising the nucleic acid according to claim 3.

5. A transformant obtained by transformation using the expression cassette or recombinant vector according to claim 4. 6 . A method for producing a modified esterase, comprising the step of culturing the transformant according to claim 5 .

7. An enzyme agent comprising the modified esterase according to claim 1 or 2. The enzyme agent according to claim 7, which is used for producing chrysanthemic acid. 9 . A method for producing chrysanthemic acid, comprising the step of allowing the modified esterase according to claim 1 or 2 to act on ethyl chrysanthemate.

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