WO2024043203A1 - Estérase modifiée - Google Patents
Estérase modifiée Download PDFInfo
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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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- WIPO (PCT)
- Prior art keywords
- esterase
- amino acid
- modified
- seq
- present
- Prior art date
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- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- XIXADJRWDQXREU-UHFFFAOYSA-M lithium acetate Chemical compound [Li+].CC([O-])=O XIXADJRWDQXREU-UHFFFAOYSA-M 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 230000002101 lytic effect Effects 0.000 description 1
- IVSZLXZYQVIEFR-UHFFFAOYSA-N m-xylene Chemical group CC1=CC=CC(C)=C1 IVSZLXZYQVIEFR-UHFFFAOYSA-N 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 238000010297 mechanical methods and process Methods 0.000 description 1
- RCYSGAYIEFAJTG-UHFFFAOYSA-N methyl acetate;propanoic acid Chemical compound CCC(O)=O.COC(C)=O RCYSGAYIEFAJTG-UHFFFAOYSA-N 0.000 description 1
- 239000004292 methyl p-hydroxybenzoate Substances 0.000 description 1
- 235000010270 methyl p-hydroxybenzoate Nutrition 0.000 description 1
- 229940017219 methyl propionate Drugs 0.000 description 1
- 229960002216 methylparaben Drugs 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- SYSQUGFVNFXIIT-UHFFFAOYSA-N n-[4-(1,3-benzoxazol-2-yl)phenyl]-4-nitrobenzenesulfonamide Chemical class C1=CC([N+](=O)[O-])=CC=C1S(=O)(=O)NC1=CC=C(C=2OC3=CC=CC=C3N=2)C=C1 SYSQUGFVNFXIIT-UHFFFAOYSA-N 0.000 description 1
- 229910017464 nitrogen compound Inorganic materials 0.000 description 1
- 150000002830 nitrogen compounds Chemical class 0.000 description 1
- LYGJENNIWJXYER-UHFFFAOYSA-N nitromethane Chemical compound C[N+]([O-])=O LYGJENNIWJXYER-UHFFFAOYSA-N 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
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- ATKIGFUAOXHQMV-UHFFFAOYSA-N octan-1-ol Chemical compound CCCCCCCCO.CCCCCCCCO ATKIGFUAOXHQMV-UHFFFAOYSA-N 0.000 description 1
- XAQDNRLCLMAVQN-UHFFFAOYSA-N octan-2-ol Chemical compound CCCCCCC(C)O.CCCCCCC(C)O XAQDNRLCLMAVQN-UHFFFAOYSA-N 0.000 description 1
- ZJKABZNFELLAQQ-UHFFFAOYSA-N octane Chemical compound CCCCCCCC.CCCCCCCC ZJKABZNFELLAQQ-UHFFFAOYSA-N 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- JCGNDDUYTRNOFT-UHFFFAOYSA-N oxolane-2,4-dione Chemical compound O=C1COC(=O)C1 JCGNDDUYTRNOFT-UHFFFAOYSA-N 0.000 description 1
- QNGNSVIICDLXHT-UHFFFAOYSA-N para-ethylbenzaldehyde Natural products CCC1=CC=C(C=O)C=C1 QNGNSVIICDLXHT-UHFFFAOYSA-N 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- KVYJZMJPHSXEDZ-UHFFFAOYSA-N pentan-1-ol Chemical compound CCCCCO.CCCCCO KVYJZMJPHSXEDZ-UHFFFAOYSA-N 0.000 description 1
- RIDQBQWONMVCHX-UHFFFAOYSA-N pentan-3-ol Chemical compound CCC(O)CC.CCC(O)CC RIDQBQWONMVCHX-UHFFFAOYSA-N 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 229960003742 phenol Drugs 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000002728 pyrethroid Substances 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 229940107700 pyruvic acid Drugs 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 102000037983 regulatory factors Human genes 0.000 description 1
- 108091008025 regulatory factors Proteins 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000007480 sanger sequencing Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
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- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 230000003381 solubilizing effect Effects 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 239000004455 soybean meal Substances 0.000 description 1
- 238000001694 spray drying Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
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- 229950011008 tetrachloroethylene Drugs 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- DTQVDTLACAAQTR-UHFFFAOYSA-N trifluoroacetic acid Substances OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 1
- 229960004418 trolamine Drugs 0.000 description 1
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
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- 239000004474 valine Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
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- 239000012138 yeast extract Substances 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
La présente invention fournit l'une quelconque des estérases modifiées de (i) à (iii) ci-dessous. (i) Estérase modifiée comprenant une séquence d'acides aminés dans laquelle au moins un acide aminé à un site choisi dans le groupe constitué par (1) à (5) ci-dessous est substitué par un autre acide aminé dans une séquence d'acides aminés de SEQ ID NO : 1, 7 ou 35. (1) S12, (2) R25, (3) S220, (4) A313, (5) S315. (Ii) une estérase modifiée dans laquelle un ou plusieurs acides aminés (à condition que l'acide aminé au niveau du site de substitution en (i) soit exclu) sont en outre substitués ou supprimés dans l'estérase modifiée de (i), ou un ou plusieurs acides aminés sont ajoutés ou insérés dans l'estérase modifiée de (i) et dans lesquels la stabilité thermique et/ou la résistance aux solvants organiques sont améliorées par comparaison avec celles d'une estérase d'une séquence d'acides aminés de SEQ ID NO : 1, 7 ou 35. (Iii) Une estérase modifiée dans laquelle un acide aminé autre que l'acide aminé substitué en (i) est en outre substitué par un autre acide aminé dans l'estérase modifiée de (i), l'estérase modifiée ayant une identité de 70 % ou plus avec l'estérase modifiée de (i), et la stabilité thermique et/ou la résistance aux solvants organiques étant améliorées par comparaison avec celles d'une estérase d'une séquence d'acides aminés de SEQ ID NO : 1, 7 ou 35.
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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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