WO2024043203A1 - Modified esterase - Google Patents

Modified esterase Download PDF

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WO2024043203A1
WO2024043203A1 PCT/JP2023/029951 JP2023029951W WO2024043203A1 WO 2024043203 A1 WO2024043203 A1 WO 2024043203A1 JP 2023029951 W JP2023029951 W JP 2023029951W WO 2024043203 A1 WO2024043203 A1 WO 2024043203A1
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esterase
amino acid
modified
seq
present
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PCT/JP2023/029951
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French (fr)
Japanese (ja)
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聡 石原
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天野エンザイム株式会社
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids

Definitions

  • the present invention relates to a novel modified esterase. More specifically, the present invention relates to modified esterases that have improved high temperature stability and/or resistance to organic solvents than existing esterases.
  • Chrysanthemum acid is widely used as a synthetic raw material for synthetic pyrethroid insecticides.
  • There are four types of isomers of chrysanthemum ((1R,3R)-chrysanthemum acid, (1R,3S)-chrysanthemum acid, (1S,3S)-chrysanthemum acid and (1S,3R)-chrysanthemum acid).
  • (1R,3R)-chrysanthemum acid has the highest insecticidal activity.
  • chrysanthemum acid is produced using chemical synthesis or an enzymatic reaction using chrysanthemum acid esterase.
  • Patent Document 1 discloses that esterase derived from Arthrobacter globiformis specifically decomposes chrysanthemum acid ethyl ester with 1R, 3R ((+)-trans).
  • Patent Document 2 discloses an example in which an esterase derived from Arthrobacter globiformis was modified into an enzyme that specifically decomposes chrysanthemum acid ethyl ester.
  • esterase derived from Arthrobacter globiformis and esterases derived from other sources specifically describe ethyl chrysanthemum Decomposition of esters is disclosed.
  • Esterase derived from Arthrobacter globiformis has high industrial utility. In the production of chrysanthemum acid using the enzymatic reaction of the esterase, a stable esterase with high resistance to heat, organic solvents, etc. is desired. However, there is room for improvement in the temperature stability and resistance to organic solvents of wild-type esterase derived from Arthrobacter globiformis. Therefore, an object of the present invention is to provide a novel esterase having more preferable high temperature stability and/or resistance to organic solvents.
  • the present inventor attempted to create a modified esterase that is more stable at high temperatures by modifying the amino acid sequence of esterase derived from Arthrobacter globiformis using protein engineering techniques. Through trial and error, the present inventors succeeded in identifying a mutation point (amino acid residue) in the esterase that is effective in improving temperature stability, and furthermore, the present inventor succeeded in identifying a mutation point (amino acid residue) that is effective in improving the resistance to organic solvents. I found that. The present invention was completed by further research based on this knowledge. That is, the present invention is as follows.
  • amino acid sequence of SEQ ID NO: 1, 7, or 35 includes an amino acid sequence in which at least one amino acid at a site selected from the group consisting of (1) to (5) below is substituted with another amino acid.
  • modified esterase (1) S12 (2) R25 (3) S220 (4) A313 (5) S315 (ii)
  • one or more amino acids may be substituted (excluding the amino acid site substituted in (i)), added, inserted, or deleted (however, ( (excluding the amino acid site substituted in i)), and has improved temperature stability and/or resistance to organic solvents than those of the esterase consisting of the amino acid sequence of SEQ ID NO: 1, 7, or 35.
  • modified esterase (iii) In the modified esterase of (i), an amino acid other than the amino acid substituted in (i) is further substituted with another amino acid, wherein the modified esterase Esterases that have 70% or more identity with the modified esterase of (i) and have temperature stability and/or resistance to organic solvents and consist of the amino acid sequence of SEQ ID NO: 1, 7, or 35. A modified esterase that is more improved than the previous one.
  • the amino acid substitution is at least one selected from the group consisting of: (1) S12P (2) R25P (3) S220A (4) A313S (5) S315M.
  • the modified esterase of the present invention has improved temperature stability and resistance to organic solvents than the wild type esterase. Therefore, the modified esterase of the present invention can be used for chrysanthemum acid production at a higher temperature than previously. Furthermore, the modified esterase of the present invention can efficiently produce chrysanthemum acid even under conditions where an organic solvent is present.
  • amino acids are sometimes indicated by one letter.
  • Glycine G Alanine: A Valin: V Leucine: L Isoleucine: I Phenylalanine: F Tyrosine: Y Tryptophan: W Serin: S Threonine: T Cysteine: C Methionine: M Aspartic acid: D Glutamic acid: E Asparagine: N Glutamine: Q Lysine: K Arginine: R Histidine:H Proline: P
  • nonpolar amino acids include alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan.
  • Uncharged amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
  • Acidic amino acids include aspartic acid and glutamic acid.
  • Basic amino acids include lysine, arginine, and histidine.
  • substitution refers not only to artificially introduced substitutions of amino acid residues, but also to cases where amino acid residue substitutions are naturally introduced, that is, when amino acid residues are originally different. This also includes cases where In this specification, substitution of amino acid residues may be artificial or natural substitutions, but artificial substitutions are preferred.
  • mutations due to amino acid substitutions are expressed by a combination of one letter representing the amino acid residue before substitution, a number representing the position of the amino acid residue where the amino acid substitution occurs, and one letter representing the amino acid residue after substitution.
  • alanine at position 328 is replaced with glycine, it is expressed as "A328G”.
  • the symbol "/" is used when representing a combination (combination) of two mutations.
  • a combination of a mutation in which alanine at position 328 is replaced with glycine and a mutation in which alanine at position 221 is replaced by phenylalanine is expressed as "A328G/A221F".
  • a "modified esterase” is an esterase obtained by modifying a standard esterase (hereinafter sometimes referred to as "standard esterase”).
  • the reference esterase is typically an esterase having the amino acid sequence of SEQ ID NO: 1, an esterase having the amino acid sequence of SEQ ID NO: 7, or an esterase having the amino acid sequence of SEQ ID NO: 35. Note that the esterase having the amino acid sequence of SEQ ID NO: 1 is a wild-type esterase derived from Arthrobacter globiformis.
  • esterase having the amino acid sequence of SEQ ID NO: 7 is a double mutant esterase in which two amino acids (A221F/A328G) are substituted in the wild-type esterase derived from Arthrobacter globiformis.
  • esterase having the amino acid sequence of SEQ ID NO: 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 globiformis.
  • nucleic acid includes both deoxyribonucleic acid and ribonucleic acid.
  • a “nucleic acid” can be a deoxyribonucleic acid.
  • the “nucleic acid” may be a ribonucleic acid.
  • the “nucleic acid” may be a chimera of deoxyribonucleic acid and ribonucleic acid.
  • the modified esterase of the present invention is an enzyme that has reactivity to ethyl chrysanthemum and is stable even at high temperatures or in organic solvents. The result is a novel esterase that can efficiently synthesize chrysanthemum acid.
  • the modified esterase of the present invention is a standard esterase in which specific amino acids are substituted, and specifically, it contains the amino acid sequences specified in (i) to (iii) below, or It consists of:
  • the amino acid sequence of SEQ ID NO: 1, 7, or 35 includes an amino acid sequence in which at least one amino acid at a site selected from the group consisting of (1) to (5) below is substituted with another amino acid. , modified esterase; (1) S12 (2) R25 (3) S220 (4) A313 (5) S315
  • one or more amino acids may be substituted (excluding the amino acid site substituted in (i)), added, inserted, or deleted (however, ( (excluding the amino acid site substituted in i)), and has improved temperature stability and/or resistance to organic solvents than those of the esterase consisting of the amino acid sequence of SEQ ID NO: 1, 7, or 35. modified esterase;
  • modified esterase of (i) an amino acid other than the amino acid substituted in (i) is further substituted with another amino acid, wherein the modified esterase Esterases that have 70% or more identity with the modified esterase of (i) and have temperature stability and/or resistance to organic solvents and consist of the amino acid sequence of SEQ ID NO: 1, 7, or 35. A modified esterase that is more improved than the previous one.
  • the modified esterase (i) contains an amino acid sequence in which the original amino acid is substituted with another amino acid at at least one of the above-mentioned positions (1) to (5) in the standard esterase.
  • the number of amino acids substituted is usually 1 or more, preferably 2 or more, and more preferably 3 or more (eg, 3, 4, 5 or more).
  • the type of amino acid after substitution is not particularly limited as long as it achieves the desired effect of the present invention, but conservative substitution may be preferred.
  • Conservative substitutions of amino acids are well known to those skilled in the art.
  • the type of amino acid after substitution can be selected with reference to the above description of ⁇ type of amino acid>, but it is not limited thereto.
  • the number of amino acids to be substituted is 1 to 5, and the substitution site can be 1 to 5 selected from the group consisting of: (1) S12, (2) R25, (3) S220, (4) A313, and (5) S315.
  • the number of amino acids to be substituted is one, and the substitution site is as follows. (1) S12 (2) R25 (3) S220 (4) A313 (5) S315
  • the number of amino acids to be substituted is two, and the substitution site can be as follows. (4) A313, and (5) S315
  • the number of amino acids to be substituted is 4, and the substitution sites can be as follows. (2) R25, (3) S220, (4) A313, and (5) S315 (1) S12, (3) S220, (4) A313, and (5) S315
  • the number of amino acids substituted is 5, and the substitution sites can be as follows. (1) S12, (2) R25, (3) S220, (4) A313, and (5) S315
  • the modified esterase of the present invention comprises or consists of an amino acid sequence having the following amino acid substitutions in SEQ ID NO: 1, 7, or 35.
  • the modified esterase of the present invention may be a fusion of an esterase portion and another polypeptide or protein.
  • the polypeptide or protein to be fused is not particularly limited, but may include, for example, a sequence useful for purification such as multiple histidine residues, or a sequence that improves the stability of the esterase during recombinant production. Polypeptides that can be enhanced are exemplified.
  • organic solvent refers to an organic compound that is liquid at room temperature and pressure and is capable of dissolving other substances.
  • organic solvents include acetaldehyde, acetic acid, acetic anhydride, acetone, acetonitrile, acetophenone, and acetylacetate.
  • the modified esterase (ii) is one in which an amino acid modification has been further introduced into the modified esterase (i) described above.
  • the additional modification site is introduced at a site other than the amino acid substitution site specified in the modified esterase (i).
  • the additional amino acid modifications may be any one of amino acid substitutions, additions, insertions, and deletions (eg, substitution only) or two or more (eg, substitutions and insertions).
  • the number of amino acid modifications that are additionally introduced is not particularly limited as long as the desired effect of the present invention is achieved, but it is usually 1 or more, for example 1 to 80, preferably is 1 to 70 pieces, 1 to 60 pieces, 1 to 50 pieces, 1 to 40 pieces, or 1 to 30 pieces, more preferably 1 to 20 pieces, 1 to 10 pieces, 1 to 8 pieces, 1 The number is 7, 1 to 6, 1 to 5, or 1 to 4, more preferably 1 to 3, particularly preferably 1 or 2.
  • esterase of SEQ ID NO: 1, 7, or 35 has esterases at position 59 (serine residue), position 62 (lysine residue), and position 148 (tyrosine residue). residues) are considered active catalytic residues. Therefore, it may be preferable not to substitute or delete these sites.
  • the (221) position (alanine residue or phenylalanine residue), the (222) position (asparagine residue), the (298) position (phenylalanine residue), and the
  • the amino acids at position (326) (aspartic acid residue) and position (328) (alanine or glycine residue) are considered to be important amino acid residues for optical selectivity to ethyl chrysanthemum. If no change is desired, it is desirable not to introduce substitutions or deletions into these sites.
  • the modified esterase (ii) has the above-mentioned amino acid sequence, and at the same time has the same temperature stability and/or resistance to organic solvents as the reference esterase (i.e., the amino acid sequence of SEQ ID NO: 1, 7, or 35). It is characterized by being improved over those of other esterases). More specifically, the modified esterase (ii) has a T50 value (°C) of 1.0°C or higher relative to the standard esterase (esterase having the amino acid sequence of SEQ ID NO: 1, 7 or 35), preferably The improvement is 1.2°C or more, more preferably 1.5°C or more.
  • the modified esterase (ii) has a stability of 1.1 in an organic solvent (e.g., methanol or acetonitrile) relative to the standard esterase (esterase having the amino acid sequence of SEQ ID NO: 1, 7, or 35). This is an improvement of at least 1.2 times, preferably 1.2 times or more.
  • the modified esterase (ii) has the same temperature stability and/or resistance to organic solvents as the modified esterase (i).
  • the modified esterase (iii) is one in which an amino acid substitution has been further introduced into the modified esterase (i) described above. However, the amino acid substitution site that is additionally introduced is introduced at a site other than the amino acid substitution site specified in the modified esterase (i).
  • sequence identity between the modified esterase (iii) and the modified esterase (i) is not particularly limited as long as it achieves the desired effect, but is usually 70% or more, preferably 75% or more, and 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, particularly preferably 99% or more.
  • sequence identity can be determined by a method known per se.
  • An example is BL2SE of BLASTPACKAGE [sgi32 bit edition, Version 2.0.12; available from National Center for Biotechnology Information (NCBI)] q program (Tatiana A. Tatsusova, Thomas L. Madden, FEMS Microbiol. Lett., Vol. 174, p247-250, 1999).
  • NCBI National Center for Biotechnology Information
  • the parameters can be set to Gap insertion Cost value: 11 and Gap extension Cost value: 1.
  • the modified esterase (iii) is also characterized by improved temperature stability and/or resistance to organic solvents compared to the standard esterase. . That is, the modified esterase (iii) has a T50 value (°C) improved by 1.0°C or more, preferably 1.2°C or more, more preferably 1.5°C or more, compared to the standard esterase. There is. Alternatively, the modified esterase (ii) is 1.1 times more stable in an organic solvent (e.g., methanol or acetonitrile) than the standard esterase (esterase having the amino acid sequence of SEQ ID NO: 1, 7, or 35). The improvement is preferably 1.2 times or more. Alternatively, the modified esterase (iii) has the same temperature stability and/or resistance to organic solvents as the modified esterase (i).
  • an organic solvent e.g., methanol or acetonitrile
  • 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").
  • 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 encodes the modified esterases (i) to (iii) described above.
  • the nucleic acid of the present invention can be prepared by a method known per se.
  • the base sequence shown in SEQ ID NO: 13 is a base sequence encoding a reference esterase consisting of the amino acid sequence shown in SEQ ID NO: 1
  • the base sequence shown in SEQ ID NO: 19 is a base sequence that encodes the reference esterase consisting of the amino acid sequence shown in SEQ ID NO: 7.
  • the base sequence shown in SEQ ID NO: 41 is the base sequence encoding the standard esterase consisting of the amino acid sequence shown in SEQ ID NO: 35. Therefore, the nucleic acid of the present invention can be appropriately designed using SEQ ID NO: 13, 19, or 41 as a reference sequence by a method known per se. Note that codon degeneracy may be taken into consideration depending on the host in which the nucleic acid of the present invention is expressed.
  • examples of the nucleic acids of the present invention are shown in SEQ ID NOs: 14-18, 20-24, 42-46. Note that these are nucleic acid sequences encoding modified esterases created based on the standard esterase of SEQ ID NO: 1, 7, or 35.
  • SEQ ID NO: 14 S220A variant of SEQ ID NO: 1 SEQ ID NO: 15: A313S/S315M variant of SEQ ID NO: 1 SEQ ID NO: 16: R25P/S220A/A313S/S315M variant of SEQ ID NO: 1 SEQ ID NO: 17: S12P of SEQ ID NO: 1 /S220A/A313S/S315M variant SEQ ID NO: 18: S12P/R25P/S220A/A313S/S315M variant of SEQ ID NO: 1 SEQ ID NO: 20: S220A variant of SEQ ID NO: 7 SEQ ID NO: 21: A313S/S315M variant of SEQ ID NO: 7 Body SEQ ID NO: 22: R25P/S220A/A313S/S315M variant of SEQ ID NO: 7 SEQ ID NO: 23: S12P/S220A/A313S/S315M variant of SEQ ID NO
  • the nucleic acid of the present invention is not limited to the nucleic acid sequence itself encoding the modified esterase of the present invention, but has a 75% or more, preferably 80% or more, 85% or more, 90% or more, Nucleic acid sequences having homology of more preferably 95% or more, 96%, 97% or more, 98% or more, particularly preferably 99% or more, have an esterase activity equivalent to that of the modified esterase of the present invention, and a temperature
  • Nucleic acids of the present invention include nucleic acids as long as they encode polypeptides that are stable and/or resistant to organic solvents.
  • the "homology" of nucleic acid sequences is calculated using publicly available or commercially available software with an algorithm that compares a reference sequence with a query sequence. Specifically, BLAST, FASTA, GENETYX (manufactured by Software Development Co., Ltd.), etc. can be used. In determining sequence homology using these, the homology score may be determined using default parameters, or the homology score may be determined using appropriately modified parameters.
  • nucleic acids that hybridize under stringent conditions with a nucleic acid consisting of a nucleic acid sequence complementary to the nucleic acid of the present invention are also polypeptides that have an enzymatic activity equivalent to that of the modified esterase of the present invention. It is included in the nucleic acids of the present invention as long as it encodes.
  • stringent conditions refers to 0.5% SDS, 5x Denhardt's, 0.1% bovine serum albumin (BSA), 0.1% polyvinylpyrrolidone, 0.1% Ficoll.
  • the nylon membrane was coated in 6x SSC for 10 min at room temperature, in 2x SSC containing 0.1% SDS for 10 min at room temperature, and in 0.2x SSC containing 0.1% SDS for 30 min at 45°C. After washing, autoradiography can be performed to detect DNA specifically hybridized with the probe.
  • the modified esterase of the present invention may be a fusion of an esterase portion and another polypeptide or protein. Therefore, the nucleic acid of the present invention can be a base sequence encoding an esterase moiety and other polypeptides or proteins.
  • the method for expressing the nucleic acid of the present invention in a host is not particularly limited, and any method known per se may be used.
  • Expression cassette or recombinant vector also relates to an expression cassette or recombinant vector (hereinafter referred to as "cassette of the present invention", “recombinant vector of the present invention”, “cassette of the present invention or recombinant vector”) containing the nucleic acid of the present invention. vector).
  • the expression cassette or recombinant vector of the present invention can be prepared by linking a promoter and 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. .
  • the expression cassette of the present invention or the recombinant vector of the present invention may contain transcription elements such as an enhancer, CCAAT box, TATA box, SPI site, etc., as necessary, in addition to a promoter and terminator as control elements. good. These control elements only need to be operably linked to the nucleic acid of the invention. Operably linked means that various regulatory factors that regulate the nucleic acid of the present invention and the nucleic acid of the present invention are linked in a state that allows them to operate in a host cell.
  • the vector is preferably an expression vector.
  • the expression vector a vector constructed for genetic recombination from a phage, plasmid, or virus that can autonomously propagate within the host can be suitably used.
  • Such expression vectors are known.
  • commercially available expression vectors include pQE-based vectors (Qiagen Co., Ltd.), pDR540, pRIT2T (GE Healthcare Biosciences Co., Ltd.), and pET-based vectors (Merck Co., Ltd.). ) etc.
  • the expression vector may be used in an appropriate combination with the host cell. For example, when using E.
  • coli as the host cell, a combination of a pET vector and a DH5 ⁇ E. coli strain, a pET vector and a BL21(DE3) E. coli strain, Examples include a combination of strains, or a combination of pDR540 vector and JM109 E. coli strain.
  • Transformants of the present invention also provides transformants transformed using the expression cassette or recombinant vector of the present invention (hereinafter sometimes referred to as "transformants of the present invention").
  • 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) Possible to introduce expression cassettes or recombinant vectors; (2) the expression cassette or recombinant vector is stable; (3) Able to reproduce autonomously, and (4) The traits of the genes in the introduced expression cassette or recombinant vector can be expressed.
  • Such hosts include, for example, bacteria belonging to the genus Escherichia such as Escherichia coli, the genus Bacillus such as Bacillus subtilis, the genus Pseudomonas such as Pseudomonas putida; filamentous fungi, yeast, and the like. Can be mentioned. Further, animal cells, insect cells, plants, etc. may be used.
  • 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 a host.
  • the place where the nucleic acid, etc. of the present invention is introduced is not particularly limited as long as the gene of interest can be expressed, and may be on a plasmid or on the genome.
  • Specific methods for introducing the expression cassette of the present invention or the recombinant vector of the present invention include, for example, the recombinant vector method and the genome editing method. Conditions for introducing the expression cassette or recombinant vector into the host may be appropriately set depending on the type of host and the like.
  • examples include a method using competent cells treated with calcium ions, an electroporation method, and the like.
  • examples include electroporation, spheroplast method, and lithium acetate method.
  • examples include electroporation method, calcium phosphate method, and lipofection method.
  • examples include the calcium phosphate method, lipofection method, and electroporation method.
  • examples include electroporation method, Agrobacterium method, particle gun method, and PEG method.
  • Confirmation of whether the expression cassette of the present invention or the recombinant vector of the present invention has been integrated into a host can be performed by a method known per se such as PCR method, Southern hybridization method, or Northern hybridization method.
  • the expression cassette of the present invention or the recombinant vector of the present invention may be isolated and purified from the transformant.
  • the expression cassette or recombinant vector is isolated and purified based on a lysate obtained by lysing the bacterium.
  • a method for bacteriolysis for example, treatment is performed with a lytic enzyme such as lysozyme, and if necessary, protease, other enzymes, and a surfactant such as sodium lauryl sulfate (SDS) are used in combination.
  • nucleic acids from lysates can be combined. Separation and purification of nucleic acids from lysates can be carried out, for example, by appropriately combining protein removal treatment using phenol treatment and protease treatment, ribonuclease treatment, alcohol precipitation treatment, and commercially available kits.
  • Nucleic acid cleavage can be performed according to conventional methods, for example, using restriction enzyme treatment.
  • restriction enzyme for example, a type II restriction enzyme that acts on a specific nucleotide sequence is used.
  • the binding of the nucleic acid and the expression cassette or expression vector is performed using, for example, DNA ligase.
  • primers specific to the DNA of the present invention are designed and PCR is performed.
  • the amplification product obtained by PCR is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., and stained with ethidium bromide and SYBR Green solution, etc., and the amplification product is detected as a band. You can confirm that it has been converted.
  • amplification products can also be detected by performing PCR using primers that have been labeled in advance with a fluorescent dye or the like.
  • a method may 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, enzyme reaction, or the like.
  • the present invention also provides a method for producing a modified esterase (hereinafter sometimes referred to as "the production method of the present invention"), which includes the step of culturing the transformant of the present invention.
  • 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 used.
  • a solid medium or a liquid medium can be used for culturing the transformant, preferably a liquid medium. Furthermore, when performing industrial production, aerated agitation culture is preferred.
  • the nutrient source for the medium any substance required for the growth of the transformant may be used as appropriate.
  • the carbon source may be any carbon compound that can be assimilated, and examples thereof include glucose, sucrose, lactose, maltose, molasses, and pyruvic acid.
  • the nitrogen source may be any nitrogen compound that can be assimilated, such as peptone, meat extract, yeast extract, casein hydrolyzate, and soybean meal alkaline extract.
  • salts such as phosphates, carbonates, sulfates, magnesium, calcium, potassium, iron, manganese, and zinc, specific amino acids, and specific vitamins may be used as necessary. You may.
  • the culture temperature may be appropriately set within a range where the transformant of the present invention can grow and the transformant produces the modified esterase.
  • the culture temperature is about 15 to 37°C, but is not limited thereto.
  • the timing of terminating the culture may be determined at a time when the modified esterase reaches its maximum yield.
  • the culture time includes, for example, about 12 to 48 hours, but is not limited thereto.
  • the modified esterase expressed using the transformant exists inside the transformant or in the culture medium.
  • the modified esterase may be recovered by a method known per se, one example of the recovery method will be briefly explained below.
  • the transformant when the expressed modified esterase is present inside the transformant, the transformant is separated from the culture supernatant using a method known per se, such as centrifugation.
  • the culture supernatant is removed and the isolated transformants are collected.
  • the isolated transformants are treated using mechanical methods such as ultrasound or French press, or enzymatic methods such as lysozyme, and if necessary, treated with enzymes such as protease or interfaces such as sodium lauryl sulfate (SDS).
  • SDS sodium lauryl sulfate
  • the expressed modified esterase when it is present in the culture solution, it may be subjected to purification treatment as it is, but it may be subjected to purification treatment after concentrating the modified esterase in the water-soluble fraction. You can also serve it. Concentration can be performed, for example, by vacuum concentration, membrane concentration, salting out treatment, fractional precipitation using a hydrophilic organic solvent (eg, methanol, ethanol, and acetone), and the like. Furthermore, the modified esterase can be purified by appropriately combining methods such as gel filtration, adsorption chromatography, ion exchange chromatography, and affinity chromatography. The thus purified modified esterase can be pulverized by freeze-drying, vacuum drying, spray-drying, etc., and distributed on the market, if necessary.
  • a hydrophilic organic solvent eg, methanol, ethanol, and acetone
  • Enzyme agent containing modified esterase The present invention also provides an enzyme agent (hereinafter sometimes referred to as "enzyme agent of the present invention") containing the modified esterase of the present invention.
  • the amount of the modified esterase of the present invention contained in the enzyme preparation of the present invention is usually 0.001 to 100% by weight, preferably 0.01 to 100% by weight, 0.1 to 100% by weight, or 1% by weight. -100% by weight, more preferably 5-100%, 10-100%, 20-100%, 30-100%, 40-100%, or 50-100% by weight. , but not limited to.
  • the enzyme preparation of the present invention may contain only the modified esterase of the present invention, but may contain other components as long as it achieves the desired effect.
  • examples of other components include enzymes other than the modified esterase of the present invention, additives, culture residue produced in the production method of the present invention, and the like.
  • enzymes include, for example, amylase ( ⁇ -amylase, ⁇ -amylase, glucoamylase), glucosidase ( ⁇ -glucosidase, ⁇ -glucosidase), galactosidase ( ⁇ -galactosidase, ⁇ -galactosidase), protease (acidic protease, (protease, alkaline protease), peptidase (leucine peptidase, aminopeptidase), lipase, esterase, cellulase, phosphatase (acid phosphatase, alkaline phosphatase), nuclease, deaminase, oxidase, dehydrogenase, glutaminase, pectinase, catalase, dextranase, trans Examples include glutaminase, protein deamidase, pullulanase, and the like. These other enzymes may
  • additives include excipients, buffers, suspending agents, stabilizers, preservatives, preservatives, physiological saline, and the like.
  • Excipients include starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, white sugar, glycerol, and the like.
  • Buffers include phosphates, citrates, acetates, and the like.
  • Stabilizers include propylene glycol, ascorbic acid, and the like.
  • Preservatives include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like.
  • preservatives include ethanol, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol, and the like. These additives may be contained singly or in combination.
  • Examples of the culture residue include components derived from the medium, contaminant proteins, bacterial body components, and the like.
  • the form of the enzyme preparation of the present invention is not particularly limited, and examples thereof include liquid form, solid form (powder, granules, etc.), and the like.
  • the shape of the enzyme agent may be molded by a method known per se.
  • the modified esterase of the present invention which is an active ingredient of the enzyme preparation of the present invention, converts ethyl chrysanthemum to chrysanthemum acid (preferably, converts (1R,3S)-ethyl chrysanthemum to (1R,3S)-chrysanthete). It has high enzymatic activity in the conversion of 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 chrysanthemum acid (preferably (1R,3S)-chrysanthemum acid).
  • a composition containing chrysanthemum acid can be efficiently produced.
  • the substrate for the enzyme agent of the present invention will be described in detail in the "method for producing chrysanthemum acid" described below.
  • the present invention also provides a method for producing chrysanthemum acid (hereinafter referred to as the "method for producing chrysanthemum acid of the present invention"), which includes a step of causing the modified esterase of the present invention to act on ethyl chrysanthemum acid. ).
  • ethyl chrysanthemum acid serving as a substrate may be purified, or a composition containing other components (hereinafter referred to as ethyl chrysanthemum-containing composition) may be used. ).
  • highly pure (1R,3S)-ethyl chrysanthema for example, purity of 98% or more
  • the content of ethyl chrysanthemum contained in the ethyl chrysanthemum-containing composition is not particularly limited, but may be, for example, 20% or more. From the viewpoint of further increasing the ethyl chrysanthemum content in the product, the ethyl chrysanthemum content in the substrate is preferably 30% or more, more preferably 40% or more, still more preferably 45% or more. The upper limit of the content range is not particularly limited, but may be, for example, 100% or less, preferably 90% or less, and more preferably 70% or less.
  • the origin of the ethyl chrysanthemum-containing composition is not particularly limited, and examples include chemically synthesized compositions.
  • the reaction time, reaction temperature, pH of the reaction solution, solvent, etc. when allowing the modified esterase to act on the substrate are not particularly limited.
  • the reaction time is, for example, 10 minutes to 96 hours, preferably 1 hour to 72 hours, more preferably 12 hours to 48 hours.
  • the reaction temperature is, for example, 10 to 90°C, preferably 20 to 80°C, more preferably 30 to 75°C, even more preferably 40 to 70°C, even more preferably 50 to 65°C.
  • the pH of the reaction solution is, for example, 3 to 12, preferably 6 to 11, more preferably 8 to 11.
  • the solvent during the reaction may or may not contain an organic solvent.
  • Methanol and acetonitrile can be used as the organic solvent.
  • the amount of organic solvent is not particularly limited, but for example, lower limits include 10%, 20%, 30% or more, and 40% or more, and upper limits include 100% or less, 80% or less, 70% or less, and 50% or less. .
  • a chrysanthemum acid-containing composition can be produced very efficiently.
  • One embodiment of the method for producing the chrysanthemum acid-containing composition of the present invention includes the following steps (1) and (2). Note that, after step (2), a step of recovering chrysanthemum acid from the obtained chrysanthemum acid-containing composition may be added.
  • Step of providing an ethyl chrysanthemum acid-containing composition (2) Process of treating the provided ethyl chrysanthemum acid-containing composition with the modified esterase of the present invention
  • the content of chrysanthemum acid in the chrysanthemum acid-containing composition obtained using the method for producing chrysanthemum acid of the present invention is not particularly limited, but is usually 1% by weight or more, preferably 2% by weight or more, and 3% by weight.
  • the content may be 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, particularly preferably 16% by weight or more.
  • a step of optically resolving the reaction product may be added as necessary.
  • Column chromatography can be used for optical resolution.
  • An immobilized modified esterase may be used.
  • the chrysanthemum acid-containing composition is suitably used as an agricultural chemical intermediate.
  • mutant library was created by the following method. (1) Introduction of mutations/transformation PCR primers for introducing mutations (SEQ ID NOs: 25 to 34) were designed, and PCR was performed under the following conditions to introduce mutations.
  • ⁇ PCR reaction composition > (total volume 20 ⁇ L) 5 ⁇ Prime STAR GXL Buffer (Takara Bio): 4 ⁇ L dNTP Mixture (2.5mM each): 1.6 ⁇ L Template (approximately 30 ng/ ⁇ L): 0.25 ⁇ L F-primer: 0.2 ⁇ L R-primer: 0.2 ⁇ L Prime STAR GXL DNA Polymerase (Takara Bio): 0.8 ⁇ L Mix these and adjust to 20 ⁇ L with ultrapure water (Milli Q water).
  • ⁇ 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) Leave at 4°C
  • ⁇ In-fusion reaction> A reaction solution having the following composition was prepared (total volume: 10 ⁇ L) and heated at 50° C. for 15 minutes in a thermal cycler. Purified PCR solution: 2-5 ⁇ L (equivalent to 50-200 ng) Milli Q water: 3-6 ⁇ L 5 ⁇ in-fusion reagent: 2 ⁇ L
  • the substrate (para-nitrophenyl-ethyl chrysanthemum acid: 6 mg) was weighed out and suspended in 4% Triton X-100: 16 mL. The substrate was dissolved by heating to 50°C. After cooling at room temperature, 4 mL of 0.5M PIPES pH 7.0 was added and mixed. 190 ⁇ L of 0.1M PIPES pH 7.0 and 10 ⁇ L of bacteriolysis solution were mixed. 3 ⁇ L of sample + each substrate solution: 197 ⁇ L/well was added. The reaction was carried out at 37°C for 2 hours. Absorbance was measured at 416 nm using a plate reader.
  • Test example 1 Modification of wild-type esterase The following mutations were introduced into wild-type esterase. We investigated closely related esterases through a homology search and selected amino acids with no homology. As a result, mutations were introduced into the following amino acid residues (one letter representing the amino acid residue, number representing the position of the amino acid residue).
  • the enzyme activity of the produced mutant enzyme was measured, and samples that showed enzyme activity were collected. Furthermore, by repeating mutation introduction by PCR, combination mutations of multiple mutation points were created, and improvement in heat resistance was easily evaluated. As a result, the following five types of mutant enzymes had changes in heat resistance and other properties compared to the wild-type enzyme.
  • Enzyme extracts were prepared for wild-type esterase and mutant enzymes. Methanol was added to the enzyme extract to give a final concentration of 40%, and the mixture was incubated at 30°C for 3 hours. The relative activity value when methanol was added was calculated based on a sample in which an equivalent amount of ultrapure water was added instead of methanol. The relative activity value of each mutant enzyme was calculated based on the wild type activity value. All experiments were performed in triplicate and the average values were calculated (Table 3). As a result, it was confirmed that the resistance to methanol was significantly improved, especially in the quintuple mutant enzyme (S12P/R25P/S220A/A313S/S315M).
  • Test example 2 (1) Modification of (1R,3S)-specific esterase (1R,3S)-A221F/A328G double mutation-introduced esterase with high selectivity for ethyl chrysanthemum is unable to be hydrolyzed by wild-type esterase. This is an esterase ((1R,3S)-specific esterase) that is highly selective for (1R,3S)-ethyl chrysanthemum.
  • the mutation of Test Example 1 was further introduced into the above-mentioned (1R,3S)-specific esterase, and it was examined whether there was an effect of improving heat resistance (temperature stability).
  • Enzyme extracts were prepared for (1R, 3S) specific esterases and mutant enzymes. 40% of 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 (1R,3S) specific esterase. All experiments were performed in triplicate and the average values were calculated (Table 6). As a result, it was confirmed that the S220A mutant enzyme, the quadruple mutant enzyme, and the quintuple mutant enzyme exhibited remarkable improvement in resistance to acetonitrile.
  • Enzyme extracts were prepared for (1R, 3S) specific esterases and mutant enzymes. 40% of 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 with ultrapure water added. The relative activity value of each mutant enzyme was calculated based on the activity value of the (1R,3S) specific esterase. All experiments were performed in triplicate and the average values were calculated (Table 6). As a result, it was confirmed that, in particular, the quadruple mutant enzyme (R25P/S220A/A313S/S315M) showed a remarkable improvement in tolerance to methanol.
  • Test example 3 (1) Improving the thermostability of chrysanthemum esterase with quintuple mutations
  • the five amino acid substitutions that can confer thermostability to esterases which were identified through the above test examples, also confer thermostability to other esterases. Further consideration was given to whether it could be granted.
  • the amino acid substitutions identified this time were applied to the modified chrysanthemum esterase (which has five mutations (A221F/N222D/F298L/D326V/A328G) compared to the wild type esterase) produced in a previous report (WO2020/116331). We introduced this technology to see if it improved heat resistance. Table 7 shows the modified enzymes produced.
  • the pentamutated chrysanthemum acid esterase and the mutant enzyme were expressed in E. coli, and the above-mentioned 1.
  • An enzyme extract was prepared by the method (3). The enzyme extract was heated for 30 minutes at a temperature of 30, 40, 50, 60, 70, 80 or 90°C using a thermal cycler. After heating, the solid content was removed by centrifugation to obtain an enzyme sample solution. The absorbance of each sample was measured using the enzyme activity measurement method and determined as the enzyme activity. The maximum value of each sample was set as 100%, and the change in enzyme activity due to temperature was measured using relative values, and the 50% inactivation temperature (T50 value) was measured. The results are shown in Table 8. As shown in Table 8, all mutant enzymes showed a remarkable improvement in heat resistance.
  • the fivefold mutant chrysanthemum acid esterase and the mutant enzyme were expressed in Escherichia coli, and the above-mentioned 1.
  • An enzyme extract was prepared by the method (3). 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 with ultrapure water added. The relative activity value of each mutant enzyme was calculated based on the activity value of the five-fold mutant chrysanthemum acid esterase. All experiments were performed in triplicate and the average values were calculated. The results are shown in Table 10. As shown in Table 10, significant improvement in methanol tolerance was confirmed in some of the mutant enzymes.
  • chrysanthemum acid which is important as an agricultural chemical intermediate, can be efficiently produced. Therefore, the present invention is extremely useful, for example, in the field of agricultural chemical production.

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Abstract

The present invention provides any of modified esterases of (i) to (iii) below.　(i) A modified esterase including an amino acid sequence in which at least one amino acid at a site selected from the group consisting of (1) to (5) below is substituted with another amino acid in an amino acid sequence of SEQ ID NO: 1, 7, or 35: (1) S12, (2) R25, (3) S220, (4) A313, (5) S315. (ii) A modified esterase in which one or more amino acids (provided that the amino acid at the substitution site in (i) is excluded) are further substituted or deleted in the modified esterase of (i), or one or more amino acids are added or inserted into the modified esterase of (i) and in which temperature stability and/or resistance to organic solvents is improved than those of an esterase of an amino acid sequence of SEQ ID NO: 1, 7, or 35. (iii) A modified esterase in which an amino acid other than the amino acid substituted in (i) is further substituted with another amino acid in the modified esterase of (i), wherein the modified esterase has identity of 70% or more with the modified esterase of (i), and temperature stability and/or resistance to organic solvents is improved than those of an esterase of an amino acid sequence of SEQ ID NO: 1, 7, or 35.

Description

改変型エステラーゼmodified esterase
　本発明は、新規の改変型エステラーゼに関する。より具体的には、本発明は、高温安定性及び／又は有機溶媒に対しての耐性が既存のエステラーゼよりも向上した改変型エステラーゼに関する。 The present invention relates to a novel modified esterase. More specifically, the present invention relates to modified esterases that have improved high temperature stability and/or resistance to organic solvents than existing esterases.
　菊酸は合成ピレスロイド系殺虫剤の合成原料として広く利用されている。菊酸には４種類の異性体（（１Ｒ，３Ｒ）－菊酸、（１Ｒ，３Ｓ）－菊酸、（１Ｓ，３Ｓ）－菊酸及び（１Ｓ，３Ｒ）－菊酸）が存在するが、（１Ｒ，３Ｒ）－菊酸の殺虫活性が最も高い。工業的には化学合成や菊酸エステラーゼを用いた酵素反応を利用して、菊酸が製造されている。 Chrysanthemum acid is widely used as a synthetic raw material for synthetic pyrethroid insecticides. There are four types of isomers of chrysanthemum ((1R,3R)-chrysanthemum acid, (1R,3S)-chrysanthemum acid, (1S,3S)-chrysanthemum acid and (1S,3R)-chrysanthemum acid). , (1R,3R)-chrysanthemum acid has the highest insecticidal activity. Industrially, chrysanthemum acid is produced using chemical synthesis or an enzymatic reaction using chrysanthemum acid esterase.
　例えば、特許文献１には、Ａｒｔｈｒｏｂａｃｔｅｒ　ｇｌｏｂｉｆｏｒｍｉｓ由来のエステラーゼが、１Ｒ，３Ｒ（（＋）－ｔｒａｎｓ）特異的に菊酸エチルエステルを分解することが開示されている。特許文献２には、Ａｒｔｈｒｏｂａｃｔｅｒ　ｇｌｏｂｉｆｏｒｍｉｓ由来のエステラーゼを、１Ｒ，３Ｓ（（＋）－ｃｉｓ）特異的に菊酸エチルエステルを分解する酵素に改変させた事例が開示されている。また、非特許文献１には、Ａｒｔｈｒｏｂａｃｔｅｒ　ｇｌｏｂｉｆｏｒｍｉｓ由来エステラーゼやその他の由来のエステラーゼにて１Ｒ，３Ｒ（（＋）－ｔｒａｎｓ）特異的又は１Ｓ，３Ｒ（（－）－ｔｒａｎｓ）特異的に菊酸エチルエステルを分解することが開示されている。 For example, Patent Document 1 discloses that esterase derived from Arthrobacter globiformis specifically decomposes chrysanthemum acid ethyl ester with 1R, 3R ((+)-trans). Patent Document 2 discloses an example in which an esterase derived from Arthrobacter globiformis was modified into an enzyme that specifically decomposes chrysanthemum acid ethyl ester. Furthermore, in Non-Patent Document 1, esterase derived from Arthrobacter globiformis and esterases derived from other sources specifically describe ethyl chrysanthemum Decomposition of esters is disclosed.
特開平５－５６７８７号公報Japanese Patent Application Publication No. 5-56787 国際公開第２０２０／１１６３３１号International Publication No. 2020/116331
　Ａｒｔｈｒｏｂａｃｔｅｒ　ｇｌｏｂｉｆｏｒｍｉｓ由来のエステラーゼは産業上の有用性が高い。当該エステラーゼの酵素反応を利用した菊酸の製造では、熱や有機溶媒などに対する耐性が高い、安定なエステラーゼが望まれる。しかし、Ａｒｔｈｒｏｂａｃｔｅｒ　ｇｌｏｂｉｆｏｒｍｉｓ由来の野生型エステラーゼの温度安定性や有機溶媒への耐性には改善の余地がある。そこで本発明は、より好ましい高温安定性及び／又は有機溶媒への耐性を有する新規エステラーゼを提供することをその課題とする。 Esterase derived from Arthrobacter globiformis has high industrial utility. In the production of chrysanthemum acid using the enzymatic reaction of the esterase, a stable esterase with high resistance to heat, organic solvents, etc. is desired. However, there is room for improvement in the temperature stability and resistance to organic solvents of wild-type esterase derived from Arthrobacter globiformis. Therefore, an object of the present invention is to provide a novel esterase having more preferable high temperature stability and/or resistance to organic solvents.
　本発明者は、Ａｒｔｈｒｏｂａｃｔｅｒ　ｇｌｏｂｉｆｏｒｍｉｓ由来のエステラーゼのアミノ酸配列を、蛋白質工学的手法を用いて改変することで、高温でより安定な改変体エステラーゼの創作を試みた。試行錯誤の末、本発明者は、当該エステラーゼにおいて温度安定性の向上に有効な変異点（アミノ酸残基）を特定することに成功し、更に当該変異点が有機溶媒に対する耐性の向上にも有効であることを見出した。かかる知見に基づいてさらに研究を進めることによって本発明を完成するに至った。すなわち、本発明は以下の通りである。 The present inventor attempted to create a modified esterase that is more stable at high temperatures by modifying the amino acid sequence of esterase derived from Arthrobacter globiformis using protein engineering techniques. Through trial and error, the present inventors succeeded in identifying a mutation point (amino acid residue) in the esterase that is effective in improving temperature stability, and furthermore, the present inventor succeeded in identifying a mutation point (amino acid residue) that is effective in improving the resistance to organic solvents. I found that. The present invention was completed by further research based on this knowledge. That is, the present invention is as follows.
［１］
　以下の（ｉ）～（ｉｉｉ）のいずれかの改変型エステラーゼ：
　（ｉ）配列番号１、７又は３５のアミノ酸配列において、以下の（１）～（５）からなる群から選択される部位の少なくとも一つのアミノ酸が、別のアミノ酸で置換されたアミノ酸配列を含む、改変型エステラーゼ、
（１）Ｓ１２
（２）Ｒ２５
（３）Ｓ２２０
（４）Ａ３１３
（５）Ｓ３１５
　（ｉｉ）（ｉ）の改変型エステラーゼにおいて、さらに１個又は数個のアミノ酸が、置換（ただし、（ｉ）において置換されたアミノ酸部位を除く）、付加、挿入、又は欠失（ただし、（ｉ）において置換されたアミノ酸部位を除く）されており、且つ、温度安定性及び／又は有機溶媒に対しての耐性が、配列番号１、７又は３５のアミノ酸配列からなるエステラーゼのそれらよりも向上している、改変型エステラーゼ、
　（ｉｉｉ）（ｉ）の改変型エステラーゼにおいて、（ｉ）において置換されたアミノ酸以外のアミノ酸が、さらに別のアミノ酸に置換されている改変型エステラーゼであって、ここで、該改変型エステラーゼが、（ｉ）の改変型エステラーゼと７０％以上の同一性を有し、且つ、温度安定性及び／又は有機溶媒に対しての耐性が、配列番号１、７又は３５のアミノ酸配列からなるエステラーゼのそれらよりも向上している、改変型エステラーゼ。
［２］
　前記（ｉ）において、アミノ酸の置換が以下からなる群から選択される少なくとも一つである、請求項１記載の改変型エステラーゼ：
（１）Ｓ１２Ｐ
（２）Ｒ２５Ｐ
（３）Ｓ２２０Ａ
（４）Ａ３１３Ｓ
（５）Ｓ３１５Ｍ。
［３］
　［１］又は［２］記載の改変型エステラーゼをコードする核酸。
［４］
　［３］記載の核酸を含む、発現カセット又は組換えベクター。
［５］
　［４］記載の発現カセット又は組換えベクターを用いて形質転換された形質転換体。
［６］
　［５］記載の形質転換体を培養する工程を含む、改変型エステラーゼの製造方法。
［７］
　［１］又は［２］記載の改変型エステラーゼを含む、酵素剤。
［８］
　菊酸生成用である、［７］記載の酵素剤。
［９］
　菊酸エチルに、［１］又は［２］記載の改変型エステラーゼを作用させる工程を含む、菊酸の製造方法。 [1]
Any of the following modified esterases (i) to (iii):
(i) The amino acid sequence of SEQ ID NO: 1, 7, or 35 includes an amino acid sequence in which at least one amino acid at a site selected from the group consisting of (1) to (5) below is substituted with another amino acid. , modified esterase,
(1) S12
(2) R25
(3) S220
(4) A313
(5) S315
(ii) In the modified esterase of (i), one or more amino acids may be substituted (excluding the amino acid site substituted in (i)), added, inserted, or deleted (however, ( (excluding the amino acid site substituted in i)), and has improved temperature stability and/or resistance to organic solvents than those of the esterase consisting of the amino acid sequence of SEQ ID NO: 1, 7, or 35. modified esterase,
(iii) In the modified esterase of (i), an amino acid other than the amino acid substituted in (i) is further substituted with another amino acid, wherein the modified esterase Esterases that have 70% or more identity with the modified esterase of (i) and have temperature stability and/or resistance to organic solvents and consist of the amino acid sequence of SEQ ID NO: 1, 7, or 35. A modified esterase that is more improved than the previous one.
[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 [1] or [2].
[4]
[3] An expression cassette or recombinant vector comprising the nucleic acid described above.
[5]
[4] A transformant transformed using the expression cassette or recombinant vector described above.
[6]
[5] 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 according to [1] or [2].
[8]
The enzyme preparation according to [7], which is for producing chrysanthemum acid.
[9]
A method for producing chrysanthemum acid, comprising a step of causing the modified esterase described in [1] or [2] to act on ethyl chrysanthemum acid.
　本発明の改変型エステラーゼは、温度安定性及び有機溶媒への耐性が野生型エステラーゼよりも向上している。従って、本発明の改変型エステラーゼは、これまでよりも高い温度帯での菊酸製造に使用することができる。また、本発明の改変型エステラーゼは、有機溶媒が存在する条件下においても効率よく菊酸を製造することができる。 The modified esterase of the present invention has improved temperature stability and resistance to organic solvents than the wild type esterase. Therefore, the modified esterase of the present invention can be used for chrysanthemum acid production at a higher temperature than previously. Furthermore, the modified esterase of the present invention can efficiently produce chrysanthemum acid even under conditions where an organic solvent is present.
　本発明の説明に使用する用語の一部について以下に説明する。 Some of the terms used to describe the present invention will be explained below.
＜アミノ酸＞
　本明細書では、慣例に従い、アミノ酸を１文字で示す場合がある。具体的には以下のとおりである：
グリシン：Ｇ
アラニン：Ａ
バリン：Ｖ
ロイシン：Ｌ
イソロイシン：Ｉ
フェニルアラニン：Ｆ
チロシン：Ｙ
トリプトファン：Ｗ
セリン：Ｓ
スレオニン：Ｔ
システイン：Ｃ
メチオニン：Ｍ
アスパラギン酸：Ｄ
グルタミン酸：Ｅ
アスパラギン：Ｎ
グルタミン：Ｑ
リジン：Ｋ
アルギニン：Ｒ
ヒスチジン：Ｈ
プロリン：Ｐ <Amino acids>
In this specification, according to convention, amino acids are sometimes indicated by one letter. Specifically:
Glycine: G
Alanine: A
Valin: V
Leucine: L
Isoleucine: I
Phenylalanine: F
Tyrosine: Y
Tryptophan: W
Serin: S
Threonine: T
Cysteine: C
Methionine: M
Aspartic acid: D
Glutamic acid: E
Asparagine: N
Glutamine: Q
Lysine: K
Arginine: R
Histidine:H
Proline: P
＜アミノ酸配列＞
　本明細書において、表示するアミノ酸配列は、左端がＮ末端、右端がＣ末端である。 <Amino acid sequence>
In the present specification, the left end of the amino acid sequences shown is the N-terminus, and the right end is the C-terminus.
＜アミノ酸の種類＞
　本明細書において、「非極性アミノ酸」には、アラニン、バリン、ロイシン、イソロイシン、プロリン、メチオニン、フェニルアラニン、及びトリプトファンが含まれる。「非電荷アミノ酸」には、グリシン、セリン、スレオニン、システイン、チロシン、アスパラギン、及びグルタミンが含まれる。「酸性アミノ酸」には、アスパラギン酸及びグルタミン酸が含まれる。「塩基性アミノ酸」には、リジン、アルギニン、及びヒスチジンが含まれる。 <Types of amino acids>
As used herein, "nonpolar amino acids" include alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan. "Uncharged amino acids" include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. "Acidic amino acids" include aspartic acid and glutamic acid. "Basic amino acids" include lysine, arginine, and histidine.
＜置換＞
　本明細書において、「置換」とは、人為的にアミノ酸残基の置換を導入した場合のみならず、自然にアミノ酸残基の置換が導入された場合、すなわち、もともとアミノ酸残基が相違していた場合も含まれる。本明細書においては、アミノ酸残基の置換は、人為的な置換であってもよく、自然な置換であってもよいが、人為的な置換が好ましい。 <Replacement>
In this specification, "substitution" refers not only to artificially introduced substitutions of amino acid residues, but also to cases where amino acid residue substitutions are naturally introduced, that is, when amino acid residues are originally different. This also includes cases where In this specification, substitution of amino acid residues may be artificial or natural substitutions, but artificial substitutions are preferred.
　本明細書では、アミノ酸置換による変異を、置換前のアミノ酸残基を表す１文字、アミノ酸置換が生じるアミノ酸残基の位置を表す数字、及び置換後のアミノ酸残基を表す１文字の組合せで表現することとする。例えば、３２８位のアラニンがグリシンに置換される場合「Ａ３２８Ｇ」と表現される。また、二つの変異の組合せ（併用）を表す場合、記号「／」を用いる。例えば、３２８位のアラニンがグリシンに置換される変異と、２２１位のアラニンがフェニルアラニンに置換される変異の組合せであれば、「Ａ３２８Ｇ／Ａ２２１Ｆ」と表現される。 In this specification, mutations due to amino acid substitutions are expressed by a combination of one letter representing the amino acid residue before substitution, a number representing the position of the amino acid residue where the amino acid substitution occurs, and one letter representing the amino acid residue after substitution. I decided to. For example, when alanine at position 328 is replaced with glycine, it is expressed as "A328G". Furthermore, when representing a combination (combination) of two mutations, the symbol "/" is used. For example, a combination of a mutation in which alanine at position 328 is replaced with glycine and a mutation in which alanine at position 221 is replaced by phenylalanine is expressed as "A328G/A221F".
＜改変型エステラーゼ＞
　本明細書において「改変型エステラーゼ」とは、基準となるエステラーゼ（以下、「基準エステラーゼ」と称することがある）を改変して得られるエステラーゼである。本明細書において、基準エステラーゼは、典型的には、配列番号１のアミノ酸配列を有するエステラーゼか、配列番号７のアミノ酸配列を有するエステラーゼか、配列番号３５のアミノ酸配列を有するエステラーゼである。尚、配列番号１のアミノ酸配列を有するエステラーゼは、Ａｒｔｈｒｏｂａｃｔｅｒ　ｇｌｏｂｉｆｏｒｍｉｓ由来の野生型エステラーゼである。また、配列番号７のアミノ酸配列を有するエステラーゼは、Ａｒｔｈｒｏｂａｃｔｅｒ　ｇｌｏｂｉｆｏｒｍｉｓ由来の野生型のエステラーゼにおいて、２カ所のアミノ酸（Ａ２２１Ｆ／Ａ３２８Ｇ）が置換された２重変異体エステラーゼである。また、配列番号３５のアミノ酸配列を有するエステラーゼは、Ａｒｔｈｒｏｂａｃｔｅｒ　ｇｌｏｂｉｆｏｒｍｉｓ由来の野生型のエステラーゼにおいて、５カ所のアミノ酸（Ａ２２１Ｆ／Ｎ２２２Ｄ／Ｆ２９８Ｌ／Ｄ３２６Ｖ／Ａ３２８Ｇ）が置換された５重変異体エステラーゼである。 <Modified esterase>
As used herein, a "modified esterase" is an esterase obtained by modifying a standard esterase (hereinafter sometimes referred to as "standard esterase"). As used herein, the reference esterase is typically an esterase having the amino acid sequence of SEQ ID NO: 1, an esterase having the amino acid sequence of SEQ ID NO: 7, or an esterase having the amino acid sequence of SEQ ID NO: 35. Note that the esterase having the amino acid sequence of SEQ ID NO: 1 is a wild-type esterase derived from Arthrobacter globiformis. Furthermore, the esterase having the amino acid sequence of SEQ ID NO: 7 is a double mutant esterase in which two amino acids (A221F/A328G) are substituted in the wild-type esterase derived from Arthrobacter globiformis. Furthermore, the esterase having the amino acid sequence of SEQ ID NO: 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 globiformis.
＜核酸＞
　本明細書において「核酸」は、デオキシリボ核酸とリボ核酸の両方を包含する。一態様において、「核酸」は、デオキシリボ核酸であり得る。また、別の一態様において、「核酸」は、リボ核酸であり得る。また、別の一態様において、「核酸」は、デオキシリボ核酸とリボ核酸のキメラであってもよい。 <Nucleic acid>
As used herein, "nucleic acid" includes both deoxyribonucleic acid and ribonucleic acid. In one embodiment, a "nucleic acid" can be a deoxyribonucleic acid. In another embodiment, the "nucleic acid" may be a ribonucleic acid. In another embodiment, the "nucleic acid" may be a chimera of deoxyribonucleic acid and ribonucleic acid.
１．改変型エステラーゼ
　本発明の改変型エステラーゼは、菊酸エチルへの反応性を有する酵素であり、高温又は有機溶媒中においても安定である。その結果、効率よく菊酸を合成できる新規のエステラーゼである。 1. Modified esterase The modified esterase of the present invention is an enzyme that has reactivity to ethyl chrysanthemum and is stable even at high temperatures or in organic solvents. The result is a novel esterase that can efficiently synthesize chrysanthemum acid.
　本発明の改変型エステラーゼは、基準エステラーゼにおいて特定のアミノ酸を置換したものであり、具体的には、以下の（ｉ）～（ｉｉｉ）で特定されるアミノ酸配列を含むか、或いは、当該アミノ酸配列からなるものである： The modified esterase of the present invention is a standard esterase in which specific amino acids are substituted, and specifically, it contains the amino acid sequences specified in (i) to (iii) below, or It consists of:
　（ｉ）配列番号１、７又は３５のアミノ酸配列において、以下の（１）～（５）からなる群から選択される部位の少なくとも一つのアミノ酸が、別のアミノ酸で置換されたアミノ酸配列を含む、改変型エステラーゼ；
（１）Ｓ１２
（２）Ｒ２５
（３）Ｓ２２０
（４）Ａ３１３
（５）Ｓ３１５ (i) The amino acid sequence of SEQ ID NO: 1, 7, or 35 includes an amino acid sequence in which at least one amino acid at a site selected from the group consisting of (1) to (5) below is substituted with another amino acid. , modified esterase;
(1) S12
(2) R25
(3) S220
(4) A313
(5) S315
　（ｉｉ）（ｉ）の改変型エステラーゼにおいて、さらに１個又は数個のアミノ酸が、置換（ただし、（ｉ）において置換されたアミノ酸部位を除く）、付加、挿入、又は欠失（ただし、（ｉ）において置換されたアミノ酸部位を除く）されており、且つ、温度安定性及び／又は有機溶媒に対しての耐性が、配列番号１、７又は３５のアミノ酸配列からなるエステラーゼのそれらよりも向上している、改変型エステラーゼ； (ii) In the modified esterase of (i), one or more amino acids may be substituted (excluding the amino acid site substituted in (i)), added, inserted, or deleted (however, ( (excluding the amino acid site substituted in i)), and has improved temperature stability and/or resistance to organic solvents than those of the esterase consisting of the amino acid sequence of SEQ ID NO: 1, 7, or 35. modified esterase;
　（ｉｉｉ）（ｉ）の改変型エステラーゼにおいて、（ｉ）において置換されたアミノ酸以外のアミノ酸が、さらに別のアミノ酸に置換されている改変型エステラーゼであって、ここで、該改変型エステラーゼが、（ｉ）の改変型エステラーゼと７０％以上の同一性を有し、且つ、温度安定性及び／又は有機溶媒に対しての耐性が、配列番号１、７又は３５のアミノ酸配列からなるエステラーゼのそれらよりも向上している、改変型エステラーゼ。 (iii) In the modified esterase of (i), an amino acid other than the amino acid substituted in (i) is further substituted with another amino acid, wherein the modified esterase Esterases that have 70% or more identity with the modified esterase of (i) and have temperature stability and/or resistance to organic solvents and consist of the amino acid sequence of SEQ ID NO: 1, 7, or 35. A modified esterase that is more improved than the previous one.
　まず、（ｉ）の改変型エステラーゼについて説明する。
　（ｉ）の改変型エステラーゼは、基準エステラーゼにおいて、上述の（１）～（５）の少なくとも１カ所で、元のアミノ酸が別のアミノ酸に置換されたアミノ酸配列を含む。本発明のエステラーゼにおいて、置換されるアミノ酸の数は、通常１個以上、好ましくは、２個以上、さらに好ましくは３個以上（例えば、３個、４個、５個以上）であり得る。 First, the modified esterase (i) will be explained.
The modified esterase (i) contains an amino acid sequence in which the original amino acid is substituted with another amino acid at at least one of the above-mentioned positions (1) to (5) in the standard esterase. In the esterase of the present invention, the number of amino acids substituted is usually 1 or more, preferably 2 or more, and more preferably 3 or more (eg, 3, 4, 5 or more).
　元のアミノ酸の別のアミノ酸への置換に関し、置換後のアミノ酸の種類は、本発明の所望の効果を奏する限り特に限定されないが、保存的置換が好ましい場合がある。アミノ酸の保存的置換は当業者には周知技術である。一例としては、上述した＜アミノ酸の種類＞の記載を参考にして置換後のアミノ酸の種類を選択することができるが、これに限定されるものではない。 Regarding the substitution of the original amino acid with another amino acid, the type of amino acid after substitution is not particularly limited as long as it achieves the desired effect of the present invention, but conservative substitution may be preferred. Conservative substitutions of amino acids are well known to those skilled in the art. As an example, the type of amino acid after substitution can be selected with reference to the above description of <type of amino acid>, but it is not limited thereto.
　一態様において、置換されるアミノ酸は１～５個であり、置換部位は以下からなる群より選択される１～５個であり得る：
　（１）Ｓ１２、（２）Ｒ２５、（３）Ｓ２２０、（４）Ａ３１３、及び（５）Ｓ３１５。 In one embodiment, the number of amino acids to be substituted is 1 to 5, and the substitution site can be 1 to 5 selected from the group consisting of:
(1) S12, (2) R25, (3) S220, (4) A313, and (5) S315.
　別の一態様において、置換されるアミノ酸の数は１個であり、置換部位は以下である。
（１）Ｓ１２
（２）Ｒ２５
（３）Ｓ２２０
（４）Ａ３１３
（５）Ｓ３１５ In another embodiment, the number of amino acids to be substituted is one, and the substitution site is as follows.
(1) S12
(2) R25
(3) S220
(4) A313
(5) S315
　別の一態様において、置換されるアミノ酸の数は２個であり、置換部位は以下であり得る。
　（４）Ａ３１３、及び（５）Ｓ３１５ In another aspect, the number of amino acids to be substituted is two, and the substitution site can be as follows.
(4) A313, and (5) S315
　別の一態様において、置換されるアミノ酸の数は４個であり、置換部位は以下であり得る。
　（２）Ｒ２５、（３）Ｓ２２０、（４）Ａ３１３、及び（５）Ｓ３１５
　（１）Ｓ１２、（３）Ｓ２２０、（４）Ａ３１３、及び（５）Ｓ３１５ In another embodiment, the number of amino acids to be substituted is 4, and the substitution sites can be as follows.
(2) R25, (3) S220, (4) A313, and (5) S315
(1) S12, (3) S220, (4) A313, and (5) S315
　別の一態様において、置換されるアミノ酸の数は５個であり、置換部位は以下であり得る。
　（１）Ｓ１２、（２）Ｒ２５、（３）Ｓ２２０、（４）Ａ３１３、及び（５）Ｓ３１５ In another aspect, the number of amino acids substituted is 5, and the substitution sites can be as follows.
(1) S12, (2) R25, (3) S220, (4) A313, and (5) S315
　好ましい一態様において、本発明の改変型エステラーゼは、配列番号１、７又は３５において、以下のアミノ酸置換を有するアミノ酸配列を含むか、当該アミノ酸配列からなる。 In a preferred embodiment, the modified esterase of the present invention comprises or consists of an amino acid sequence having the following amino acid substitutions in SEQ ID NO: 1, 7, or 35.
＜基準エステラーゼにおいて１アミノ酸置換を有する改変型エステラーゼ＞
（Ｅ－１）Ｓ１２Ｐ
（Ｅ－２）Ｒ２５Ｐ
（Ｅ－３）Ｓ２２０Ａ
（Ｅ－４）Ａ３１３Ｓ
（Ｅ－５）Ｓ３１５Ｍ <Modified esterase having one amino acid substitution in standard esterase>
(E-1) S12P
(E-2) R25P
(E-3) S220A
(E-4) A313S
(E-5)S315M
＜基準エステラーゼにおいて２アミノ酸置換を有する改変型エステラーゼ＞
（Ｅ－６）Ａ３１３Ｓ／Ｓ３１５Ｍ <Modified esterase having two amino acid substitutions in standard esterase>
(E-6) A313S/S315M
＜基準エステラーゼにおいて４アミノ酸置換を有する改変型エステラーゼ＞
（Ｅ－７）Ｒ２５Ｐ／Ｓ２２０Ａ／Ａ３１３Ｓ／Ｓ３１５Ｍ
（Ｅ－８）Ｓ１２Ｐ／Ｓ２２０Ａ／Ａ３１３Ｓ／Ｓ３１５Ｍ <Modified esterase having 4 amino acid substitutions in standard esterase>
(E-7) R25P/S220A/A313S/S315M
(E-8) S12P/S220A/A313S/S315M
＜基準エステラーゼにおいて５アミノ酸置換を有する改変型エステラーゼ＞
（Ｅ－９）Ｓ１２Ｐ／Ｒ２５Ｐ／Ｓ２２０Ａ／Ａ３１３Ｓ／Ｓ３１５Ｍ <Modified esterase having 5 amino acid substitutions in standard esterase>
(E-9)S12P/R25P/S220A/A313S/S315M
　一態様において、本発明の改変型エステラーゼは、エステラーゼ部分と他のポリペプチドやタンパク質とが融合したものであってもよい。本発明の改変型エステラーゼにおいて、融合されるポリペプチドやタンパク質としては、特に限定されないが、例えば、多重ヒスチジン残基のような精製に役立つ配列や、組換え生産の際に、エステラーゼの安定性を高め得るポリペプチドが例示される。 In one embodiment, the modified esterase of the present invention may be a fusion of an esterase portion and another polypeptide or protein. In the modified esterase of the present invention, the polypeptide or protein to be fused is not particularly limited, but may include, for example, a sequence useful for purification such as multiple histidine residues, or a sequence that improves the stability of the esterase during recombinant production. Polypeptides that can be enhanced are exemplified.
　尚、本明細書において、「有機溶媒」とは、常温常圧において液体であり、他の物質を溶解させ得る有機化合物を意味する。有機溶媒としては、例えば、アセトアルデヒド（Ａｃｅｔａｌｄｅｈｙｄｅ）、酢酸（Ａｃｅｔｉｃ　Ａｃｉｄ）、無水酢酸（Ａｃｅｔｉｃ　Ａｎｈｙｄｒｉｄｅ）、アセトン（Ａｃｅｔｏｎｅ）、アセトニトリル（Ａｃｅｔｏｎｉｔｒｉｌｅ）、アセトフェノン（Ａｃｅｔｏｐｈｅｎｏｎｅ）、アセチルアセトン（Ａｃｅｔｙｌａｃｅｔｏｎｅ）、アリルアルコール（Ａｌｌｙｌ　Ａｌｃｏｈｏｌ）、エタノールアミン（Ｅｔｈａｎｏｌａｍｉｎｅ）、アニリン（Ａｎｉｌｉｎｅ）、ベンズアルデヒド（Ｂｅｎｚａｌｄｅｈｙｄｅ）、ベンゼン（Ｂｅｎｚｅｎｅ）、ベンジルアルコール（Ｂｅｎｚｙｌ　Ａｌｃｏｈｏｌ）、安息香酸ベンジル（Ｂｅｎｚｙｌ　Ｂｅｎｚｏａｔｅ）、１－ブタノール（１－Ｂｕｔａｎｏｌ）、２－ブタノール（２－Ｂｕｔａｎｏｌ）、メチルエチルケトン（Ｍｅｔｈｙｌ　Ｅｔｈｙｌ　Ｋｅｔｏｎｅ）、酢酸ブチル（Ｂｕｔｙｌ　Ａｃｅｔａｔｅ）、２－メチル－２－プロパノール（２－Ｍｅｔｈｙｌ　－２－ｐｒｏｐａｎｏｌ）、ジブチルエーテル（Ｄｉｂｕｔｙｌ　ｅｔｈｅｒ）、二硫化炭素（Ｃａｒｂｏｎ　Ｄｉｓｕｌｆｉｄｅ）、クロロホルム（Ｃｈｌｏｒｏｆｏｒｍ）、エピクロロヒドリン（Ｅｐｉｃｈｌｏｒｏｈｙｄｒｉｎ）、ｏ－クレゾール（ｏ－Ｃｒｅｓｏｌ）、ｍ－クレゾール（ｍ－Ｃｒｅｓｏｌ）、ｐ－クレゾール（ｐ－Ｃｒｅｓｏｌ）、シクロヘキサン（Ｃｙｃｌｏｈｅｘａｎｅ）、シクロヘキサノール（Ｃｙｃｌｏｈｅｘａｎｏｌ）、１，２－ジクロロエタン（１，２－Ｄｉｃｈｌｏｒｏｅｔｈａｎｅ）、ジクロロメタン（Ｄｉｃｈｌｏｒｏｍｅｔｈａｎｅ）、炭酸ジエチル（Ｄｉｅｔｈｙｌ　Ｃａｒｂｏｎａｔｅ）、ジエチレングリコール（Ｄｉｅｔｈｙｌｅｎｅ　Ｇｌｙｃｏｌ）、ジエチレングリコールモノブチルエーテル（Ｄｉｅｔｈｙｌｅｎｅ　Ｇｌｙｃｏｌ　Ｍｏｎｏｂｕｔｙｌ　Ｅｔｈｅｒ）、ブチルカルビトールアセテート（Ｂｕｔｙｌ　ｃａｒｂｉｔｏｌ　ａｃｅｔａｔｅ）、ジエチレングリコールモノエチルエーテル（Ｄｉｅｔｈｙｌｅｎｅ　Ｇｌｙｃｏｌ　Ｍｏｎｏｅｔｈｙｌ　Ｅｔｈｅｒ）、ジエチレングリコールモノエチルエーテルアセテート（Ｄｉｅｔｈｙｌｅｎｅ　Ｇｌｙｃｏｌ　Ｍｏｎｏｅｔｈｙｌ　Ｅｔｈｅｒ　Ａｃｅｔａｔｅ）、ジエチレングリコールモノメチルエーテル（Ｄｉｅｔｈｙｌｅｎｅ　Ｇｌｙｃｏｌ　Ｍｏｎｏｍｅｔｈｙｌ　Ｅｔｈｅｒ）、ジエチルエーテル（Ｄｉｅｔｈｙｌ　Ｅｔｈｅｒ）、ジメチルアセトアミド（Ｄｉｍｅｔｈｙｌａｃｅｔａｍｉｄｅ）、Ｎ、Ｎ－ジメチルホルムアミド（Ｎ，Ｎ－Ｄｉｍｅｔｈｙｌｆｏｒｍａｍｉｄｅ）、ジメチルスルホキシド（Ｄｉｍｅｔｈｙｌ　Ｓｕｌｆｏｘｉｄｅ）、１，４－ジオキサン（１，４－Ｄｉｏｘａｎｅ）、エタノール（Ｅｔｈａｎｏｌ）、無水エタノール（Ａｂｓｏｌｕｔｅ　Ｅｔｈａｎｏｌ）、酢酸エチル（Ｅｔｈｙｌ　Ａｃｅｔａｔｅ）、安息香酸エチル（Ｅｔｈｙｌ　Ｂｅｎｚｏａｔｅ）、２－クロロエタノール（２－Ｃｈｌｏｒｏｅｔｈａｎｏｌ）、エチレングリコール（Ｅｔｈｙｌｅｎｅ　Ｇｌｙｃｏｌ）、１，２－ジメトキシエタン（１，２－Ｄｉｍｅｔｈｏｘｙｅｔｈａｎｅ）、２－ブトキシエタノール（２－Ｂｕｔｏｘｙｅｔｈａｎｏｌ）、エチレングリコールモノブチルエーテルアセテート（Ｅｔｈｙｌｅｎｅ　Ｇｌｙｃｏｌ　Ｍｏｎｏｂｕｔｙｌ　Ｅｔｈｅｒ　Ａｃｅｔａｔｅ）、２－エトキシエタノール（２－Ｅｔｈｏｘｙｅｔｈａｎｏｌ）、エチレングリコールモノエチルエーテルアセテート（Ｅｔｈｙｌｅｎｅ　Ｇｌｙｃｏｌ　Ｍｏｎｏｅｔｈｙｌ　Ｅｔｈｅｒ　Ａｃｅｔａｔｅ）、エチレングリコールモノメチルエーテルアセテート（Ｅｔｈｙｌｅｎｅ　Ｇｌｙｃｏｌ　Ｍｏｎｏｍｅｔｈｙｌ　Ｅｔｈｅｒ　Ａｃｅｔａｔｅ）、ギ酸エチル（Ｅｔｈｙｌ　Ｆｏｒｍａｔｅ）、２－エチルヘキサノール（２－Ｅｔｈｙｌｈｅｘａｎｏｌ）、アセト酢酸エチル（Ｅｔｈｙｌ　Ａｃｅｔｏａｃｅｔａｔｅ）、３－オキソブタン酸エチル（Ｅｔｈｙｌ－３－Ｏｘｏｂｕｔａｎａｔｅ）、プロピオン酸エチル（Ｅｔｈｙｌ　Ｐｒｏｐｉｏｎａｔｅ）、ホルムアミド（Ｆｏｒｍａｍｉｄｅ）、ギ酸（Ｆｏｒｍｉｃ　Ａｃｉｄ）、フルフリルアルコール（Ｆｕｒｆｕｒｙｌ　Ａｌｃｏｈｏｌ）、グリセロール（Ｇｌｙｃｅｒｏｌ）、ヘプタン（Ｈｅｐｔａｎｅ）、ヘキサン（Ｈｅｘａｎｅ）、１－ヘキサノール（１－Ｈｅｘａｎｏｌ）、リグロイン（Ｌｉｇｒｏｉｎ）、２，６－ルチジン（２，６－Ｌｕｔｉｄｉｎｅ）、メタノール（Ｍｅｔｈａｎｏｌ）、２－メトキシエタノール（２－Ｍｅｔｈｏｘｙｅｔｈａｎｏｌ）、酢酸メチル（Ｍｅｔｈｙｌ　Ａｃｅｔａｔｅ）、２－メチル－２－ブタノール（２－Ｍｅｔｈｙｌ－２－ｂｕｔａｎｏｌ）、３－メチル－１－ブタノール（３－Ｍｅｔｈｙｌ－１－ｂｕｔａｎｏｌ）、酢酸イソアミル（Ｉｓｏａｍｙｌ　Ａｃｅｔａｔｅ）、プロピオン酸メチル（Ｍｅｔｈｙｌ　Ｐｒｏｐｉｏｎａｔｅ）、トリエタノールアミン（Ｔｒｉｅｔｈａｎｏｌａｍｉｎｅ）、ニトロベンゼン（Ｎｉｔｒｏｂｅｎｚｅｎｅ）、ニトロメタン（Ｎｉｔｒｏｍｅｔｈａｎｅ）、ｎ－オクタン（ｎ－Ｏｃｔａｎｅ）、１－オクタノール（１－Ｏｃｔａｎｏｌ）、２－オクタノール（２－Ｏｃｔａｎｏｌ）、ペンタン（Ｐｅｎｔａｎｅ）、１－ペンタノール（１－Ｐｅｎｔａｎｏｌ）、３－ペンタノール（３－Ｐｅｎｔａｎｏｌ）、酢酸ｎ－ペンチル（ｎ－Ｐｅｎｔｙｌ　Ａｃｅｔａｔｅ）、石油ベンジン（Ｐｅｔｒｏｌｅｕｍ　Ｂｅｎｚｉｎｅ）、フェノール（Ｐｈｅｎｏｌ）、１－プロパノール（１－Ｐｒｏｐａｎｏｌ）、酢酸ノルマルプロピル（ｎ－Ｐｒｏｐｙｌ　Ａｃｅｔａｔｅ）、プロピレングリコール（Ｐｒｏｐｙｌｅｎｅ　Ｇｌｙｃｏｌ）、酸化プロピレン（Ｐｒｏｐｙｌｅｎｅ　Ｏｘｉｄｅ）、メチルオキシラン（Ｍｅｔｈｙｌｏｘｉｒａｎｅ）、１，２－エポキシプロパン（１，２－Ｅｐｏｘｙｐｒｏｐａｎｅ）、ｎ－プロピルエーテル（ｎ－Ｐｒｏｐｙｌ　Ｅｔｈｅｒ）、ピリジン（Ｐｙｒｉｄｉｎｅ）、１，１，２，２－テトラクロロエタン（１，１，２，２－Ｔｅｔｒａｃｈｌｏｒｏｅｔｈａｎｅ）、テトラクロロエチレン（Ｔｅｔｒａｃｈｌｏｒｏｅｔｈｙｌｅｎｅ）、テトラヒドロフラン（Ｔｅｔｒａｈｙｄｒｏｆｕｒａｎ）、テトラリン（Ｔｅｔｒａｌｉｎ）、トルエン（Ｔｏｌｕｅｎｅ）、１，１，２－トリクロロエタン（１，１，２－Ｔｒｉｃｈｌｏｒｏｅｔｈａｎｅ）、１，１，２－ＴＣＡ、トリクロロエチレン（Ｔｒｉｃｈｌｏｒｏｅｔｈｙｌｅｎｅ）、トリエチルアミン（Ｔｒｉｅｔｈｙｌａｍｉｎｅ）、ＴＥＡ、トリフルオロ酢酸（Ｔｒｉｆｌｕｏｒｏａｃｅｔｉｃ　Ａｃｉｄ）、ＴＦＡ、キシレン（Ｘｙｌｅｎｅ）、ｏ－キシレン（ｏ－Ｘｙｌｅｎｅ）、ｍ－キシレン（ｍ－Ｘｙｌｅｎｅ）、又は、ｐ－キシレン（ｐ－Ｘｙｌｅｎｅ）等が挙げられるが、これらに限定されない。好ましい一態様において、有機溶媒は、アセトニトリル及びメタノールであり得る。 In this specification, the term "organic solvent" refers to an organic compound that is liquid at room temperature and pressure and is capable of dissolving other substances. Examples of organic solvents include acetaldehyde, acetic acid, acetic anhydride, acetone, acetonitrile, acetophenone, and acetylacetate. Acetylacetone, Allyl Alcohol ), Ethanolamine, Aniline, Benzaldehyde, Benzene, Benzyl Alcohol, Benzyl Benzoate, 1 -Butanol (1-Butanol), 2-Butanol (2-Butanol), Methyl Ethyl Ketone, Butyl Acetate, 2-Methyl-2-propanol, Dibutyl ether, Disulfide Carbon Disulfide ), Chloroform, Epichlorohydrin, o-Cresol, m-Cresol, p-Cresol, Cyclohexane, Cyclohexanol ( Cyclohexanol), 1,2-Dichloroethane, Dichloromethane, Diethyl Carbonate, Diethylene Glycol, Diethylene Glycol Monobutylene Diethylene Glycol Monobutyl Ether, Butyl Carbitol Acetate carbitol acetate), Diethylene Glycol Monoethyl Ether, Diethylene Glycol Monoethyl Ether Acetate), Diethylene Glycol Monomethyl Ether, Diethyl Ether, Dimethylacetamide , N, N -N -dimethylholmamide (N, N -DimethylFormime), dimethyl sulfoxide, 1,4 -dioxan (1,4 -dioxane), ethanol (Ethanol), absolute ethanol (ABSO). LUTE ETHANOL), ethyl acetate ( Ethyl Acetate, Ethyl Benzoate, 2-Chloroethanol, Ethylene Glycol, 1,2-Dimethoxyethane, 2- Butoxyethanol (2- Ethylene Glycol Monobutyl Ether Acetate, 2-Ethoxyethanol (2-Ethoxyethanol), Ethylene Glycol Monobutyl Ether Acetate Glycol Monoethyl Ether Acetate), Ethylene Glycol Monomethyl Ether Ethyl Formate, 2-Ethylhexanol, Ethyl Acetate, Ethyl-3-Oxobutanate, Ethyl Propionate Ethyl Propionate), Formamide (Formamide), Formic Acid, Furfuryl Alcohol, Glycerol, Heptane, Hexane, 1-Hexanol, Ligroin ,2,6 -Lutidine (2,6-Lutidine), Methanol (Methanol), 2-Methoxyethanol (2-Methoxyethanol), Methyl Acetate (Methyl Acetate), 2-Methyl-2-butanol (2-Methyl-2-butanol), 3 -Methyl-1-butanol (3-Methyl-1-butanol), Isoamyl Acetate, Methyl Propionate, Triethanolamine, Nitrobenzene, Nitromethane, n- Octane (n-Octane), 1-Octanol (1-Octanol), 2-Octanol (2-Octanol), Pentane (Pentane), 1-Pentanol (1-Pentanol), 3-Pentanol (3-Pentanol), n-Pentyl Acetate, Petroleum Benzine, Phenol, 1-Propanol, n-Propyl Acetate, Propylene Glycol Glycol), oxidation Propylene Oxide, Methyloxirane, 1,2-Epoxypropane, n-Propyl Ether, Pyridine, 1,1,2,2- Tetrachloroethane (1,1,2,2-Tetrachloroethane), Tetrachloroethylene, Tetrahydrofuran, Tetralin, Toluene, 1,1,2-Trichloroethane (1,1,2) -Trichloroethane) , 1,1,2 -TCA, Trichloroethylene, Triethylamine, TEA, TrifluoroCetic ACID, TFA, XYLENE, XYLENE, O -xylene, M -Xylene (M) Examples include, but are not limited to, -Xylene), p-xylene (p-Xylene), and the like. In one preferred embodiment, the organic solvent may be acetonitrile and methanol.
　次に、（ｉｉ）の改変型エステラーゼについて説明する。
　（ｉｉ）の改変型エステラーゼは、上述した（ｉ）の改変型エステラーゼに対して、さらにアミノ酸の改変が導入されたものである。但し、追加で導入される改変の部位は、（ｉ）の改変型エステラーゼにおいて特定されるアミノ酸置換部位以外の部位に導入される。追加で導入されるアミノ酸の改変は、アミノ酸の置換、付加、挿入、及び欠失のいずれか１種（例えば、置換のみ）又は２種以上（例えば、置換と挿入）であってよい。（ｉｉ）の改変型エステラーゼにおいて、追加で導入されるアミノ酸の改変の数は、本発明の所望の効果を奏する限り特に限定されないが、通常１個以上であり、例えば、１～８０個、好ましくは、１～７０個、１～６０個、１～５０個、１～４０個、又は１～３０個であり、より好ましくは、１～２０個、１～１０個、１～８個、１～７個、１～６個、１～５個、又は１～４個であり、更に好ましくは、１～３個、特に好ましくは１又は２個である。 Next, the modified esterase (ii) will be explained.
The modified esterase (ii) is one in which an amino acid modification has been further introduced into the modified esterase (i) described above. However, the additional modification site is introduced at a site other than the amino acid substitution site specified in the modified esterase (i). The additional amino acid modifications may be any one of amino acid substitutions, additions, insertions, and deletions (eg, substitution only) or two or more (eg, substitutions and insertions). In the modified esterase (ii), the number of amino acid modifications that are additionally introduced is not particularly limited as long as the desired effect of the present invention is achieved, but it is usually 1 or more, for example 1 to 80, preferably is 1 to 70 pieces, 1 to 60 pieces, 1 to 50 pieces, 1 to 40 pieces, or 1 to 30 pieces, more preferably 1 to 20 pieces, 1 to 10 pieces, 1 to 8 pieces, 1 The number is 7, 1 to 6, 1 to 5, or 1 to 4, more preferably 1 to 3, particularly preferably 1 or 2.
　尚、理論に拘束されることを望むものではないが、配列番号１、７又は３５のエステラーゼは、第５９位（セリン残基）、第６２位（リジン残基）及び第１４８位（チロシン酸残基）が、活性触媒残基と考えられている。従って、これらの部位を置換又は欠失させないことが好ましい場合があり得る。より好ましくは、配列番号１、７又は３５における第（２２１）位（アラニン残基又はフェニルアラニン残基）、第（２２２）位（アスパラギン残基）、第（２９８）位（フェニルアラニン残基）、第（３２６）位（アスパラギン酸残基）、第（３２８）位（アラニン又はグリシン残基）のアミノ酸は、菊酸エチルに対する光学選択性に重要なアミノ酸残基と考えられることから、光学選択性を変えたくない場合はこれらの部位には置換又は欠失を導入しないことが望ましい。 Although not wishing to be bound by theory, the esterase of SEQ ID NO: 1, 7, or 35 has esterases at position 59 (serine residue), position 62 (lysine residue), and position 148 (tyrosine residue). residues) are considered active catalytic residues. Therefore, it may be preferable not to substitute or delete these sites. More preferably, the (221) position (alanine residue or phenylalanine residue), the (222) position (asparagine residue), the (298) position (phenylalanine residue), and the The amino acids at position (326) (aspartic acid residue) and position (328) (alanine or glycine residue) are considered to be important amino acid residues for optical selectivity to ethyl chrysanthemum. If no change is desired, it is desirable not to introduce substitutions or deletions into these sites.
　また、（ｉｉ）の改変型エステラーゼは、上述のアミノ酸配列を有すると同時に、温度安定性及び／又は有機溶媒に対しての耐性が、基準エステラーゼ（即ち、配列番号１、７又は３５のアミノ酸配列からなるエステラーゼ）のそれらよりも向上していることを特徴とする。より具体的には、（ｉｉ）の改変型エステラーゼは、基準エステラーゼ（配列番号１、７又は３５のアミノ酸配列を有するエステラーゼ）に対してＴ５０値（℃）が１．０℃以上、好ましくは、１．２℃以上、より好ましくは、１．５℃以上向上している。又は、（ｉｉ）の改変型エステラーゼは、基準エステラーゼ（配列番号１、７又は３５のアミノ酸配列を有するエステラーゼ）に対して、有機溶媒（例、メタノール又はアセトニトリル等）中の安定性が１．１倍以上、好ましくは１．２倍以上、向上している。或いは、（ｉｉ）の改変型エステラーゼは、（ｉ）の改変型エステラーゼと同等の温度安定性及び／又は有機溶媒に対しての耐性を有する。 In addition, the modified esterase (ii) has the above-mentioned amino acid sequence, and at the same time has the same temperature stability and/or resistance to organic solvents as the reference esterase (i.e., the amino acid sequence of SEQ ID NO: 1, 7, or 35). It is characterized by being improved over those of other esterases). More specifically, the modified esterase (ii) has a T50 value (°C) of 1.0°C or higher relative to the standard esterase (esterase having the amino acid sequence of SEQ ID NO: 1, 7 or 35), preferably The improvement is 1.2°C or more, more preferably 1.5°C or more. Alternatively, the modified esterase (ii) has a stability of 1.1 in an organic solvent (e.g., methanol or acetonitrile) relative to the standard esterase (esterase having the amino acid sequence of SEQ ID NO: 1, 7, or 35). This is an improvement of at least 1.2 times, preferably 1.2 times or more. Alternatively, the modified esterase (ii) has the same temperature stability and/or resistance to organic solvents as the modified esterase (i).
　次に、（ｉｉｉ）の改変型エステラーゼについて説明する。
　（ｉｉｉ）の改変型エステラーゼは、上述した（ｉ）の改変型エステラーゼに対して、さらにアミノ酸の置換が導入されたものである。但し、追加で導入されるアミノ酸の置換の部位は、（ｉ）の改変型エステラーゼにおいて特定されるアミノ酸置換部位以外の部位に導入される。 Next, the modified esterase (iii) will be explained.
The modified esterase (iii) is one in which an amino acid substitution has been further introduced into the modified esterase (i) described above. However, the amino acid substitution site that is additionally introduced is introduced at a site other than the amino acid substitution site specified in the modified esterase (i).
　（ｉｉｉ）の改変型エステラーゼと（ｉ）の改変型エステラーゼの間の配列同一性は、所望の効果を奏する限り特に限定されないが、通常７０％以上であり、好ましくは７５％以上、８０％以上、又は８５％以上であり、より好ましくは９０％以上、９５％以上、９６％以上、９７％以上、又は９８％以上であり、特に好ましくは９９％以上である。 The sequence identity between the modified esterase (iii) and the modified esterase (i) is not particularly limited as long as it achieves the desired effect, but is usually 70% or more, preferably 75% or more, and 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, particularly preferably 99% or more.
　尚、配列同一性は自体公知の方法により特定することができる。一例としては、ＢＬＡＳＴＰＡＣＫＡＧＥ［ｓｇｉ３２ ｂｉｔ ｅｄｉｔｉｏｎ，Ｖｅｒｓｉｏｎ ２．０．１２；ａｖａｉｌａｂｌｅ ｆｒｏｍ Ｎａｔｉｏｎａｌ Ｃｅｎｔｅｒ ｆｏｒ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ Ｉｎｆｏｒｍａｔｉｏｎ（ＮＣＢＩ）］のｂｌ２ｓｅｑ ｐｒｏｇｒａｍ（Ｔａｔｉａｎａ Ａ．Ｔａｔｓｕｓｏｖａ，Ｔｈｏｍａｓ Ｌ．Ｍａｄｄｅｎ，ＦＥＭＳ Ｍｉｃｒｏｂｉｏｌ．Ｌｅｔｔ．，Ｖｏｌ．１７４，ｐ２４７－２５０，１９９９）により得ることができる。本方法を採用する場合、パラメータは、Ｇａｐ ｉｎｓｅｒｔｉｏｎ Ｃｏｓｔ ｖａｌｕｅ：１１、Ｇａｐ ｅｘｔｅｎｓｉｏｎ Ｃｏｓｔ ｖａｌｕｅ：１に設定することができる。 Incidentally, sequence identity can be determined by a method known per se. An example is BL2SE of BLASTPACKAGE [sgi32 bit edition, Version 2.0.12; available from National Center for Biotechnology Information (NCBI)] q program (Tatiana A. Tatsusova, Thomas L. Madden, FEMS Microbiol. Lett., Vol. 174, p247-250, 1999). When this method is adopted, the parameters can be set to Gap insertion Cost value: 11 and Gap extension Cost value: 1.
　（ｉｉｉ）の改変型エステラーゼも、（ｉｉ）の改変型エステラーゼと同様に、温度安定性及び／又は有機溶媒に対しての耐性が、基準エステラーゼのそれらよりも向上していることを特徴とする。即ち、（ｉｉｉ）の改変型エステラーゼは、基準エステラーゼに対してＴ５０値（℃）が１．０℃以上、好ましくは、１．２℃以上、より好ましくは、１．５℃以上、向上している。又は、（ｉｉ）の改変型エステラーゼは、基準エステラーゼ（配列番号１、７又は３５のアミノ酸配列を有するエステラーゼ）に対して、有機溶媒（例えば、メタノール又はアセトニトリル）中の安定性が１．１倍以上、好ましくは１．２倍以上、向上している。或いは、（ｉｉｉ）の改変型エステラーゼは、（ｉ）の改変型エステラーゼと同等の温度安定性及び／又は有機溶媒に対しての耐性を有する。 Similar to the modified esterase (ii), the modified esterase (iii) is also characterized by improved temperature stability and/or resistance to organic solvents compared to the standard esterase. . That is, the modified esterase (iii) has a T50 value (°C) improved by 1.0°C or more, preferably 1.2°C or more, more preferably 1.5°C or more, compared to the standard esterase. There is. Alternatively, the modified esterase (ii) is 1.1 times more stable in an organic solvent (e.g., methanol or acetonitrile) than the standard esterase (esterase having the amino acid sequence of SEQ ID NO: 1, 7, or 35). The improvement is preferably 1.2 times or more. Alternatively, the modified esterase (iii) has the same temperature stability and/or resistance to organic solvents as the modified esterase (i).
２．改変型エステラーゼをコードする核酸
　本発明はまた、本発明の改変型エステラーゼをコードする核酸（以下、「本発明の核酸」と称することがある）を提供する。 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").
　本発明の核酸は、本発明の改変型エステラーゼをコードする核酸である。本発明の核酸は、上記で述べた（ｉ）～（ｉｉｉ）の改変型エステラーゼをコードする核酸であれば特に限定されない。 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 encodes the modified esterases (i) to (iii) described above.
　本発明の核酸は自体公知の方法により調製することができる。一例としては、配列番号１３に示される塩基配列は、配列番号１に示すアミノ酸配列からなる基準エステラーゼをコードする塩基配列であり、配列番号１９に示される塩基配列は、配列番号７に示すアミノ酸配列からなる基準エステラーゼをコードする塩基配列であり、配列番号４１に示される塩基配列は、配列番号３５に示すアミノ酸配列からなる基準エステラーゼをコードする塩基配列である。従って、本発明の核酸は、自体公知の方法により、配列番号１３、１９又は４１を基準の配列として適宜設計することができる。尚、本発明の核酸を発現させる宿主に応じて、コドン縮重が考慮され得る。 The nucleic acid of the present invention can be prepared by a method known per se. As an example, the base sequence shown in SEQ ID NO: 13 is a base sequence encoding a reference esterase consisting of the amino acid sequence shown in SEQ ID NO: 1, and the base sequence shown in SEQ ID NO: 19 is a base sequence that encodes the reference esterase consisting of the amino acid sequence shown in SEQ ID NO: 7. The base sequence shown in SEQ ID NO: 41 is the base sequence encoding the standard esterase consisting of the amino acid sequence shown in SEQ ID NO: 35. Therefore, the nucleic acid of the present invention can be appropriately designed using SEQ ID NO: 13, 19, or 41 as a reference sequence by a method known per se. Note that codon degeneracy may be taken into consideration depending on the host in which the nucleic acid of the present invention is expressed.
　一態様において、本発明の核酸の一例を、配列番号１４～１８、２０～２４、４２～４６に示す。尚、これらは配列番号１、７又は３５の基準エステラーゼに基づいて作成された改変型エステラーゼをコードする核酸配列である。 In one embodiment, examples of the nucleic acids of the present invention are shown in SEQ ID NOs: 14-18, 20-24, 42-46. Note that these are nucleic acid sequences encoding modified esterases created based on the standard esterase of SEQ ID NO: 1, 7, or 35.
配列番号１４：配列番号１のＳ２２０Ａ変異体
配列番号１５：配列番号１のＡ３１３Ｓ／Ｓ３１５Ｍ変異体
配列番号１６：配列番号１のＲ２５Ｐ／Ｓ２２０Ａ／Ａ３１３Ｓ／Ｓ３１５Ｍ変異体
配列番号１７：配列番号１のＳ１２Ｐ／Ｓ２２０Ａ／Ａ３１３Ｓ／Ｓ３１５Ｍ変異体
配列番号１８：配列番号１のＳ１２Ｐ／Ｒ２５Ｐ／Ｓ２２０Ａ／Ａ３１３Ｓ／Ｓ３１５Ｍ変異体
配列番号２０：配列番号７のＳ２２０Ａ変異体
配列番号２１：配列番号７のＡ３１３Ｓ／Ｓ３１５Ｍ変異体
配列番号２２：配列番号７のＲ２５Ｐ／Ｓ２２０Ａ／Ａ３１３Ｓ／Ｓ３１５Ｍ変異体
配列番号２３：配列番号７のＳ１２Ｐ／Ｓ２２０Ａ／Ａ３１３Ｓ／Ｓ３１５Ｍ変異体
配列番号２４：配列番号７のＳ１２Ｐ／Ｒ２５Ｐ／Ｓ２２０Ａ／Ａ３１３Ｓ／Ｓ３１５Ｍ変異体
配列番号４２：配列番号３５のＳ２２０Ａ変異体
配列番号４３：配列番号３５のＡ３１３Ｓ／Ｓ３１５Ｍ変異体
配列番号４４：配列番号３５のＲ２５Ｐ／Ｓ２２０Ａ／Ａ３１３Ｓ／Ｓ３１５Ｍ変異体
配列番号４５：配列番号３５のＳ１２Ｐ／Ｓ２２０Ａ／Ａ３１３Ｓ／Ｓ３１５Ｍ変異体
配列番号４６：配列番号３５のＳ１２Ｐ／Ｒ２５Ｐ／Ｓ２２０Ａ／Ａ３１３Ｓ／Ｓ３１５Ｍ変異体 SEQ ID NO: 14: S220A variant of SEQ ID NO: 1 SEQ ID NO: 15: A313S/S315M variant of SEQ ID NO: 1 SEQ ID NO: 16: R25P/S220A/A313S/S315M variant of SEQ ID NO: 1 SEQ ID NO: 17: S12P of SEQ ID NO: 1 /S220A/A313S/S315M variant SEQ ID NO: 18: S12P/R25P/S220A/A313S/S315M variant of SEQ ID NO: 1 SEQ ID NO: 20: S220A variant of SEQ ID NO: 7 SEQ ID NO: 21: A313S/S315M variant of SEQ ID NO: 7 Body SEQ ID NO: 22: R25P/S220A/A313S/S315M variant of SEQ ID NO: 7 SEQ ID NO: 23: S12P/S220A/A313S/S315M variant of SEQ ID NO: 7 SEQ ID NO: 24: S12P/R25P/S220A/A313S of SEQ ID NO: 7 /S315M variant SEQ ID NO: 42: S220A variant of SEQ ID NO: 35 SEQ ID NO: 43: A313S/S315M variant of SEQ ID NO: 35 SEQ ID NO: 44: R25P/S220A/A313S/S315M variant of SEQ ID NO: 35 SEQ ID NO: 45: Sequence S12P/S220A/A313S/S315M variant of number 35 SEQ ID NO: 46: S12P/R25P/S220A/A313S/S315M variant of SEQ ID NO: 35
　尚、本発明の核酸は、本発明の改変型エステラーゼをコードする核酸配列そのものに限定されるものではなく、当該核酸配列と７５％以上、好ましくは８０％以上、８５％以上、９０％以上、より好ましくは９５％以上、９６％、９７％以上、９８％以上、特に好ましくは９９％以上の相同性を有する核酸配列も、それが本発明の改変型エステラーゼと同等のエステラーゼ活性、並びに、温度安定性及び／又は有機溶媒に対しての耐性を有するポリペプチドをコードする限り、本発明の核酸に含まれる。 The nucleic acid of the present invention is not limited to the nucleic acid sequence itself encoding the modified esterase of the present invention, but has a 75% or more, preferably 80% or more, 85% or more, 90% or more, Nucleic acid sequences having homology of more preferably 95% or more, 96%, 97% or more, 98% or more, particularly preferably 99% or more, have an esterase activity equivalent to that of the modified esterase of the present invention, and a temperature Nucleic acids of the present invention include nucleic acids as long as they encode polypeptides that are stable and/or resistant to organic solvents.
　ここで、核酸配列の「相同性」は、基準配列を照会配列として比較するアルゴリズムをもった公開又は市販されているソフトウェアを用いて計算される。具体的には、ＢＬＡＳＴ、ＦＡＳＴＡ、又はＧＥＮＥＴＹＸ（ソフトウエア開発株式会社製）等を用いることができる。これらを用いた配列相同性の決定においては、デフォルトパラメータを用いて相同性スコアを決定してもよく、適宜改変されたパラメータを用いて相同性スコアを決定してもよい。 Here, the "homology" of nucleic acid sequences is calculated using publicly available or commercially available software with an algorithm that compares a reference sequence with a query sequence. Specifically, BLAST, FASTA, GENETYX (manufactured by Software Development Co., Ltd.), etc. can be used. In determining sequence homology using these, the homology score may be determined using default parameters, or the homology score may be determined using appropriately modified parameters.
　また、本発明の核酸に対して相補的な核酸配列からなる核酸とストリンジェントな条件下でハイブリダイズする核酸も、それらが本発明の改変型エステラーゼが有する酵素活性と同等の活性を有するポリペプチドをコードする限り、本発明の核酸に含まれる。ここで、「ストリンジェントな条件下」とは、０．５％ＳＤＳ、５×デンハルト〔Ｄｅｎｈａｒｄｔ’ｓ、０．１％ウシ血清アルブミン（ＢＳＡ）、０．１％ポリビニルピロリドン、０．１％フィコール４００〕および１００μｇ／ｍｌサケ精子ＤＮＡを含む６×ＳＳＣ（１×ＳＳＣは、０．１５Ｍ　ＮａＣｌ、０．０１５Ｍクエン酸ナトリウム、ｐＨ７．０）中で、５０℃～６５℃で４時間～一晩保温する条件をいう。ストリンジェントな条件下でのハイブリダイゼーションは、具体的には、以下の手法によって行われる。即ち、ＤＮＡライブラリ又はｃＤＮＡライブラリを固定化したナイロン膜を作成し、６×ＳＳＣ、０．５％ＳＤＳ、５×デンハルツ、１００μｇ／ｍｌサケ精子ＤＮＡを含むプレハイブリダイゼーション溶液中、６５℃でナイロン膜をブロッキングする。その後、３２Ｐでラベルした各プローブを加えて、６５℃で一晩保温する。このナイロン膜を６×ＳＳＣ中、室温で１０分間、０．１％ＳＤＳを含む２×ＳＳＣ中、室温で１０分間、０．１％ＳＤＳを含む０．２×ＳＳＣ中、４５℃で３０分間洗浄した後、オートラジオグラフィーをとり、プローブと特異的にハイブリダイズしているＤＮＡを検出することができる。 Furthermore, nucleic acids that hybridize under stringent conditions with a nucleic acid consisting of a nucleic acid sequence complementary to the nucleic acid of the present invention are also polypeptides that have an enzymatic activity equivalent to that of the modified esterase of the present invention. It is included in the nucleic acids of the present invention as long as it encodes. Here, "stringent conditions" refers to 0.5% SDS, 5x Denhardt's, 0.1% bovine serum albumin (BSA), 0.1% polyvinylpyrrolidone, 0.1% Ficoll. 400] and 100 μg/ml salmon sperm DNA in 6× SSC (1× SSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) at 50°C to 65°C for 4 hours to overnight. Conditions for keeping warm. Specifically, hybridization under stringent conditions is performed by the following method. That is, a nylon membrane with a DNA library or cDNA library immobilized thereon was created, and the nylon membrane was heated at 65°C in a prehybridization solution containing 6x SSC, 0.5% SDS, 5x Denharz, and 100 μg/ml salmon sperm DNA. Blocking. Thereafter, each probe labeled with 32P is added and kept at 65°C overnight. The nylon membrane was coated in 6x SSC for 10 min at room temperature, in 2x SSC containing 0.1% SDS for 10 min at room temperature, and in 0.2x SSC containing 0.1% SDS for 30 min at 45°C. After washing, autoradiography can be performed to detect DNA specifically hybridized with the probe.
　一態様において、上述した通り、本発明の改変型エステラーゼは、エステラーゼ部分と他のポリペプチドやタンパク質とが融合したものであり得る。従って、本発明の核酸は、エステラーゼ部分と他のポリペプチドやタンパク質をコードする塩基配列であり得る。 In one embodiment, as described above, the modified esterase of the present invention may be a fusion of an esterase portion and another polypeptide or protein. Therefore, the nucleic acid of the present invention can be a base sequence encoding an esterase moiety and other polypeptides or proteins.
　本発明の核酸を宿主内で発現させる方法は、特に限定されず、自体公知の方法を用いればよい。 The method for expressing the nucleic acid of the present invention in a host is not particularly limited, and any method known per se may be used.
３．発現カセット又は組換えベクター
　本発明はまた、本発明の核酸を含む発現カセット又は組換えベクター（以下、「本発明のカセット」、「本発明の組換えベクター」、「本発明のカセット又は組換えベクター」と称することがある）を提供する。 3. Expression cassette or recombinant vector The present invention also relates to an expression cassette or recombinant vector (hereinafter referred to as "cassette of the present invention", "recombinant vector of the present invention", "cassette of the present invention or recombinant vector") containing the nucleic acid of the present invention. vector).
　本発明の発現カセット又は組換えベクターは、本発明の核酸にプロモーター及びターミネーターを連結することにより、又は、発現ベクターに本発明の発現カセット若しくは本発明の核酸を挿入することにより調製することができる。 The expression cassette or recombinant vector of the present invention can be prepared by linking a promoter and 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. .
　本発明の発現カセット又は本発明の組換えベクターには、制御因子として、プロモーター及びターミネーターの他、必要に応じて、エンハンサー、ＣＣＡＡＴボックス、ＴＡＴＡボックス、ＳＰＩ部位等の転写要素が含まれていてもよい。これらの制御因子は、本発明の核酸に作動可能に連結されていればよい。作動可能に連結とは、本発明の核酸を調節する種々の制御因子と本発明の核酸が、宿主細胞中で作動し得る状態で連結されることをいう。 The expression cassette of the present invention or the recombinant vector of the present invention may contain transcription elements such as an enhancer, CCAAT box, TATA box, SPI site, etc., as necessary, in addition to a promoter and terminator as control elements. good. These control elements only need to be operably linked to the nucleic acid of the invention. Operably linked means that various regulatory factors that regulate the nucleic acid of the present invention and the nucleic acid of the present invention are linked in a state that allows them to operate in a host cell.
　本発明の組換えベクターにおいて、ベクターは、好ましくは発現ベクターである。発現ベクターとしては、宿主内で自律的に増殖し得るファージ、プラスミド、又はウイルスから遺伝子組換え用として構築されたベクターが好適に使用できる。かかる発現ベクターは公知であり、例えば、商業的に入手可能な発現ベクターとしては、ｐＱＥ系ベクター（株式会社キアゲン）、ｐＤＲ５４０、ｐＲＩＴ２Ｔ（ＧＥヘルスケアバイオサイエンス株式会社）、ｐＥＴ系ベクター（メルク株式会社）等が挙げられる。発現ベクターは、宿主細胞との適切な組み合わせを選んで使用すればよく、例えば、大腸菌を宿主細胞とする場合には、ｐＥＴ系ベクターとＤＨ５α大腸菌株の組み合わせ、ｐＥＴ系ベクターとＢＬ２１（ＤＥ３）大腸菌株の組み合わせ、又はｐＤＲ５４０ベクターとＪＭ１０９大腸菌株の組み合わせ等が挙げられる。 In the recombinant vector of the present invention, the vector is preferably an expression vector. As the expression vector, a vector constructed for genetic recombination from a phage, plasmid, or virus that can autonomously propagate within the host can be suitably used. Such expression vectors are known. For example, commercially available expression vectors include pQE-based vectors (Qiagen Co., Ltd.), pDR540, pRIT2T (GE Healthcare Biosciences Co., Ltd.), and pET-based vectors (Merck Co., Ltd.). ) etc. The expression vector may be used in an appropriate combination with the host cell. For example, when using E. coli as the host cell, a combination of a pET vector and a DH5α E. coli strain, a pET vector and a BL21(DE3) E. coli strain, Examples include a combination of strains, or a combination of pDR540 vector and JM109 E. coli strain.
４．形質転換体
　本発明はまた、本発明の発現カセット又は組換えベクターを用いて形質転換された形質転換体（以下、「本発明の形質転換体」と称することがある）を提供する。 4. Transformants The present invention also provides transformants transformed using the expression cassette or recombinant vector of the present invention (hereinafter sometimes referred to as "transformants of the present invention").
　本発明の形質転換体の製造に使用される宿主としては、以下の（１）～（４）の特徴を備えるものであれば、特に限定されない：
（１）発現カセット又は組換えベクターの導入が可能、
（２）発現カセット又は組換えベクターが安定、
（３）自律増殖可能、且つ、
（４）導入した発現カセット又は組換えベクター中の遺伝子の形質を発現できる。かかる宿主としては、例えば、大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ）等のエッシェリヒア属、バチルス・ズブチリス（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ）等のバチルス属、シュードモナス・プチダ（Ｐｓｅｕｄｏｍｏｎａｓ ｐｕｔｉｄａ）等のシュードモナス属に属する細菌；糸状菌、酵母等が挙げられる。また、動物細胞、昆虫細胞、又は植物等であってもよい。 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) Possible to introduce expression cassettes or recombinant vectors;
(2) the expression cassette or recombinant vector is stable;
(3) Able to reproduce autonomously, and
(4) The traits of the genes in the introduced expression cassette or recombinant vector can be expressed. Such hosts include, for example, bacteria belonging to the genus Escherichia such as Escherichia coli, the genus Bacillus such as Bacillus subtilis, the genus Pseudomonas such as Pseudomonas putida; filamentous fungi, yeast, and the like. Can be mentioned. Further, animal cells, insect cells, plants, etc. may be used.
　本発明の形質転換体は、宿主に、本発明の核酸、本発明の発現カセット、又は本発明の組換えベクターを導入することによって調製することができる。本発明の核酸等を導入する場所も、目的の遺伝子が発現できる限り特に限定されず、プラスミド上であってもよいし、ゲノム上であってもよい。本発明の発現カセット又は本発明の組換えベクターを導入する具体的な方法としては、例えば、組換えベクター法、ゲノム編集法が挙げられる。宿主に発現カセット又は組換えベクターを導入する条件は、宿主の種類等に応じて適宜設定すればよい。宿主が細菌の場合であれば、例えば、カルシウムイオン処理によるコンピテントセルを用いる方法及びエレクトロポレーション法等が挙げられる。宿主が酵母の場合であれば、例えば、電気穿孔法（エレクトロポレーション法）、スフェロプラスト法及び酢酸リチウム法等が挙げられる。宿主が動物細胞の場合であれば、例えば、エレクトロポレーション法、リン酸カルシウム法及びリポフェクション法等が挙げられる。宿主が昆虫細胞の場合であれば、例えば、リン酸カルシウム法、リポフェクション法及びエレクトロポレーション法等が挙げられる。宿主が植物胞の場合であれば、例えば、エレクトロポレーション法、アグロバクテリウム法、パーティクルガン法、及びＰＥＧ法等が挙げられる。 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 a host. The place where the nucleic acid, etc. of the present invention is introduced is not particularly limited as long as the gene of interest can be expressed, and may be on a plasmid or on the genome. Specific methods for introducing the expression cassette of the present invention or the recombinant vector of the present invention include, for example, the recombinant vector method and the genome editing method. Conditions for introducing the expression cassette or recombinant vector into the host may be appropriately set depending on the type of host and the like. If the host is a bacterium, examples include a method using competent cells treated with calcium ions, an electroporation method, and the like. When the host is yeast, examples include electroporation, spheroplast method, and lithium acetate method. When the host is an animal cell, examples include electroporation method, calcium phosphate method, and lipofection method. When the host is an insect cell, examples include the calcium phosphate method, lipofection method, and electroporation method. When the host is a plant vesicle, examples include electroporation method, Agrobacterium method, particle gun method, and PEG method.
　本発明の発現カセット又は本発明の組換えベクターが宿主に組み込まれたか否かの確認は、ＰＣＲ法、サザンハイブリダイゼーション法、又はノーザンハイブリダイゼーション法等の自体公知の方法により行うことができる。 Confirmation of whether the expression cassette of the present invention or the recombinant vector of the present invention has been integrated into a host can be performed by a method known per se such as PCR method, Southern hybridization method, or Northern hybridization method.
　ＰＣＲ法によって本発明の発現カセット又は本発明の組換えベクターが宿主に組み込まれたか否かを確認する場合、例えば、形質転換体からゲノムＤＮＡ又は発現カセット又は組換えベクターを分離・精製すればよい。発現カセット又は組換えベクターの分離・精製は、例えば、宿主が細菌の場合、細菌を溶菌して得られる溶菌物に基づいて行われる。溶菌の方法としては、例えばリゾチームなどの溶菌酵素により処理が施され、必要に応じてプロテアーゼ、及び他の酵素並びにラウリル硫酸ナトリウム（ＳＤＳ）等の界面活性剤が併用される。 When confirming whether the expression cassette of the present invention or the recombinant vector of the present invention has been integrated into a host by the PCR method, for example, genomic DNA, the expression cassette, or the recombinant vector may be isolated and purified from the transformant. . For example, when the host is a bacterium, the expression cassette or recombinant vector is isolated and purified based on a lysate obtained by lysing the bacterium. As a method for bacteriolysis, for example, treatment is performed with a lytic enzyme such as lysozyme, and if necessary, protease, other enzymes, and a surfactant such as sodium lauryl sulfate (SDS) are used in combination.
　更に、凍結融解およびフレンチプレス処理のような物理的破砕方法を組み合わせてもよい。溶菌物からの核酸の分離・精製は、例えば、フェノール処理およびプロテアーゼ処理による除蛋白処理、リボヌクレアーゼ処理、アルコール沈殿処理並びに市販のキットを適宜組み合わせることにより行うことができる。 Additionally, physical crushing methods such as freeze-thaw and French press treatments may be combined. Separation and purification of nucleic acids from lysates can be carried out, for example, by appropriately combining protein removal treatment using phenol treatment and protease treatment, ribonuclease treatment, alcohol precipitation treatment, and commercially available kits.
　核酸の切断は、常法に従い、例えば制限酵素処理を用いて行うことができる。制限酵素としては、例えば特定のヌクレオチド配列に作用するＩＩ型制限酵素を用いる。核酸と発現カセット又は発現ベクターとの結合は、例えばＤＮＡリガーゼを用いて行う。 Nucleic acid cleavage can be performed according to conventional methods, for example, using restriction enzyme treatment. As the restriction enzyme, for example, a type II restriction enzyme that acts on a specific nucleotide sequence is used. The binding of the nucleic acid and the expression cassette or expression vector is performed using, for example, DNA ligase.
　その後、分離・精製したＤＮＡを鋳型として、本発明のＤＮＡに特異的なプライマーを設計してＰＣＲを行う。ＰＣＲにより得られた増幅産物についてアガロースゲル電気泳動、ポリアクリルアミドゲル電気泳動、キャピラリー電気泳動等を行い、臭化エチジウムおよびＳＹＢＲ Ｇｒｅｅｎ液等により染色し、そして増幅産物をバンドとして検出することにより、形質転換されたことを確認することができる。 Thereafter, using the isolated and purified DNA as a template, primers specific to the DNA of the present invention are designed and PCR is performed. The amplification product obtained by PCR is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., and stained with ethidium bromide and SYBR Green solution, etc., and the amplification product is detected as a band. You can confirm that it has been converted.
　また、予め蛍光色素等により標識したプライマーを用いてＰＣＲを行い、増幅産物を検出することもできる。さらに、マイクロプレート等の固相に増幅産物を結合させ、蛍光および酵素反応等により増幅産物を確認する方法を採用してもよい。 Alternatively, amplification products can also be detected by performing PCR using primers that have been labeled in advance with a fluorescent dye or the like. Furthermore, a method may 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, enzyme reaction, or the like.
５．改変型エステラーゼの製造方法
　本発明はまた、本発明の形質転換体を培養する工程を含む、改変型エステラーゼの製造方法（以下、「本発明の製造方法」と称することがある）を提供する。 5. Method for producing a modified esterase The present invention also provides a method for producing a modified esterase (hereinafter sometimes referred to as "the production method of the present invention"), which includes the step of culturing the transformant of the present invention.
　本発明の製造方法に用いられる培養条件は、用いられる形質転換体の栄養生理的性質を考慮して適宜設定すればよい。 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 used.
　形質転換体の培養は固形培地又は液体培地を用いることができるが、好ましくは液体培地であり得る。また、工業的製造を行う場合は、通気攪拌培養が好ましい。培地の栄養源としては、形質転換体の生育に必要とされる物質を適宜使用すればよい。炭素源としては、資化可能な炭素化合物であればよく、例えば、グルコース、スクロース、ラクトース、マルトース、糖蜜、ピルビン酸等が挙げられる。窒素源としては、資化可能な窒素化合物であればよく、例えば、ペプトン、肉エキス、酵母エキス、カゼイン加水分解物、大豆粕アルカリ抽出物が挙げられる。炭素源及び窒素源の他に、例えば、リン酸塩、炭酸塩、硫酸塩、マグネシウム、カルシウム、カリウム、鉄、マンガンおよび亜鉛などの塩類、特定のアミノ酸並びに特定のビタミンなどを必要に応じて使用してもよい。 A solid medium or a liquid medium can be used for culturing the transformant, preferably a liquid medium. Furthermore, when performing industrial production, aerated agitation culture is preferred. As the nutrient source for the medium, any substance required for the growth of the transformant may be used as appropriate. The carbon source may be any carbon compound that can be assimilated, and examples thereof include glucose, sucrose, lactose, maltose, molasses, and pyruvic acid. The nitrogen source may be any nitrogen compound that can be assimilated, such as peptone, meat extract, yeast extract, casein hydrolyzate, and soybean meal alkaline extract. In addition to carbon and nitrogen sources, salts such as phosphates, carbonates, sulfates, magnesium, calcium, potassium, iron, manganese, and zinc, specific amino acids, and specific vitamins may be used as necessary. You may.
　培養温度は、本発明の形質転換体が生育可能であり、且つ当該形質転換体が改変型エステラーゼを産生する範囲で適宜設定すればよい。一態様において、培養温度は１５～３７℃程度であるが、これらに限定されない。培養を終了するタイミングとしては、改変型エステラーゼが最高収量に達するタイミングを見計らって完了すればよい。培養時間は、例えば、約１２～４８時間が挙げられるが、これらに限定されない。 The culture temperature may be appropriately set within a range where the transformant of the present invention can grow and the transformant produces the modified esterase. In one embodiment, the culture temperature is about 15 to 37°C, but is not limited thereto. The timing of terminating the culture may be determined at a time when the modified esterase reaches its maximum yield. The culture time includes, for example, about 12 to 48 hours, but is not limited thereto.
　形質転換体を用いて発現させた改変型エステラーゼは、形質転換体の内部又は培養培地中に存在する。改変型エステラーゼの回収は自体公知の方法により行えばよいが、以下に回収方法の一例を簡潔に説明する。 The modified esterase expressed using the transformant exists inside the transformant or in the culture medium. Although the modified esterase may be recovered by a method known per se, one example of the recovery method will be briefly explained below.
　一態様において、発現させた改変型エステラーゼが、形質転換体の内部に存在する場合、形質転換体を遠心分離等の自体公知の方法を用いて培養上清から分離する。培養上清を除去し、分離された形質転換体を回収する。分離された形質転換体は、超音波やフレンチプレスといった機械的方法、或いはリゾチーム等の酵素的な方法を用いて処理し、必要に応じてプロテアーゼ等の酵素やラウリル硫酸ナトリウム（ＳＤＳ）等の界面活性剤を使用することにより可溶化することで、改変型エステラーゼを含む水溶性画分を得ることができる。 In one embodiment, when the expressed modified esterase is present inside the transformant, the transformant is separated from the culture supernatant using a method known per se, such as centrifugation. The culture supernatant is removed and the isolated transformants are collected. The isolated transformants are treated using mechanical methods such as ultrasound or French press, or enzymatic methods such as lysozyme, and if necessary, treated with enzymes such as protease or interfaces such as sodium lauryl sulfate (SDS). By solubilizing using an activator, a water-soluble fraction containing the modified esterase can be obtained.
　また、別の態様において、発現させた改変型エステラーゼが培養液中に存在する場合は、そのまま精製処理に供してもよいが、該水溶性画分中の改変型エステラーゼを濃縮した後に精製処理に供してもよい。濃縮は、例えば、減圧濃縮、膜濃縮、塩析処理、親水性有機溶媒（例えば、メタノール、エタノールおよびアセトン）による分別沈殿法等により行うことができる。また、改変型エステラーゼの精製処理は、例えば、ゲルろ過、吸着クロマトグラフィー、イオン交換クロマトグラフィー、アフィニティクロマトグラフィー等の方法を適宜組み合わせることによって行うことができる。かくして精製された改変型エステラーゼは、必要に応じて、凍結乾燥、真空乾燥、スプレードライ等により粉末化して市場に流通させることができる。 In another embodiment, when the expressed modified esterase is present in the culture solution, it may be subjected to purification treatment as it is, but it may be subjected to purification treatment after concentrating the modified esterase in the water-soluble fraction. You can also serve it. Concentration can be performed, for example, by vacuum concentration, membrane concentration, salting out treatment, fractional precipitation using a hydrophilic organic solvent (eg, methanol, ethanol, and acetone), and the like. Furthermore, the modified esterase can be purified by appropriately combining methods such as gel filtration, adsorption chromatography, ion exchange chromatography, and affinity chromatography. The thus purified modified esterase can be pulverized by freeze-drying, vacuum drying, spray-drying, etc., and distributed on the market, if necessary.
６．改変型エステラーゼを含む酵素剤
　本発明はまた、本発明の改変型エステラーゼを含む、酵素剤（以下、「本発明の酵素剤」と称することがある）を提供する。 6. Enzyme agent containing modified esterase The present invention also provides an enzyme agent (hereinafter sometimes referred to as "enzyme agent of the present invention") containing the modified esterase of the present invention.
　本発明の酵素剤に含まれる本発明の改変型エステラーゼの量は、通常０．００１～１００重量％であり、好ましくは、０．０１～１００重量％、０．１～１００重量％、又は１～１００重量％であり、より好ましくは５～１００重量％、１０～１００重量％、２０～１００重量％、３０～１００重量％、４０～１００重量％、又は５０～１００重量％であり得るが、これらに限定されない。 The amount of the modified esterase of the present invention contained in the enzyme preparation of the present invention is usually 0.001 to 100% by weight, preferably 0.01 to 100% by weight, 0.1 to 100% by weight, or 1% by weight. -100% by weight, more preferably 5-100%, 10-100%, 20-100%, 30-100%, 40-100%, or 50-100% by weight. , but not limited to.
　本発明の酵素剤は、本発明の改変型エステラーゼのみを含むものであってもよいが、所望の効果を奏する限り、他の成分を含んでいてもよい。他の成分としては、本発明の改変型エステラーゼ以外の他の酵素、添加剤、本発明の製造方法において生じた培養残渣等が挙げられる。 The enzyme preparation of the present invention may contain only the modified esterase of the present invention, but may contain other components as long as it achieves the desired effect. Examples of other components include enzymes other than the modified esterase of the present invention, additives, culture residue produced in the production method of the present invention, and the like.
　他の酵素としては、例えば、アミラーゼ（α－アミラーゼ、β－アミラーゼ、グルコアミラーゼ）、グルコシダーゼ（α－グルコシダーゼ、β－グルコシダーゼ）、ガラクトシダーゼ（α－ガラクトシダーゼ、β－ガラクトシダーゼ）、プロテアーゼ（酸性プロテアーゼ、中性プロテアーゼ、アルカリプロテアーゼ）、ペプチダーゼ（ロイシンペプチダーゼ、アミノペプチダーゼ）、リパーゼ、エステラーゼ、セルラーゼ、ホスファターゼ（酸性ホスファターゼ、アルカリホスファターゼ）、ヌクレアーゼ、デアミナーゼ、オキシダーゼ、デヒドロゲナーゼ、グルタミナーゼ、ペクチナーゼ、カタラーゼ、デキストラナーゼ、トランスグルタミナーゼ、蛋白質脱アミド酵素、プルラナーゼ等が挙げられる。これらの他の酵素は、１種を単独で含んでもよいし、複数種の組み合わせで含んでもよい。 Other enzymes include, for example, amylase (α-amylase, β-amylase, glucoamylase), glucosidase (α-glucosidase, β-glucosidase), galactosidase (α-galactosidase, β-galactosidase), protease (acidic protease, (protease, alkaline protease), peptidase (leucine peptidase, aminopeptidase), lipase, esterase, cellulase, phosphatase (acid phosphatase, alkaline phosphatase), nuclease, deaminase, oxidase, dehydrogenase, glutaminase, pectinase, catalase, dextranase, trans Examples include glutaminase, protein deamidase, pullulanase, and the like. These other enzymes may be contained singly or in combination.
　添加剤としては、賦形剤、緩衝剤、懸濁剤、安定剤、保存剤、防腐剤、生理食塩水等が挙げられる。賦形剤としては、デンプン、デキストリン、マルトース、トレハロース、乳糖、Ｄ－グルコース、ソルビトール、Ｄ－マンニトール、白糖、グリセロール等が挙げられる。緩衝剤としては、リン酸塩、クエン酸塩、酢酸塩等が挙げられる。安定剤としては、プロピレングリコール、アスコルビン酸等が挙げられる。保存剤としては、フェノール、塩化ベンザルコニウム、ベンジルアルコール、クロロブタノール、メチルパラベン等が挙げられる。防腐剤としては、エタノール、塩化ベンザルコニウム、パラオキシ安息香酸、クロロブタノール等が挙げられる。これらの添加剤は、１種を単独で含んでもよいし、複数種の組み合わせで含んでもよい。 Examples of additives include excipients, buffers, suspending agents, stabilizers, preservatives, preservatives, physiological saline, and the like. Excipients include starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, white sugar, glycerol, and the like. Buffers include phosphates, citrates, acetates, and the like. Stabilizers include propylene glycol, ascorbic acid, and the like. Preservatives include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like. Examples of preservatives include ethanol, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol, and the like. These additives may be contained singly or in combination.
　培養残渣としては、培地由来の成分、夾雑タンパク質、菌体成分等が挙げられる。 Examples of the culture residue include components derived from the medium, contaminant proteins, bacterial body components, and the like.
　本発明の酵素剤の形態は特に限定されず、例えば、液状、固形状（粉末、顆粒等）等が挙げられる。酵素剤の形状は、自体公知の方法により成型すればよい。 The form of the enzyme preparation of the present invention is not particularly limited, and examples thereof include liquid form, solid form (powder, granules, etc.), and the like. The shape of the enzyme agent may be molded by a method known per se.
　本発明の酵素剤の有効成分である本発明の改変型エステラーゼは、菊酸エチルの菊酸への変換（好ましくは、（１Ｒ，３Ｓ）－菊酸エチルの（１Ｒ，３Ｓ）－菊酸への変換）において高い酵素活性を有する。従って、本発明の酵素剤の一態様において、本発明の酵素剤は、菊酸（好ましくは（１Ｒ，３Ｓ）－菊酸）の生成用の酵素剤である。 The modified esterase of the present invention, which is an active ingredient of the enzyme preparation of the present invention, converts ethyl chrysanthemum to chrysanthemum acid (preferably, converts (1R,3S)-ethyl chrysanthemum to (1R,3S)-chrysanthete). It has high enzymatic activity in the conversion of 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 chrysanthemum acid (preferably (1R,3S)-chrysanthemum acid).
　本発明の酵素剤を適切な条件下において、菊酸エチルを含有する組成物に作用させることにより、効率よく菊酸を含有する組成物を製造することができる。尚、本発明の酵素剤の基質については、後述する「菊酸の製造方法」において詳述される。 By allowing the enzyme agent of the present invention to act on a composition containing ethyl chrysanthemum acid under appropriate conditions, a composition containing chrysanthemum acid can be efficiently produced. The substrate for the enzyme agent of the present invention will be described in detail in the "method for producing chrysanthemum acid" described below.
７．菊酸の製造方法
　本発明はまた、菊酸エチルに、本発明の改変型エステラーゼを作用させる工程を含む、菊酸の製造方法（以下、「本発明の菊酸の製造方法」と称することがある）を提供する。 7. Method for producing chrysanthemum acid The present invention also provides a method for producing chrysanthemum acid (hereinafter referred to as the "method for producing chrysanthemum acid of the present invention"), which includes a step of causing the modified esterase of the present invention to act on ethyl chrysanthemum acid. ).
　本発明の菊酸の製造方法において、基質となる菊酸エチルは、精製されたものであってよく、或いは、他の成分を含む組成物（以下、菊酸エチル含有組成物」と称することがある）であってもよい。菊酸エチル含有組成物は、純度の高い（１Ｒ，３Ｓ）－菊酸エチル（例えば純度が９８％以上）を使用しても、或いは（１Ｒ，３Ｒ）－菊酸エチル、（１Ｓ，３Ｓ）－菊酸エチル及び（１Ｓ，３Ｒ）－菊酸エチルが共存した状態の基質（例えば、（１Ｒ，３Ｒ）：（１Ｓ，３Ｓ）：（１Ｒ，３Ｓ）：（１Ｓ，３Ｒ）＝３：３：２：２の混合菊酸エチル等）を使用してもよい。 In the method for producing chrysanthemum acid of the present invention, ethyl chrysanthemum acid serving as a substrate may be purified, or a composition containing other components (hereinafter referred to as ethyl chrysanthemum-containing composition) may be used. ). The ethyl chrysanthemum-containing composition can be prepared by using highly pure (1R,3S)-ethyl chrysanthema (for example, purity of 98% or more), or by using (1R,3R)-ethyl chrysanthemum, (1S,3S) -Substrate in which ethyl chrysanthemum acidate and (1S,3R)-ethyl chrysanthemumate coexist (for example, (1R,3R):(1S,3S):(1R,3S):(1S,3R)=3:3 :2:2 mixed ethyl chrysanthemum acid, etc.) may also be used.
　菊酸エチル含有組成物に含まれる菊酸エチル含有率としては特に限定されないが、例えば２０％以上が挙げられる。生成物中の菊酸エチル含有率をより一層高める観点から、当該基質における菊酸エチル含有率としては、好ましくは３０％以上、より好ましくは４０％以上、さらに好ましくは４５％以上が挙げられる。当該含有量範囲の上限としては特に限定されないが、例えば１００％以下、好ましくは９０％以下、より好ましくは７０％以下が挙げられる。菊酸エチル含有組成物の由来については特に限定されず、例えば、化学合成したものが挙げられる。 The content of ethyl chrysanthemum contained in the ethyl chrysanthemum-containing composition is not particularly limited, but may be, for example, 20% or more. From the viewpoint of further increasing the ethyl chrysanthemum content in the product, the ethyl chrysanthemum content in the substrate is preferably 30% or more, more preferably 40% or more, still more preferably 45% or more. The upper limit of the content range is not particularly limited, but may be, for example, 100% or less, preferably 90% or less, and more preferably 70% or less. The origin of the ethyl chrysanthemum-containing composition is not particularly limited, and examples include chemically synthesized compositions.
　菊酸を製造できる限り、基質（例えば、菊酸エチル含有組成物）に改変型エステラーゼを作用させるときの反応時間、反応温度、反応溶液のｐＨ、溶媒等も特に限定されない。反応時間は、例えば１０分～９６時間、好ましくは１時間～７２時間、より好ましくは１２時間～４８時間である。反応温度は、例えば１０～９０℃、好ましくは２０～８０℃、より好ましくは３０～７５℃、更に好ましくは４０～７０℃、一層好ましくは５０～６５℃が挙げられる。反応溶液のｐＨは、例えば３～１２、好ましくは６～１１、より好ましくは８～１１が挙げられる。反応中の溶媒としては、有機溶媒を含んでいても、含んでいなくても良い。有機溶媒としては、メタノールやアセトニトリルを用いることができる。有機溶媒の量は特に限定されないが、例えば下限として１０％、２０％、３０％以上、４０％以上が挙げられ、上限として１００％以下、８０％以下、７０％以下、５０％以下が挙げられる。これらの反応条件は、使用する原料や目的とする生成物によって適宜選択される。尚、最適な反応条件は予備実験を通して決定すればよい。 As long as chrysanthemum acid can be produced, the reaction time, reaction temperature, pH of the reaction solution, solvent, etc. when allowing the modified esterase to act on the substrate (for example, a composition containing ethyl chrysanthemum acid) are not particularly limited. The reaction time is, for example, 10 minutes to 96 hours, preferably 1 hour to 72 hours, more preferably 12 hours to 48 hours. The reaction temperature is, for example, 10 to 90°C, preferably 20 to 80°C, more preferably 30 to 75°C, even more preferably 40 to 70°C, even more preferably 50 to 65°C. The pH of the reaction solution is, for example, 3 to 12, preferably 6 to 11, more preferably 8 to 11. The solvent during the reaction may or may not contain an organic solvent. Methanol and acetonitrile can be used as the organic solvent. The amount of organic solvent is not particularly limited, but for example, lower limits include 10%, 20%, 30% or more, and 40% or more, and upper limits include 100% or less, 80% or less, 70% or less, and 50% or less. . These reaction conditions are appropriately selected depending on the raw materials used and the desired product. Note that the optimal reaction conditions may be determined through preliminary experiments.
　本発明の菊酸の製造方法を用いることで、菊酸含有組成物を非常に効率よく製造することができる。本発明の菊酸含有組成物の製造方法の一態様は、以下の工程（１）及び（２）を含む。尚、工程（２）の後に、得られた菊酸含有組成物から菊酸を回収する工程を追加しても良い。
　（１）菊酸エチル含有組成物を提供する工程
　（２）提供された菊酸エチル含有組成物を本発明の改変型エステラーゼで処理する工程 By using the method for producing chrysanthemum acid of the present invention, a chrysanthemum acid-containing composition can be produced very efficiently. One embodiment of the method for producing the chrysanthemum acid-containing composition of the present invention includes the following steps (1) and (2). Note that, after step (2), a step of recovering chrysanthemum acid from the obtained chrysanthemum acid-containing composition may be added.
(1) Step of providing an ethyl chrysanthemum acid-containing composition (2) Process of treating the provided ethyl chrysanthemum acid-containing composition with the modified esterase of the present invention
　本発明の菊酸の製造方法を用いて得られた菊酸含有組成物における菊酸の含有率は、特に限定されないが、通常１重量％以上であり、好ましくは２重量％以上、３重量％以上、４重量％以上、又は５重量％以上であり、より好ましくは１０重量％以上、１２重量％以上、又は１４重量％以上であり、特に好ましくは１６重量％以上であり得る。 The content of chrysanthemum acid in the chrysanthemum acid-containing composition obtained using the method for producing chrysanthemum acid of the present invention is not particularly limited, but is usually 1% by weight or more, preferably 2% by weight or more, and 3% by weight. The content may be 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, particularly preferably 16% by weight or more.
　反応産物は必要に応じて光学分割する工程を追加しても良い。光学分割にはカラムクロマトグラフィーを用いることができる。改変型エステラーゼは固定化したものを用いても良い。 A step of optically resolving the reaction product may be added as necessary. Column chromatography can be used for optical resolution. An immobilized modified esterase may be used.
　尚、菊酸含有組成物は、農薬中間体として好適に使用される。 Incidentally, the chrysanthemum acid-containing composition is suitably used as an agricultural chemical intermediate.
　以下の実施例において本発明を更に具体的に説明するが、本発明はこれらの例によってなんら限定されるものではない。 The present invention will be explained in more detail in the following examples, but the present invention is not limited to these examples in any way.
　Ａｒｔｈｒｏｂａｃｔｅｒ　ｇｌｏｂｉｆｏｒｍｉｓ由来の野生型エステラーゼ（配列番号１）の２重変異体であり、菊酸エチルエステルに対して高い反応性を有する配列番号７のエステラーゼは、（１Ｒ，３Ｓ）－菊酸エチルに対する高い選択性を有する（１Ｒ，３Ｓ）特異的エステラーゼである。そこで、当該（１Ｒ，３Ｓ）特異的エステラーゼと野生型エステラーゼを基準エステラーゼとして、蛋白質工学的手法により耐熱性と有機溶媒中での安定性が向上した改変型エステラーゼの作成を試みた。詳細な手順を以下に示す。 The esterase of SEQ ID NO: 7, which is a double mutant of the wild type esterase (SEQ ID NO: 1) derived from Arthrobacter globiformis and has high reactivity to ethyl chrysanthemum ester, has a high reactivity to (1R,3S)-ethyl chrysanthemum ester. It is a (1R,3S) specific esterase with selectivity. Therefore, using the (1R,3S)-specific esterase and the wild-type esterase as reference esterases, we attempted to create a modified esterase with improved heat resistance and stability in organic solvents using protein engineering techniques. Detailed steps are shown below.
１．変異体ライブラリの作製
　以下の方法で変異体ライブラリを作製した。
（１）変異の導入・形質転換
　変異導入用ＰＣＲプライマー（配列番号２５～３４）を設計し、以下の条件でＰＣＲを行い、変異を導入した。 1. Creation of mutant library A mutant library was created by the following method.
(1) Introduction of mutations/transformation PCR primers for introducing mutations (SEQ ID NOs: 25 to 34) were designed, and PCR was performed under the following conditions to introduce mutations.
＜鋳型プラスミド＞
　ｐＥＴ２１ｂ（基準となるエステラーゼの遺伝子配列を導入）約３０ｎｇ／μＬ <Template plasmid>
pET21b (introducing the standard esterase gene sequence) approximately 30 ng/μL
＜ＰＣＲ反応組成＞（総量２０μＬ）
５×Ｐｒｉｍｅ　ＳＴＡＲ　ＧＸＬ　Ｂｕｆｆｅｒ（タカラバイオ）： ４μＬ　
ｄＮＴＰ Ｍｉｘｔｕｒｅ （２．５ｍＭ ｅａｃｈ）： １．６μＬ
鋳型（約３０ ｎｇ/μＬ）： ０．２５μＬ
Ｆ－プライマー： ０．２μＬ
Ｒ－プライマー： ０．２μＬ
Ｐｒｉｍｅ ＳＴＡＲ ＧＸＬ ＤＮＡ Ｐｏｌｙｍｅｒａｓｅ （タカラバイオ）： ０．８μＬ
　これらを混合し、超純水（Ｍｉｌｌｉ　Ｑ水）で２０μＬに調整。 <PCR reaction composition> (total volume 20 μL)
5×Prime STAR GXL Buffer (Takara Bio): 4μL
dNTP Mixture (2.5mM each): 1.6μL
Template (approximately 30 ng/μL): 0.25μL
F-primer: 0.2μL
R-primer: 0.2μL
Prime STAR GXL DNA Polymerase (Takara Bio): 0.8μL
Mix these and adjust to 20 μL with ultrapure water (Milli Q water).
＜ＰＣＲ条件＞
（１）９８℃、１分
（２）＜９８℃、１０秒、６０℃、１５秒、６８℃、２分＞×２０サイクル
（３）６８℃、５分
（４）４℃で静置 <PCR conditions>
(1) 98℃, 1 minute (2) <98℃, 10 seconds, 60℃, 15 seconds, 68℃, 2 minutes>×20 cycles (3) 68℃, 5 minutes (4) Leave at 4℃
　ＰＣＲ反応液２０μＬにＤｐｎＩ　０．５μＬを添加して処理（３７℃、３時間以上）した。ＤＮＡ精製キットＮｕｃｌｅｏＳｐｉｎ　Ｇｅｌ　ａｎｄ　ＰＣＲ　Ｃｌｅａｎ－ｕｐ（タカラバイオ（株）製）でＤＮＡを精製した。 0.5 μL of DpnI was added to 20 μL of the PCR reaction solution and treated (37° C., 3 hours or more). DNA was purified using the DNA purification kit NucleoSpin Gel and PCR Clean-up (manufactured by Takara Bio Inc.).
＜ｉｎ－ｆｕｓｉｏｎ反応＞
　以下の組成の反応液を調製し（総量１０μＬ）、サーマルサイクラーで５０℃１５分加温した。
精製済みＰＣＲ溶液：２～５μＬ（５０～２００ｎｇ相当量）
Ｍｉｌｌｉ　Ｑ水：３～６μＬ
５×ｉｎ－ｆｕｓｉｏｎ試薬：２μＬ <In-fusion reaction>
A reaction solution having the following composition was prepared (total volume: 10 μL) and heated at 50° C. for 15 minutes in a thermal cycler.
Purified PCR solution: 2-5 μL (equivalent to 50-200 ng)
Milli Q water: 3-6μL
5× in-fusion reagent: 2 μL
　ｉｎ－ｆｕｓｉｏｎ反応液２μＬをＥ．ｃｏｌｉ　ＢＬ２１（ＤＥ３）（ニッポンジーン社）１８μＬに混合し、形質転換を行った。３７℃２０分間の回復培養後に、形質転換溶液を、アンピシリン（最終濃度１００μｇ／ｍＬ）を添加したＬＢ寒天培地プレート（インビトロジェン（株）製）に塗布して、３７℃で１８時間培養した。（前培養）。 Add 2 μL of the in-fusion reaction solution to E. The mixture was mixed with 18 μL of E. coli BL21 (DE3) (Nippon Gene) and transformed. After recovery culture at 37°C for 20 minutes, the transformation solution was applied to an LB agar plate (manufactured by Invitrogen Co., Ltd.) supplemented with ampicillin (final concentration 100 μg/mL), and cultured at 37°C for 18 hours. (preculture).
（２）配列の確認
　前培養後の各変異株を、アンピシリン（最終濃度１００μｇ／ｍＬ）を添加したＬＢ培地（インビトロジェン（株）製）に植菌した。３７℃で１８時間培養して菌体培養液とした。遠心分離で菌体を回収して、プラスミドミニプレップキットＮｕｃｌｅｏＳｐｉｎ　Ｐｌａｓｍｉｄ　ＥａｓｙＰｕｒｅ（タカラバイオ社）でそれぞれのプラスミドを精製した。サンガーシーケンスにより遺伝子のＤＮＡ配列を確認した。 (2) Confirmation of Sequence Each precultured mutant strain was inoculated into LB medium (manufactured by Invitrogen Co., Ltd.) supplemented with ampicillin (final concentration 100 μg/mL). The cells were cultured at 37°C for 18 hours to obtain a bacterial cell culture solution. Bacterial cells were collected by centrifugation, and each plasmid was purified using a plasmid miniprep kit, NucleoSpin Plasmid EasyPure (Takara Bio Inc.). The DNA sequence of the gene was confirmed by Sanger sequencing.
（３）酵素抽出液の調製
　前培養後の各変異株を、アンピシリン（最終濃度１００μｇ／ｍＬ）を添加したＴＢ培地（インビトロジェン（株）製）１ｍＬに植菌した。３３℃で４８時間培養し（培養開始２４時間の時点でＩＰＴＧ（最終濃度 ０．１ｍＭ）を添加）、遠心分離（３，０００ ｇ× １０分）した。上澄みを除去して菌体を回収した後、溶菌剤にて菌体を溶菌（２５℃、２時間以上）させて酵素を抽出した。溶菌液を遠心分離（３，０００ ｇ × １０分）後、上澄みを回収して酵素抽出液とした。 (3) Preparation of enzyme extract Each precultured mutant strain was inoculated into 1 mL of TB medium (manufactured by Invitrogen Co., Ltd.) supplemented with ampicillin (final concentration 100 μg/mL). The cells were cultured at 33°C for 48 hours (IPTG (final concentration 0.1 mM) was added 24 hours after the start of culture) and centrifuged (3,000 g x 10 minutes). After removing the supernatant and collecting the bacterial cells, the bacterial cells were lysed using a lytic agent (25° C., 2 hours or more) to extract the enzyme. After centrifuging the lysate (3,000 g x 10 minutes), the supernatant was collected and used as an enzyme extract.
（４）活性測定
　各酵素は以下の活性測定方法にて活性を測定して評価した。 (4) Activity measurement Each enzyme was evaluated by measuring the activity using the following activity measurement method.
活性測定方法
　基質（パラニトロフェニル-菊酸エチル：６ｍｇ）を計り取り、４％　Ｔｒｉｔｏｎ　Ｘ－１００：１６ｍＬ に懸濁した。５０℃ に加温して、基質を溶解させた。室温にて放冷後、０．５Ｍ ＰＩＰＥＳ ｐＨ７．０ を４ｍＬ添加、混合した。０．１Ｍ ＰＩＰＥＳ　ｐＨ７．０：１９０μＬと溶菌液１０μＬを混合した。サンプル３μＬ＋各基質溶液：
１９７μＬ／ウェルを添加した。３７℃ にて２時間反応させた。プレートリーダーで４１６ｎｍにて、吸光度を測定した。 Activity Measuring Method The substrate (para-nitrophenyl-ethyl chrysanthemum acid: 6 mg) was weighed out and suspended in 4% Triton X-100: 16 mL. The substrate was dissolved by heating to 50°C. After cooling at room temperature, 4 mL of 0.5M PIPES pH 7.0 was added and mixed. 190 μL of 0.1M PIPES pH 7.0 and 10 μL of bacteriolysis solution were mixed. 3 μL of sample + each substrate solution:
197 μL/well was added. The reaction was carried out at 37°C for 2 hours. Absorbance was measured at 416 nm using a plate reader.
（５）組合せ変異体の作製
　得られた変異体を鋳型として、上記（１）と同様の操作でＰＣＲを行った。ＰＣＲ産物を回収し、上記（３）と同様の操作で酵素抽出液を得た。 (5) Preparation of combination mutants Using the obtained mutants as templates, PCR was performed in the same manner as in (1) above. The PCR product was collected and an enzyme extract was obtained in the same manner as in (3) above.
２．試験例１
（１）野生型エステラーゼの改変
　野生型エステラーゼに以下の変異を導入した。ホモロジー検索から近縁のエステラーゼを調査して、相同性のないアミノ酸を選択した。その結果、下記のアミノ酸残基（アミノ酸残基を表す１文字、アミノ酸残基の位置を表す数字）に変異を導入した。 2. Test example 1
(1) Modification of wild-type esterase The following mutations were introduced into wild-type esterase. We investigated closely related esterases through a homology search and selected amino acids with no homology. As a result, mutations were introduced into the following amino acid residues (one letter representing the amino acid residue, number representing the position of the amino acid residue).
Q4、S12、R25、V64、F77、A93、H107、Y123、L128、C161、H184、Q208、H216、S220、L242、A245、M254、V286、F292、C303、A313、S315、S363 Q4, S12, R25, V64, F77, A93, H107, Y123, L128, C161, H184, Q208, H216, S220, L242, A245, M254, V286, F292, C303, A313, S315, S363
　作製した変異酵素の酵素活性を測定して、酵素活性を示したサンプルを集めた。さらに、ＰＣＲによる変異導入を繰り返すことで、複数変異点の組み合わせ変異を作製して、耐熱性向上を簡易的に評価した。その結果、以下の５種類の変異酵素が、野生型の酵素と比較して耐熱性などに変化があった。 The enzyme activity of the produced mutant enzyme was measured, and samples that showed enzyme activity were collected. Furthermore, by repeating mutation introduction by PCR, combination mutations of multiple mutation points were created, and improvement in heat resistance was easily evaluated. As a result, the following five types of mutant enzymes had changes in heat resistance and other properties compared to the wild-type enzyme.
　そこで、それぞれの変異酵素について詳細な評価を行った。 Therefore, we conducted a detailed evaluation of each mutant enzyme.
（２）耐熱性（温度安定性）の評価
　上述の１．（３）の方法で酵素抽出液を調製した。酵素抽出液を、サーマルサイクラーを用いて、３０、４０、５０、６０、７０、８０又は９０℃で３０分間加温した。加温後に遠心分離により固形分を取り除き、酵素サンプル液とした。酵素活性測定方法により、それぞれのサンプルの吸光度を測定することで酵素活性を測定した。各サンプルの最大値を１００％として、相対値で温度による酵素活性の変化を測定し、５０％失活温度（Ｔ５０値）を計測した（表２）。その結果、最大で３．４１℃の耐熱性の向上が見られた。 (2) Evaluation of heat resistance (temperature stability) 1. An enzyme extract was prepared by the method (3). 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 content was removed by centrifugation to obtain an enzyme sample solution. Enzyme activity was measured by measuring the absorbance of each sample using an enzyme activity measurement method. With the maximum value of each sample as 100%, changes in enzyme activity due to temperature were measured using relative values, and the 50% inactivation temperature (T50 value) was measured (Table 2). As a result, an improvement in heat resistance of up to 3.41°C was observed.
（３）アセトニトリル耐性評価
　野生型及び変異型酵素について酵素抽出液を調製した。酵素抽出液に、終濃度が３０％量となるようにアセトニトリルを加えて、３０℃で３時間インキュベートした。アセトニトリルの代わりに等量の超純水を加えたサンプルを基準にして、アセトニトリルを添加した場合の相対活性値を計算した。実験はすべて３回行い、平均値を計算した（表３）。その結果、特にＳ２２０Ａの変異型酵素や４重変異型酵素（Ｓ１２Ｐ／Ｓ２２０Ａ／Ａ３１３Ｓ／Ｓ３１５Ｍ）においてアセトニトリルに対する耐性における顕著な向上が確認された。 (3) Acetonitrile tolerance evaluation Enzyme extracts were prepared for wild-type and mutant enzymes. Acetonitrile was added to the enzyme extract so that the final concentration was 30%, and the mixture was incubated at 30°C for 3 hours. The relative activity value when acetonitrile was added was calculated based on a sample in which an equivalent amount of ultrapure water was added instead of acetonitrile. All experiments were performed in triplicate and the average values were calculated (Table 3). As a result, it was confirmed that the resistance to acetonitrile was significantly improved, especially in the S220A mutant enzyme and the quadruple mutant enzyme (S12P/S220A/A313S/S315M).
（４）メタノール耐性評価
　野生型エステラーゼおよび変異酵素について、酵素抽出液を調製した。酵素抽出液に、終濃度が４０％量となるようにメタノールを加えて、３０℃で３時間インキュベートした。メタノールの代わりに等量の超純水を加えたサンプルを基準にして、メタノールを添加した場合の相対活性値を計算した。野生型の活性値を基準に、それぞれの変異酵素の相対活性値を計算した。実験はすべて３回行い、平均値を計算した（表３）。その結果、特に５重変異酵素（Ｓ１２Ｐ／Ｒ２５Ｐ／Ｓ２２０Ａ／Ａ３１３Ｓ／Ｓ３１５Ｍ）において、メタノールに対する耐性における顕著な向上が確認された。 (4) Methanol tolerance evaluation Enzyme extracts were prepared for wild-type esterase and mutant enzymes. Methanol was added to the enzyme extract to give a final concentration of 40%, and the mixture was incubated at 30°C for 3 hours. The relative activity value when methanol was added was calculated based on a sample in which an equivalent amount of ultrapure water was added instead of methanol. The relative activity value of each mutant enzyme was calculated based on the wild type activity value. All experiments were performed in triplicate and the average values were calculated (Table 3). As a result, it was confirmed that the resistance to methanol was significantly improved, especially in the quintuple mutant enzyme (S12P/R25P/S220A/A313S/S315M).
３．試験例２
（１）（１Ｒ，３Ｓ）特異的エステラーゼの改変
　（１Ｒ，３Ｓ）－菊酸エチルに選択性の高いエステラーゼとしてＡ２２１Ｆ／Ａ３２８Ｇの二重変異を導入したエステラーゼは、野生型エステラーゼでは加水分解が出来ない（１Ｒ，３Ｓ）－菊酸エチルに選択性の高いエステラーゼ（（１Ｒ，３Ｓ）特異的エステラーゼ）である。上記（１Ｒ，３Ｓ）特異的エステラーゼに更に試験例１の変異を導入して、耐熱性（温度安定性）の向上効果があるかを検討した。Ａ２２１Ｆ／Ａ３２８Ｇの変異が導入された塩基配列（配列番号１９）をテンプレートにして、ＰＣＲによる変異導入と配列確認を行った。複数の変異点はＰＣＲを繰り返して変異点を導入した。５種類の変異酵素を作製した（表４）。 3. Test example 2
(1) Modification of (1R,3S)-specific esterase (1R,3S)-A221F/A328G double mutation-introduced esterase with high selectivity for ethyl chrysanthemum is unable to be hydrolyzed by wild-type esterase. This is an esterase ((1R,3S)-specific esterase) that is highly selective for (1R,3S)-ethyl chrysanthemum. The mutation of Test Example 1 was further introduced into the above-mentioned (1R,3S)-specific esterase, and it was examined whether there was an effect of improving heat resistance (temperature stability). Using the nucleotide sequence (SEQ ID NO: 19) into which the A221F/A328G mutation was introduced as a template, mutation introduction and sequence confirmation were performed by PCR. Multiple mutation points were introduced by repeating PCR. Five types of mutant enzymes were created (Table 4).
（２）耐熱性（温度安定性）評価
　（１Ｒ，３Ｓ）特異的エステラーゼおよび変異酵素について、酵素抽出液を調製した。酵素抽出液に、サーマルサイクラーにより、３０、４０、５０、６０、７０、８０又は９０℃で３０分間加温した。加温後に遠心分離により固形分を取り除き、酵素サンプル液とした。酵素活性測定方法により、それぞれのサンプルの吸光度を測定することで酵素活性を測定した。各サンプルの最大値を１００％として、相対値で温度による酵素活性の変化を測定し、５０％失活温度（Ｔ５０値）を計測した（表５）。その結果、最大で１．７２℃の耐熱性向上が見られた。 (2) Evaluation of heat resistance (temperature stability) Enzyme extracts were prepared for the (1R, 3S) specific esterase and the mutant enzyme. 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 content was removed by centrifugation to obtain an enzyme sample solution. Enzyme activity was measured by measuring the absorbance of each sample using an enzyme activity measurement method. With the maximum value of each sample as 100%, changes in enzyme activity due to temperature were measured using relative values, and the 50% inactivation temperature (T50 value) was measured (Table 5). As a result, an improvement in heat resistance of up to 1.72°C was observed.
（３）アセトニトリル耐性評価
　（１Ｒ，３Ｓ）特異的エステラーゼおよび変異酵素について、酵素抽出液を調製した。酵素抽出液に、４０％量の超純水またはアセトニトリルを加えて、３０℃で３時間インキュベートした。超純水を加えたサンプルを基準にして、アセトニトリルを添加した場合の相対活性値を計算した。（１Ｒ，３Ｓ）特異的エステラーゼの活性値を基準に、それぞれの変異酵素の相対活性値を計算した。実験はすべて３回行い、平均値を計算した（表６）。その結果、特に、Ｓ２２０Ａ変異酵素、４重変異酵素、５重変異酵素で、アセトニトリルに対する耐性における顕著な向上が確認された。 (3) Acetonitrile resistance evaluation Enzyme extracts were prepared for (1R, 3S) specific esterases and mutant enzymes. 40% of 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 (1R,3S) specific esterase. All experiments were performed in triplicate and the average values were calculated (Table 6). As a result, it was confirmed that the S220A mutant enzyme, the quadruple mutant enzyme, and the quintuple mutant enzyme exhibited remarkable improvement in resistance to acetonitrile.
（４）メタノール耐性評価
　（１Ｒ，３Ｓ）特異的エステラーゼおよび変異酵素について、酵素抽出液を調製した。酵素抽出液に、４０％量の超純水またはメタノール加えて、３０℃で３時間インキュベートした。超純水を加えたサンプルを基準にして、メタノールを添加した場合の相対活性値を計算した。（１Ｒ，３Ｓ）特異的エステラーゼの活性値を基準に、それぞれの変異酵素の相対活性値を計算した。実験はすべて３回行い、平均値を計算した（表６）。その結果、特に、４重変異酵素（Ｒ２５Ｐ／Ｓ２２０Ａ／Ａ３１３Ｓ／Ｓ３１５Ｍ）で、メタノールに対する耐性における顕著な向上が確認された。 (4) Methanol tolerance evaluation Enzyme extracts were prepared for (1R, 3S) specific esterases and mutant enzymes. 40% of 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 with ultrapure water added. The relative activity value of each mutant enzyme was calculated based on the activity value of the (1R,3S) specific esterase. All experiments were performed in triplicate and the average values were calculated (Table 6). As a result, it was confirmed that, in particular, the quadruple mutant enzyme (R25P/S220A/A313S/S315M) showed a remarkable improvement in tolerance to methanol.
４．試験例３
（１）五重変異を有する菊酸エステラーゼの耐熱性向上
　上述の試験例によって同定された、エステラーゼに耐熱性を付与することができる５つのアミノ酸置換が、別のエステラーゼに対しても耐熱性を付与できるかをさらに検討した。
　既報（ＷＯ２０２０／１１６３３１）にて作製された改変菊酸エステラーゼ（野生型エステラーゼに対して五重変異（Ａ２２１Ｆ／Ｎ２２２Ｄ／Ｆ２９８Ｌ／Ｄ３２６Ｖ／Ａ３２８Ｇ）を有する）に対して、今回同定されたアミノ酸置換を導入し、耐熱性が向上するかを確認した。作製した改変酵素を表７に示す。 4. Test example 3
(1) Improving the thermostability of chrysanthemum esterase with quintuple mutations The five amino acid substitutions that can confer thermostability to esterases, which were identified through the above test examples, also confer thermostability to other esterases. Further consideration was given to whether it could be granted.
The amino acid substitutions identified this time were applied to the modified chrysanthemum esterase (which has five mutations (A221F/N222D/F298L/D326V/A328G) compared to the wild type esterase) produced in a previous report (WO2020/116331). We introduced this technology to see if it improved heat resistance. Table 7 shows the modified enzymes produced.
　五重変異菊酸エステラーゼおよび変異酵素について、大腸菌で発現させ、上述の１．（３）の方法で酵素抽出液を調製した。酵素抽出液をサーマルサイクラーにより、３０、４０、５０、６０、７０、８０又は９０℃のそれぞれの温度で３０分間加温した。加温後に遠心分離により固形分を取り除き、酵素サンプル液とした。酵素活性測定方法により、それぞれのサンプルの吸光度を測定して、酵素活性とした。各サンプルの最大値を１００％として、相対値で温度による酵素活性の変化を測定して、５０％失活温度（Ｔ５０値）を計測した。結果を表８に示す。表８に示される通り、全ての変異酵素において、耐熱性の顕著な向上が見られた。 The pentamutated chrysanthemum acid esterase and the mutant enzyme were expressed in E. coli, and the above-mentioned 1. An enzyme extract was prepared by the method (3). The enzyme extract was heated for 30 minutes at a temperature of 30, 40, 50, 60, 70, 80 or 90°C using a thermal cycler. After heating, the solid content was removed by centrifugation to obtain an enzyme sample solution. The absorbance of each sample was measured using the enzyme activity measurement method and determined as the enzyme activity. The maximum value of each sample was set as 100%, and the change in enzyme activity due to temperature was measured using relative values, and the 50% inactivation temperature (T50 value) was measured. The results are shown in Table 8. As shown in Table 8, all mutant enzymes showed a remarkable improvement in heat resistance.
（２）アセトニトリル耐性向上
　五重変異菊酸エステラーゼおよび変異酵素について、大腸菌で発現させ、上述の１．（３）の方法で酵素抽出液を調製した。酵素抽出液に、３０％の超純水またはアセトニトリルを加えて、３０℃で３時間インキュベートした。超純水を加えたサンプルを基準にして、アセトニトリルを添加した場合の相対活性値を計算した。五重変異菊酸エステラーゼの活性値を基準に、それぞれの変異酵素の相対活性値を計算した。実験はすべて３回行い、平均値を計算した。結果を表９に示す。表９に示される通り、全ての変異酵素でアセトニトリルに対する耐性における顕著な向上が確認された。 (2) Improving acetonitrile tolerance The five-fold mutant chrysanthemum esterase and the mutant enzyme were expressed in Escherichia coli, and the above-mentioned 1. An enzyme extract was prepared by the method (3). 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 five-fold mutant chrysanthemum acid esterase. All experiments were performed in triplicate and the average values were calculated. The results are shown in Table 9. As shown in Table 9, remarkable improvement in resistance to acetonitrile was confirmed for all mutant enzymes.
（３）メタノール耐性向上
　五重変異菊酸エステラーゼおよび変異酵素について、大腸菌で発現させ、上述の１．（３）の方法で酵素抽出液を調製した。酵素抽出液に、３０％の超純水またはメタノール加えて、３０℃で３時間インキュベートした。超純水を加えたサンプルを基準にして、メタノールを添加した場合の相対活性値を計算した。五重変異菊酸エステラーゼの活性値を基準に、それぞれの変異酵素の相対活性値を計算した。実験はすべて３回行い、平均値を計算した。結果を表１０に示す。表１０に示される通り、一部の変異酵素で、メタノールに対する耐性における顕著な向上が確認された。 (3) Improving methanol tolerance The fivefold mutant chrysanthemum acid esterase and the mutant enzyme were expressed in Escherichia coli, and the above-mentioned 1. An enzyme extract was prepared by the method (3). 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 with ultrapure water added. The relative activity value of each mutant enzyme was calculated based on the activity value of the five-fold mutant chrysanthemum acid esterase. All experiments were performed in triplicate and the average values were calculated. The results are shown in Table 10. As shown in Table 10, significant improvement in methanol tolerance was confirmed in some of the mutant enzymes.
　本発明の改変型エステラーゼを用いれば、農薬中間体として重要な菊酸を効率よく製造することができる。従って、本発明は、例えば、農薬製造分野において極めて有用である。 By using the modified esterase of the present invention, chrysanthemum acid, which is important as an agricultural chemical intermediate, can be efficiently produced. Therefore, the present invention is extremely useful, for example, in the field of agricultural chemical production.
　本出願は、日本で出願された特願２０２２－１３２０８２（出願日：２０２２年８月２２日）を基礎としており、その内容は本明細書に全て包含されるものである。 This application is based on Japanese Patent Application No. 2022-132082 (filing date: August 22, 2022) filed in Japan, the contents of which are fully included in this specification.

Claims (9)

  1. 　以下の（ｉ）～（ｉｉｉ）のいずれかの改変型エステラーゼ：
    　（ｉ）配列番号１、７又は３５のアミノ酸配列において、以下の（１）～（５）からなる群から選択される部位の少なくとも一つのアミノ酸が、別のアミノ酸で置換されたアミノ酸配列を含む、改変型エステラーゼ、
    （１）Ｓ１２
    （２）Ｒ２５
    （３）Ｓ２２０
    （４）Ａ３１３
    （５）Ｓ３１５
    　（ｉｉ）（ｉ）の改変型エステラーゼにおいて、さらに１個又は数個のアミノ酸が、置換（ただし、（ｉ）において置換されたアミノ酸部位を除く）、付加、挿入、又は欠失（ただし、（ｉ）において置換されたアミノ酸部位を除く）されており、且つ、温度安定性及び／又は有機溶媒に対しての耐性が、配列番号１、７又は３５のアミノ酸配列からなるエステラーゼのそれらよりも向上している、改変型エステラーゼ、
    　（ｉｉｉ）（ｉ）の改変型エステラーゼにおいて、（ｉ）において置換されたアミノ酸以外のアミノ酸が、さらに別のアミノ酸に置換されている改変型エステラーゼであって、ここで、該改変型エステラーゼが、（ｉ）の改変型エステラーゼと７０％以上の同一性を有し、且つ、温度安定性及び／又は有機溶媒に対しての耐性が、配列番号１、７又は３５のアミノ酸配列からなるエステラーゼのそれらよりも向上している、改変型エステラーゼ。     Any of the following modified esterases (i) to (iii):
    (i) The amino acid sequence of SEQ ID NO: 1, 7, or 35 includes an amino acid sequence in which at least one amino acid at a site selected from the group consisting of (1) to (5) below is substituted with another amino acid. , modified esterase,
    (1) S12
    (2) R25
    (3) S220
    (4) A313
    (5) S315
    (ii) In the modified esterase of (i), one or more amino acids may be substituted (excluding the amino acid site substituted in (i)), added, inserted, or deleted (however, ( (excluding the amino acid site substituted in i)), and has improved temperature stability and/or resistance to organic solvents than those of the esterase consisting of the amino acid sequence of SEQ ID NO: 1, 7, or 35. modified esterase,
    (iii) In the modified esterase of (i), an amino acid other than the amino acid substituted in (i) is further substituted with another amino acid, wherein the modified esterase Esterases that have 70% or more identity with the modified esterase of (i) and have temperature stability and/or resistance to organic solvents and consist of the amino acid sequence of SEQ ID NO: 1, 7, or 35. A modified esterase that is more improved than the previous one.
  2. 　前記（ｉ）において、アミノ酸の置換が以下からなる群から選択される少なくとも一つである、請求項１記載の改変型エステラーゼ：
    （１）Ｓ１２Ｐ
    （２）Ｒ２５Ｐ
    （３）Ｓ２２０Ａ
    （４）Ａ３１３Ｓ
    （５）Ｓ３１５Ｍ。     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. 　請求項１又は２記載の改変型エステラーゼをコードする核酸。 A nucleic acid encoding the modified esterase according to claim 1 or 2.
  4. 　請求項３記載の核酸を含む、発現カセット又は組換えベクター。 An expression cassette or recombinant vector comprising the nucleic acid according to claim 3.
  5. 　請求項４記載の発現カセット又は組換えベクターを用いて形質転換された形質転換体。 A transformant transformed using the expression cassette or recombinant vector according to claim 4.
  6. 　請求項５記載の形質転換体を培養する工程を含む、改変型エステラーゼの製造方法。 A method for producing a modified esterase, comprising the step of culturing the transformant according to claim 5.
  7. 　請求項１又は２記載の改変型エステラーゼを含む、酵素剤。 An enzyme preparation comprising the modified esterase according to claim 1 or 2.
  8. 　菊酸生成用である、請求項７記載の酵素剤。 The enzyme agent according to claim 7, which is for producing chrysanthemum acid.
  9. 　菊酸エチルに、請求項１又は２記載の改変型エステラーゼを作用させる工程を含む、菊酸の製造方法。 A method for producing chrysanthemum acid, comprising a step of causing the modified esterase according to claim 1 or 2 to act on ethyl chrysanthemum acid.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020116331A1 (en) * 2018-12-06 2020-06-11 天野エンザイム株式会社 Modified chrysanthemic acid esterase

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020116331A1 (en) * 2018-12-06 2020-06-11 天野エンザイム株式会社 Modified chrysanthemic acid esterase

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MARCUS SCHÜTTE ; SUSANNE FETZNER: "EstA from Arthrobacter nitroguajacolicus Rü61a, a Thermo and Solvent-Tolerant Carboxylesterase Related to Class C β-Lactamases", CURRENT MICROBIOLOGY, SPRINGER-VERLAG, NE, vol. 54, no. 3, 8 February 2007 (2007-02-08), Ne , pages 230 - 236, XP019490105, ISSN: 1432-0991, DOI: 10.1007/s00284-006-0438-2 *

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