WO2008047819A1 - Novel ester hydrolase, gene encoding the same, and use thereof - Google Patents

Novel ester hydrolase, gene encoding the same, and use thereof Download PDF

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Publication number
WO2008047819A1
WO2008047819A1 PCT/JP2007/070225 JP2007070225W WO2008047819A1 WO 2008047819 A1 WO2008047819 A1 WO 2008047819A1 JP 2007070225 W JP2007070225 W JP 2007070225W WO 2008047819 A1 WO2008047819 A1 WO 2008047819A1
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Prior art keywords
ibuprofen
optically active
ester
sequence
polypeptide
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PCT/JP2007/070225
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French (fr)
Japanese (ja)
Inventor
Teruaki Takesue
Noriyuki Kizaki
Yoshihiko Yasohara
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Kaneka Corporation
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Publication of WO2008047819A1 publication Critical patent/WO2008047819A1/en

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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)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/003—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions
    • C12P41/005—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions by esterification of carboxylic acid groups in the enantiomers or the inverse reaction
    • 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
    • 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
    • C12P7/62—Carboxylic acid esters

Definitions

  • Novel ester hydrolases genes encoding them, and methods for using them
  • the present invention relates to an ester hydrolase having an activity of asymmetrically hydrolyzing an ester compound to produce an optically active carboxylic acid or to leave an optically active ester, DNA encoding the enzyme,
  • the present invention relates to a vector having DNA and a transformed cell transformed with this vector.
  • it is useful as a catalyst in the production of optically active ibuprofen, which is a compound useful as a synthetic material for pharmaceutical and agricultural chemicals.
  • Porcine liver-derived esterases and the like are generally used for such purposes, but they are expensive and have a limited supply amount, which is disadvantageous for industrial use. .
  • a method of using a microorganism-derived esterase instead of porcine liver esterase has also been tried.
  • each has a characteristic substrate specificity, so the selectivity and reaction rate vary greatly depending on the compound applied.
  • Patent Document 1 As part of the enzymatic synthesis method, a method has been proposed to reduce the catalyst cost by using genetically modified microorganisms (Patent Document 1), but the enzyme used in this method has an optimal reaction temperature of 25- 4 It is hard to say that it is a practical method as low as 5 ° C. Therefore, development of a more efficient method for synthesizing (S) -ibuprofen, which is an alternative to these methods, has been desired! /.
  • Patent Document 1 JP-A-8-242853
  • Non-Patent Document 1 S. Adams, et al., J. Pharm. Pharmac, 28, 256 (1976)
  • Non-patent document 2 A. J. Hutt and J. Caldwell, Clinical Pharmacokinetics., 9, 371 (1984)
  • Non-patent document 3 Williams, Tsuji, et al., Biochem. Pharmac, 35, 3403 (1986)
  • Non-patent document 4 J. Caldwell and MV Marsh, Biochem. Pharmac, 32, 1667 (1983)
  • Non-patent document 5 Duan, G. and Chen, JY, Biotechnol. Lett., 16, 1065 (1994)
  • Non-patent document 6 Mustranta, A., J. Org. Chem., 59, 4410 (1994)
  • Non-Patent Document 7 Lee, W. ⁇ ., Et al., J. Ferment. Bioeng., 80, 613 (1995)
  • the inventors of the present application have released a novel ester hydrolase having a high optimal reaction temperature of 60 to 70 ° C from a fine cattle belonging to the genus Rhodococcus. Furthermore, it was found that by using this enzyme, (S) -ibuprofen can be efficiently produced from racemic ibuprofen methyl, and the present invention was completed.
  • the present invention is an ester hydrolase which is a polypeptide according to any one of (1) to (3) below.
  • (1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 in the Sequence Listing;
  • the present invention is an ester hydrolase having the following physicochemical properties (1) to (5).
  • Inhibitor Inhibited by silver ion, mercury ion, and odoacetic acid, not inhibited by PMSF.
  • the present invention is a DNA encoding the ester hydrolase.
  • the present invention is a DN sequence consisting of the base sequence described in any of (1) to (4) below.
  • the present invention is a vector having the DNA.
  • the present invention also relates to a transformed cell transformed with the vector.
  • the present invention is an optically active carboxylate compound and / or remaining by reacting the ester hydrolase of the present invention and / or the transformed cell of the present invention with an ester compound.
  • a novel ester hydrolase a DNA encoding the same, a vector having the DNA, a transformant transformed with the vector, an optically active carboxylic acid compound using them, and / or Alternatively, a method for producing an optically active ester compound is provided.
  • the organism used as the origin of the ester hydrolase of the present invention is not particularly limited, and examples thereof include fine cattle belonging to the genus Rhodococcus, and in particular, Rhodococcus sp. KNK0401 preferable.
  • Rhodococcus sp. KNK0401 The mycological properties of Rhodococcus sp. KNK0401 are described below.
  • Oxidase Yinsei
  • Rhodococcus sp (The original deposit date was transferred to the international deposit under the Budapest Treaty (September 21, 2007)).
  • the medium used for culturing the microorganism producing the enzyme of the present invention is not particularly limited as long as the microorganism can grow.
  • a normal liquid nutrient medium containing a carbon source, nitrogen source, inorganic salts, organic nutrients and the like can be used.
  • purification of the enzyme from the microorganism producing the enzyme of the present invention can be carried out by a conventional method.
  • the enzyme of the present invention can be isolated from Rhodococcus sp. KNK0401 as follows, for example. First, the strain is cultured in an appropriate medium, and the cells are collected from the culture solution by centrifugation. The cells are crushed with, for example, Dynomill (Dyno-Mill), and the cell residue is removed by centrifugation to obtain a cell-free extract. For this cell-free extract, columns such as salting out (ammonium sulfate precipitation, sodium phosphate precipitation, etc.), solvent precipitation (protein fraction precipitation with acetone or ethanol, etc.), dialysis, gel filtration, ion exchange, reverse phase, etc.
  • salting out ammonium sulfate precipitation, sodium phosphate precipitation, etc.
  • solvent precipitation protein fraction precipitation with acetone or ethanol, etc.
  • dialysis gel filtration, ion exchange, reverse phase, etc.
  • the enzyme can be purified by performing treatments such as chromatography and ultrafiltration alone or in combination. Ester hydrolysis activity is performed by measuring the increase in absorbance at 405 nm at 30 ° C by adding the substrate p-nitrophenyl acetate ImM and enzyme to lOOmM phosphate phosphate buffer (pH 7.0). obtain. Under these reaction conditions, the activity of hydrolyzing l ⁇ mol of p-nitrotrophyl acetate per minute is defined as lu nit.
  • an enzyme is "inhibited" by a specific compound means that when a specific compound is added to a reaction solution of the enzyme at a concentration of ImM or less, the enzyme is not added. The activity decreases to% or less!
  • the molecular weight of the enzyme was measured using Superdex 200 HR 10/30 (lOmml. D. X 30cm) Performed by gel filtration analysis using a column (GE Healthcare Biosciences). As eluent, 10 mM potassium phosphate buffer ( ⁇ 7 ⁇ 0) containing 0.2 M NaCl is used. Subunit molecular weight is calculated from the relative mobility of standard proteins by electrophoresis on 10% SDS-polyacrylamide gel under reducing conditions (reducing agent: 2% ( ⁇ / ⁇ ) 2-mercaptoethanol). To be determined.
  • the ester hydrolase of the present invention has, for example, the following physicochemical properties (1) to (5).
  • Inhibitor Inhibited by silver ion, mercury ion, and odoacetic acid, not inhibited by PMSF.
  • the enzyme having almost the same properties as the enzyme of the present invention may be a natural enzyme or a recombinant enzyme.
  • the recombinant enzyme can be obtained by introducing a gene of an enzyme derived from the genus Rhodococcus determined by the following method into an appropriate host.
  • a mutant enzyme in which one or several amino acids in the amino acid sequence of the enzyme are substituted, deleted, inserted or added is added. It is also possible to produce it.
  • Mutant enzymes thus obtained are also included in the present invention as long as they have the activity of acting on ibuprofen methyl to produce (R) -ibuprofen and leave (S) -ibuprofenmethyl.
  • the number of amino acids to be deleted, replaced, inserted or added is preferably 40 or less, more preferably 25 or less, still more preferably 10 or less, most preferably 5 or 4 , 3 or 2 or less.
  • a polypeptide having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing acts on ibuprofen methyl to produce (R) -ibuprofen (S)-
  • a polypeptide having the activity of leaving buprofen methyl is also included in the polypeptide of the present invention.
  • a polypeptide having 85% or more sequence identity with the amino acid sequence of SEQ ID NO: 2 in the sequence listing is a force S included in the polypeptide of the present invention, and its sequence identity is preferably 90% or more, more preferably 95% or more. More than 98% is more preferable. More than 99% is more preferable.
  • sequence identity of amino acid sequences is determined by comparing the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing with the evaluated amino acid sequence and the number of positions where the amino acid matches in both sequences. Divided by the number of mino acids and then multiplied by 100.
  • the enzyme of the present invention purified as described above can be denatured under appropriate conditions and then digested with an appropriate endopeptidase to obtain a peptide fragment constituting the enzyme of the present invention.
  • These peptide fragments are purified by reverse phase HPLC using, for example, a YMC-Pack SIL-06 (manufactured by YMC) column, and then subjected to a protein sequencer, whereby the partial amino acid sequence of the enzyme of the present invention is obtained.
  • YMC-Pack SIL-06 manufactured by YMC
  • a PCR (Polymerase Chain Reaction) primer can be synthesized based on the partial amino acid sequence information obtained.
  • chromosomal DNA of the microorganism is prepared from the microorganism that is the origin of the ester hydrolase of the present invention by, for example, the method of Murray et al. (Nucleic Acids Res., 8: 4321-4325 (1980)). obtain.
  • PCR can be performed using the PCR primers described above to amplify a part of the DNA encoding the enzyme (core arrangement IJ) and determine the nucleotide sequence.
  • the base sequence can be determined by a dideoxy chain termination method or the like, and can be performed using, for example, ABI 3130xlDNA Sequencer (Applied Biosystems).
  • the chromosomal DNA of the microorganism is digested with a restriction enzyme whose recognition sequence does not exist in the core sequence, and the generated DNA fragment is A cage DNA for reverse PCR (Nucleic Acids Res., 16: 8186 (1988)) is prepared by self-cyclization with T4 ligase.
  • a primer that acts as a starting point for DNA synthesis is synthesized outside the core sequence, and the peripheral region of the core sequence is amplified by inverse PCR.
  • the base sequence of the entire coding region of the target enzyme can be read.
  • DNA encoding the polypeptide of the present invention for example, DNA consisting of the base sequence shown in SEQ ID NO: 1 in the sequence listing, or a base sequence complementary to the base sequence shown in SEQ ID NO: 1 in the sequence listing And DNA encoding a polypeptide that has the activity of hybridizing under stringent conditions and acting on ibuprofen phenmethyl to produce (R) -ibuprofen and (S) -ibuprofenmethyl to remain. The power to raise S.
  • DNA encoding a polypeptide having the activity of leaving ibuprofen methyl refers to a colony under stringent conditions using a DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 1 in the Sequence Listing as a probe. DNA obtained by using the 'noisy hybridization method, plaque' hybridization method, or Southern hybridization method, etc. and acting on ibuprofen methyl to produce (R) -ibuprofen.
  • S refers to DNA encoding a polypeptide having the activity of leaving ibuprofen methyl.
  • noisy Pre-Daisyong is Molecular Cloning, A laboratory manual, second edition
  • DNA that hybridizes under stringent conditions means, for example, hybridization at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which colony or plaque-derived DNA is immobilized. After dialysis, filter at 65 ° C using 2x SSC solution (1x SSC solution consists of 150mM sodium chloride, 15mM sodium citrate). It is possible to increase the DNA that can be obtained by washing. Preferably washed with 0.5 times SSC solution at 65 ° C, more preferably washed with 0.2 times SSC solution at 65 ° C, more preferably 0.1 times SSC at 65 ° C This DNA can be obtained by washing with a solution.
  • the DNA that can be hybridized under the above conditions is 85% or more, preferably 90% or more, more preferably 95% or more, and still more preferably 98%, with the DNA represented by SEQ ID NO: 1.
  • SEQ ID NO: 1 the DNA represented by SEQ ID NO: 1.
  • sequence identity means that two DNAs to be compared are optimally aligned and both nucleic acid bases (eg, A, T, C, G, U, or I) are both aligned. The number of matching positions in this sequence is divided by the total number of comparison bases, and this result is expressed by multiplying by 100.
  • Sequence identity can be calculated, for example, using the following sequence analysis tool: GCG Wise onsin Package (Program Manual ror he Wisconsin Package, Version 8, September 1994, enetics Computer Group, 575 Science Drive Medison , Wisconsin, USA 53711; Rice, P. (1996) Program Manual for EGCG Package, Peter Rice, The Sanger Centre, Hinxton Hall, Cambridge, CB10 IRQ, England), and the ExPASy World Wide Web for molecular biology Geneva University Hospital and University or ueneva, en eva, Switzerland.
  • GCG Wise onsin Package Program Manual ror he Wisconsin Package, Version 8, September 1994, enetics Computer Group, 575 Science Drive Medison , Wisconsin, USA 53711
  • Rice, P. (1996) Program Manual for EGCG Package, Peter Rice, The Sanger Centre, Hinxton Hall, Cambridge, CB10 IRQ, England), and the ExPASy World Wide Web for molecular biology Geneva University Hospital and University or ueneva, en eva, Switzerland.
  • the ester hydrolase is expressed in an appropriate host microorganism.
  • Any vector can be used as long as it is available.
  • examples of such vector DNA include plasmid vector 1, phage vector, cosmid vector and the like.
  • a shuttle vector that can exchange genes with other host strains can also be used.
  • such vector DNA is operably linked to promoters (lacUV5 promoter, trp promoter, trc promoter, tac promoter, lpp promoter, tufB promoter, recA promoter motor, PL promoter, etc.), enhancer sequences, etc.
  • pUCNT WO94 / 03613
  • This plasmid pUCNT is 1 Since it has an insertion site such as Ndel or EcoRI site downstream of the ac promoter, it can be suitably used.
  • the obtained recombinant plasmid having ester hydrolase can be introduced into a host cell by a conventional method.
  • host cells bacteria, yeasts, filamentous fungi, plant cells, animal cells and the like can be used.
  • the use of E. coli is particularly preferred.
  • the introduction of the plasmid into the host can be performed by methods well known to those skilled in the art, for example, a method including a step of mixing a recombinant host cell with a competent host cell, a conjugative transfer using a helper plasmid.
  • the plasmid introduced into the host can replicate autonomously as an episome, or all or part of it can be integrated into the chromosome and replicated together with the chromosome.
  • optically active carboxylic acid (S) -ibuprofen is obtained, for example, as follows.
  • ibuprofen methyl may be used as the substrate.
  • the reaction can be carried out by adding the substrate ibuprofen methyl, a microorganism culture or a treated product thereof in an appropriate solvent, and stirring under pH adjustment.
  • the reaction is carried out at a temperature of 10 ° C to 70 ° C, pH 4 to 10;
  • the substrate concentration is 0.1% to 90% (W / V), but the substrate can be added continuously.
  • the reaction can be carried out batchwise or continuously.
  • the treated product of microorganisms is, for example, a crude extract, cultured cells, freeze-dried organisms, acetone-dried organisms, or a ground product of these cells, and the catalytic activity of ester hydrolase. Means the remaining item.
  • ibuprofen methyl is subjected to treatment such as centrifugation and filtration as necessary when microorganisms are used, etc. to remove cell suspensions, and then extracted with an organic solvent such as ethyl acetate or toluene.
  • Dehydrate with a dehydrating agent such as sodium sulfate remove the organic solvent under reduced pressure, and It can be purified by performing a treatment such as distillation or chromatography (for example, silica gel column chromatography).
  • a treatment such as distillation or chromatography (for example, silica gel column chromatography).
  • the obtained optically active ibuprofen methyl can be chemically hydrolyzed by a conventional method. For example, it can be hydrolyzed by stirring under conditions of a strong base in an appropriate solvent.
  • Quantification of ibuprofen methyl can be performed by gas chromatography using TC-FFAP (manufactured by GL Sciences Inc.), chromatography at a column temperature of 80 ° C to 200 ° C, and detection by FID. .
  • the culture was performed for 3 days under the condition of 0.5 L / min.
  • the pH was adjusted to 7.0 with a 5N aqueous sodium hydroxide solution.
  • the cells were collected from the culture solution by centrifugation. In this way, 470 g of wet cells of the strain was obtained.
  • the wet cells were suspended in 2 L of 10 mM potassium phosphate buffer (pH 7.0) and heat-treated by stirring for 20 minutes in a 50 ° C constant temperature bath.
  • the cells were crushed with Dynomill (Dyno-Mill).
  • the cell residue was removed from the crushed cell by centrifugation to obtain 2100 ml of a cell-free extract.
  • the resulting precipitate was removed by centrifugation.
  • ester hydrolase activity is basically performed by measuring lOOmM potassium phosphate buffer.
  • the substrate p-nitrophenyl acetate ImM and enzyme were added to (pH 7.0), and the increase in absorbance at a wavelength of 405 nm was measured at 30 ° C.
  • the reaction product was identified by gas chromatography using ibuprofen instead of the substrate P-nitrophenyl acetate.
  • the molecular weight of the enzyme is measured using a Superdex 200 HR 10/30 (lOmml. D. X 30cm) column (manufactured by GE Healthcare Biosciences Inc.) and contains 0.2M sodium chloride as the eluent. When 10 mM potassium phosphate buffer (pH 7.0) was used, it was about 57,000.
  • the molecular weight of the subunit of the enzyme is calculated from the relative mobility of the standard protein after electrophoresis on 10% SDS-polyacrylamide gel under reducing conditions (reducing agent: 2% (V / v) 2-mercaptoethanol). Was determined by As a result, the molecular weight of the subunit of this enzyme was about 37,000. [0054] [Table 1]
  • Rhodococcus sp. KNK0401 Chromosomal DNA was extracted in accordance with the method of Nakuryoku et al., Murray et al. (Nucleic Acids Res., 8: 4321-4325 (1980)).
  • the purified ester hydrolase obtained as in Example 1 was denatured in the presence of 8M urea and then digested with acrymopacter-derived lysyl endopeptidase (manufactured by Wako Pure Chemical Industries, Ltd.). The sequence of the fragment was determined by the Edman method. Considering the DNA sequence expected from this amino acid sequence, two kinds of PCR primers 5'—CCRTGR TTRTANCCYTCCCA-3 ′ (primer 1: SEQ ID NO: 3), 5′—GARGCNG TNAGYGTNGAYGG-3 ′ (primer 2: Sequence listing SEQ ID NO: 4) was synthesized.
  • Primer 1 and Primer 2 2 types of primers 50 pmol each, chromosomal DNA 240 ng, d NTP 10 nmol each, ExTaq (manufactured by Takara Bio Inc.) 1. Prepare ExTaq buffer solution 50 1 containing 3 U, and heat denaturation ( 97 ° C, 30 seconds), annealing (50 ° C, 1 minute), extension reaction (72 ° C, 1 minute) for 30 cycles. After cooling to 4 ° C, amplified DNA was confirmed by agarose genomic electrophoresis. .
  • the amplified DNA was subcloned into pT7Blue Vector (Novagen), and its nucleotide sequence was determined. From this result and the result of the core sequence, the entire nucleotide sequence of DNA encoding the ester hydrolase was determined. The entire base sequence and the deduced amino acid sequence encoded by the DNA are shown in SEQ ID NO: 2 in the sequence listing.
  • primers (primer 5 and primer 6) 50 pmol each, Rhodococcus sp. KNK0401 chromosomal DNA 15 ng, dNTP 10 nmol each, ExTaq (manufactured by Takara Bio Inc.) 1.
  • ExTaq buffer containing 3 U Prepare 501, heat denaturation (97 ° C, 30 seconds), annealing (60 ° C, 1 minute), extension reaction (72 ° C, 5 minutes) for 30 cycles, and after cooling to 4 ° C The amplified DNA was confirmed by agarose gel electrophoresis.
  • This amplified fragment was digested with Ndel and EcoRI and inserted into the Ndel and EcoRI sites downstream of the lac promoter of the plasmid pUCNT (WO94 / 03613) to obtain a recombinant vector pNTHR.
  • the recombinant vector pNTHR obtained in Example 4 was mixed with DNA Ligation Kit Ver. 2.1 I solution (manufactured by Takara Bio Inc.) and incubated at 16 ° C. for 30 minutes. The resulting reaction solution was added to Escherichia coli HB101 combined cell (manufactured by Takara Bio Inc.), incubated on ice for 30 minutes, incubated at 42 ° C for 45 seconds, and then cooled on ice for 2 minutes, Recombinant E. coli HB101 (pNTHR) was obtained.
  • Escherichia coli HB101 (pNTHR), a transformant obtained in this way, has the accession number FERM P-20271, dated October 22, 2004, Tsukuba Sakai Higashi 1-chome, 1-chome, Ibaraki, Japan 1 No. (Postal code: 305-8566) Deposited at the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary.
  • Recombinant Escherichia coli HB101 (pNTHR) obtained in Example 5 was mixed with 2 XYT medium containing 120 ⁇ g / ml ampicillin (batato'tryptone 1 ⁇ 6% (w / v), butato'yeast extract 1 ⁇ 0% (w / v), NaClO. 5% (w / v), pH 7.0), and the resulting culture broth was sonicated to obtain a cell-free extract. The ester hydrolysis activity of this cell-free extract was measured by the method described in Example 2.
  • E. coli HB101 (pNTHR) showed a clear increase in ester hydrolysis activity as compared to E. coli HB101 (pUCNT), which is a transformant of only the vector plasmid.
  • the recombinant Escherichia coli HB101 (pNTHR) obtained in Example 5 was inoculated into 50 ml of 2X YT medium sterilized in a 500 ml volumetric flask and cultured with shaking at 37 ° C for 48 hours. The resulting culture solution to give a P H7.
  • Cell-free extract by adjusting to sonication at 0. 10 mg of racemic ibuprofen methyl was added to 1 ml of the cell-free extract and shaken at 30 ° C. for 12 hours. After the conversion reaction, the reaction solution is saturated with ammonium sulfate, extracted with ethyl acetate, and remains.
  • CHIRALCEL ⁇ J—H manufactured by Daicel Chemical Industries, Ltd.
  • flow rate 1. Oml / min.
  • elution time R-form 30 minutes, S-form 25 minutes
  • the cells were obtained from 1 L of the Rhodococcus sp. KNK0401 culture solution obtained by the method described in Example 1 by centrifugation, and racemic into 250 ml of OOmM potassium phosphate buffer (pH 8.0). 2.5g of ibuprofen methinore was added and stirred for 25 days at 40 ° C. After the conversion reaction, the reaction solution was saturated with ammonium sulfate, extracted with ethyl acetate, and the remaining ibuprofen methyl and the produced ibuprofen were analyzed by the method described in Example 7. As a result, 750 mg of ibuprofen was produced.

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Abstract

Disclosed are: a polypeptide having an activity of hydrolyzing an ester, which is isolated from a microorganism belonging the genus Rhodococcus; DNA encoding the polypeptide; a transformant capable of producing the polypeptide; and a method for producing an optically active carboxylic acid or an optically active ester by reacting the polypeptide or the transformant with an ester. It becomes possible to efficiently producing an optically active compound (e.g., optically active ibuprofen) which is useful as a pharmaceutical agent or an intermediate for a pharmaceutical agent.

Description

明 細 書  Specification
新規エステル加水分解酵素、およびこれをコードする遺伝子、ならびにこ れらの利用法  Novel ester hydrolases, genes encoding them, and methods for using them
技術分野  Technical field
[0001] 本発明はエステル化合物を不斉的に加水分解して、光学活性カルボン酸を生成も しくは光学活性エステルを残存させる活性を有するエステル加水分解酵素、この酵 素をコードする DNA、この DNAを有するベクター、このベクターで形質転換された 形質転換細胞に関する。特に医薬 '農薬の合成材料として有用な化合物である光学 活性イブプロフェンを製造する際に触媒として有用である。  [0001] The present invention relates to an ester hydrolase having an activity of asymmetrically hydrolyzing an ester compound to produce an optically active carboxylic acid or to leave an optically active ester, DNA encoding the enzyme, The present invention relates to a vector having DNA and a transformed cell transformed with this vector. In particular, it is useful as a catalyst in the production of optically active ibuprofen, which is a compound useful as a synthetic material for pharmaceutical and agricultural chemicals.
背景技術  Background art
[0002] 近年、酵素を有機合成に応用する試みが盛んに行われている。特に、高い基質選 択性を有するエステラーゼを用い、ラセミ体のエステル化合物から立体選択的加水 分解反応により、 1種の異性体を取得する方法や、プロキラルな化合物から位置選択 的加水分解反応によりキラルな化合物を製造する反応、あるいは、それらの逆反応を 利用した立体選択的なエステルの生産方法などは、工業的に有用性が高い。  [0002] In recent years, many attempts have been made to apply enzymes to organic synthesis. In particular, esterase with high substrate selectivity is used to obtain one isomer by stereoselective hydrolysis from a racemic ester compound, or chiral by regioselective hydrolysis from a prochiral compound. Reactions for producing such compounds, or stereoselective ester production methods utilizing their reverse reactions are highly industrially useful.
[0003] このような目的に供されるエステラーゼとしては、豚肝臓由来エステラーゼなどが一 般的であるが、高価であり、且つ供給量にも制限があるため、工業的使用には不利 である。豚肝臓エステラーゼの代わりに微生物由来のエステラーゼを用いる方法も試 みられている。生産する生物の起源が異なる場合、各々特徴的な基質特異性を有す るため、適用する化合物によって選択性や反応速度は大きく異なる。  [0003] Porcine liver-derived esterases and the like are generally used for such purposes, but they are expensive and have a limited supply amount, which is disadvantageous for industrial use. . A method of using a microorganism-derived esterase instead of porcine liver esterase has also been tried. When the origins of the organisms to be produced differ, each has a characteristic substrate specificity, so the selectivity and reaction rate vary greatly depending on the compound applied.
[0004] 現在、非ステロイド系抗炎症剤であるイブプロフェンはラセミ体の形で広く使用され ているが、両ェナンチォマーのうち、（S)体は (R)体に比較して約 150倍活性が高く (非特許文献 1、 2)、また、（R)体は脂肪組織中に蓄積しやすぐ脂質代謝異常や膜 機能の破壊をまねくことが知られている（非特許文献 3、 4)。従って、（S)—イブプロ フェンのみの抗炎症剤の開発が盛んに行われており、そのための ） イブプロフエ ンの効率的な合成法が広く検討されて!/、る。  [0004] Currently, ibuprofen, a non-steroidal anti-inflammatory drug, is widely used in racemic form, but among both enantiomers, the (S) form is approximately 150 times more active than the (R) form. High (Non-patent Documents 1 and 2), and (R) body is known to accumulate in adipose tissue and immediately cause abnormal lipid metabolism and destruction of membrane function (Non-Patent Documents 3 and 4). Therefore, (S) -ibuprofen-only anti-inflammatory agents have been actively developed, and an efficient method for synthesizing ibuprofen has been widely studied!
[0005] (S) イブプロフェンの合成法として、グリニァ増炭反応やシャープレス酸化反応を 鍵反応とする有機合成的手法や、液体クロマトグラフィーによる分割法が提案されて いる。し力、しながら、有機合成的手法では、副反応進行にともなう純度の低下などの 問題があり、また、液体クロマトグラフィーによる方法では大量の有機溶媒の使用が 避けられず、結果的にコスト高になる。一方で、酵素的エステル化反応によりラセミ体 イブプロフェンを光学分割する方法 (非特許文献 5、 6)、酵素的加水分解反応により ラセミ体イブプロフェンエステルを光学分割する方法 (非特許文献 7)などが提案され ているが、これらの酵素的合成手法では触媒コストが問題となる。酵素的合成手法の 一部では、遺伝子組換え微生物を利用することにより触媒コストを低減する方法（特 許文献 1)も提案されているが、この手法で用いられる酵素は反応最適温度が 25〜4 5°Cと低く実用的な手法とは言い難い。従って、これら手法に代わる、より効率的な（S )一イブプロフェンの合成方法の開発が望まれて!/、た。 [0005] (S) As a method for synthesizing ibuprofen, Grignard carbon addition reaction or sharpened oxidation reaction Organic synthetic methods for key reactions and resolution methods using liquid chromatography have been proposed. However, organic synthetic methods have problems such as a decrease in purity as the side reaction progresses, and liquid chromatography methods inevitably use large amounts of organic solvents, resulting in high costs. become. On the other hand, methods of optical resolution of racemic ibuprofen by enzymatic esterification (Non-patent Documents 5 and 6), methods of optical resolution of racemic ibuprofen ester by enzymatic hydrolysis (Non-patent Document 7), etc. are proposed. However, catalyst costs are a problem with these enzymatic synthesis methods. As part of the enzymatic synthesis method, a method has been proposed to reduce the catalyst cost by using genetically modified microorganisms (Patent Document 1), but the enzyme used in this method has an optimal reaction temperature of 25- 4 It is hard to say that it is a practical method as low as 5 ° C. Therefore, development of a more efficient method for synthesizing (S) -ibuprofen, which is an alternative to these methods, has been desired! /.
特許文献 1：特開平 8— 242853  Patent Document 1: JP-A-8-242853
非特許文献 1 : S. Adams, et al.， J. Pharm. Pharmac, 28， 256 (1976)  Non-Patent Document 1: S. Adams, et al., J. Pharm. Pharmac, 28, 256 (1976)
非特許文献 2 : A. J. Hutt and J. Caldwell, Clinical Pharmacokinetics., 9， 371 (1984) 非特許文献 3 : Williams, Κ·， et al.， Biochem. Pharmac , 35， 3403 (1986)  Non-patent document 2: A. J. Hutt and J. Caldwell, Clinical Pharmacokinetics., 9, 371 (1984) Non-patent document 3: Williams, Tsuji, et al., Biochem. Pharmac, 35, 3403 (1986)
非特許文献 4 : J.Caldwell and M. V. Marsh, Biochem. Pharmac, 32， 1667 (1983) 非特許文献 5 : Duan， G. and Chen, J. Y.， Biotechnol. Lett., 16， 1065 (1994) 非特許文献 6 : Mustranta, A. , J. Org. Chem., 59， 4410 (1994)  Non-patent document 4: J. Caldwell and MV Marsh, Biochem. Pharmac, 32, 1667 (1983) Non-patent document 5: Duan, G. and Chen, JY, Biotechnol. Lett., 16, 1065 (1994) Non-patent document 6: Mustranta, A., J. Org. Chem., 59, 4410 (1994)
非特許文献 7 : Lee, W. Η·， et al.， J. Ferment. Bioeng., 80， 613 (1995)  Non-Patent Document 7: Lee, W. Η., Et al., J. Ferment. Bioeng., 80, 613 (1995)
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0006] 本願発明者らは、反応最適温度が 60〜70°Cと高い新規なエステル加水分解酵素 をロドコッカス（Rhodococcus)属に属する微牛物より,,出した。さらに、この酵素を使 用することにより、ラセミ体イブプロフェンメチルから効率良く（S)—イブプロフェンを 製造し得ることを見出し、本願発明を完成した。 [0006] The inventors of the present application have released a novel ester hydrolase having a high optimal reaction temperature of 60 to 70 ° C from a fine cattle belonging to the genus Rhodococcus. Furthermore, it was found that by using this enzyme, (S) -ibuprofen can be efficiently produced from racemic ibuprofen methyl, and the present invention was completed.
課題を解決するための手段  Means for solving the problem
[0007] 本願発明は、以下の（1)から（3)のいずれかに記載のポリペプチドであるエステル 加水分解酵素である。 (1) 配列表の配列番号 2に記載のアミノ酸配列からなるポリペプチド；[0007] The present invention is an ester hydrolase which is a polypeptide according to any one of (1) to (3) below. (1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 in the Sequence Listing;
(2) 配列表の配列番号 2に記載のアミノ酸配列において 1または数個のアミノ酸が 欠失、置換、揷入または付加されたアミノ酸配列を有し、かつイブプロフェンメチルを 不斉的に加水分解して (R)—イブプロフェンを生成し（S)—イブプロフェンメチルを 残存させる活性を有するポリペプチド； (2) It has an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing and asymmetrically hydrolyzes ibuprofen methyl. A polypeptide having the activity of producing (R) -ibuprofen and leaving (S) -ibuprofen methyl;
(3) 配列表の配列番号 2に記載のアミノ酸配列と 85%以上の配列同一性を有し、 かつイブプロフェンメチルを不斉的に加水分解して (R)—イブプロフェンを生成し（S )一イブプロフェンメチルを残存させる活性を有するポリペプチド。  (3) It has a sequence identity of 85% or more with the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, and ibuprofen methyl is asymmetrically hydrolyzed to produce (R) -ibuprofen (S). A polypeptide having an activity of leaving ibuprofen methyl.
[0008] また、本願発明は、以下の（1)から（5)の理化学的性質を有するエステル加水分解 酵素である。  [0008] The present invention is an ester hydrolase having the following physicochemical properties (1) to (5).
(1)作用：イブプロフェンメチルに作用し、 (R)—イブプロフェンを生成し（S)—イブプ 口フェンメチルを残存させる。  (1) Action: Acts on ibuprofen methyl to produce (R) -ibuprofen and (S) -ibupofenmethyl.
(2)至適 ρΗ : ρΗ8· 0〜9· 0である。  (2) Optimum ρΗ: ρΗ8 · 0 to 9 · 0.
(3)作用至適温度： 60〜70°Cである。  (3) Optimum temperature of action: 60-70 ° C.
(4)阻害剤:銀イオン、水銀イオン、ョード酢酸により阻害され、 PMSFに阻害されな い。  (4) Inhibitor: Inhibited by silver ion, mercury ion, and odoacetic acid, not inhibited by PMSF.
(5)分子量:ゲル濾過分析において約 57, 000、 SDSポリアクリルアミド電気泳動分 析 ίこおレヽて約 37, 000である。  (5) Molecular weight: about 57,000 in gel filtration analysis, about 37,000 in SDS polyacrylamide electrophoresis analysis.
[0009] また、本願発明は、前記エステル加水分解酵素をコードする DNAである。  [0009] The present invention is a DNA encoding the ester hydrolase.
[0010] また、本願発明は、以下の（1)から（4)のいずれかに記載の塩基配列からなる DN Αである。 [0010] The present invention is a DN sequence consisting of the base sequence described in any of (1) to (4) below.
(1) 配列表の配列番号 1に記載の塩基配列；  (1) the nucleotide sequence set forth in SEQ ID NO: 1 in the sequence listing;
(2) 配列表の配列番号 2に記載のアミノ酸配列からなるポリペプチドをコードする塩 基配列；  (2) a base sequence encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 in the Sequence Listing;
(3) 配列表の配列番号 1に記載の塩基配列と相補的な塩基配列とストリンジェント な条件下でハイブリダィズし、かつイブプロフェンメチルを不斉的に加水分解して (R) イブプロフェンを生成し ） イブプロフェンメチルを残存させる活性を有するポリ ペプチドをコードする塩基配列； (4) 配列表の配列番号 1に記載の塩基配列と 85%以上の配列同一性を有し、かつ イブプロフェンメチルを不斉的に加水分解して (R)—イブプロフェンを生成し（S)—ィ ブプロフェンメチルを残存させる活性を有するポリペプチドをコードする塩基配列。 (3) Hybridize under stringent conditions with a base sequence complementary to the base sequence shown in SEQ ID NO: 1 in the sequence listing and asymmetrically hydrolyze ibuprofen methyl to produce (R) ibuprofen) A base sequence encoding a polypeptide having an activity of leaving ibuprofen methyl; (4) It has 85% or more sequence identity with the nucleotide sequence set forth in SEQ ID NO: 1 in the Sequence Listing, and asymmetrically hydrolyzes ibuprofen methyl to produce (R) —ibuprofen (S) — A base sequence encoding a polypeptide having an activity of leaving ibuprofen methyl.
[0011] また、本願発明は、前記 DNAを有するベクターである。 [0011] The present invention is a vector having the DNA.
[0012] また、本願発明は、前記ベクターにより形質転換された形質転換細胞である。 [0012] The present invention also relates to a transformed cell transformed with the vector.
[0013] さらに、本願発明は、本願発明のエステル加水分解酵素、および/または、本願発 明の形質転換細胞をエステル化合物に作用させ、生成する光学活性カルボン酸化 合物、および/または、残存する光学活性エステル化合物を採取することを特徴とす る、光学活性カルボン酸化合物、および/または、光学活性エステル化合物の製造 方法である。 [0013] Furthermore, the present invention is an optically active carboxylate compound and / or remaining by reacting the ester hydrolase of the present invention and / or the transformed cell of the present invention with an ester compound. An optically active carboxylic acid compound and / or a method for producing an optically active ester compound, comprising collecting an optically active ester compound.
発明の効果  The invention's effect
[0014] 本発明により、新規なエステル加水分解酵素、それをコードする DNA、その DNA を有するベクター、そのベクターで形質転換された形質転換体、およびそれらを利用 した光学活性カルボン酸化合物、および/または、光学活性エステル化合物の製造 方法が提供される。  [0014] According to the present invention, a novel ester hydrolase, a DNA encoding the same, a vector having the DNA, a transformant transformed with the vector, an optically active carboxylic acid compound using them, and / or Alternatively, a method for producing an optically active ester compound is provided.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0015] 以下、更に詳細に本願発明を説明する。 [0015] Hereinafter, the present invention will be described in more detail.
[0016] (酵素の精製） [0016] (Purification of enzyme)
本願発明のエステル加水分解酵素の起源として用いられる生物は特に限定されな いが、例えば、ロドコッカス（Rhodococcus)属に属する微牛物が挙げられ、特に、ロド コッカス.エスピー（Rhodococcus sp.) KNK0401が好ましい。  The organism used as the origin of the ester hydrolase of the present invention is not particularly limited, and examples thereof include fine cattle belonging to the genus Rhodococcus, and in particular, Rhodococcus sp. KNK0401 preferable.
[0017] ロドコッカス.エスピー (Rhodococcus sp.) KNK0401の菌学的性質を以下に記載 する。 [0017] The mycological properties of Rhodococcus sp. KNK0401 are described below.
1.細胞形態：不規則桿菌（培養 24時間： 0. 8 X 3. 0〜4. 0 m、培養 72時間： 0. 8 X I . 0〜丄. 5 ;U mノで rod— coccus cyclefcり  1. Cell morphology: irregular gonococci (culture 24 hours: 0.8 X 3.0 to 4.0 m, culture 72 hours: 0.8 XI. 0 to 丄. 5; U m no rod— coccus cyclefc
2.グラム染色：陽性  2. Gram staining: positive
3.胞子形成：陰性  3. Sporulation: Negative
4.運動性：陰性 5.コロニー形成：栄養寒天培地で 30°C、 48時間培養後、円形、周縁やや波状、凸 状、やや光沢あり、クリーム色のコロニーを形成 4.Mobility: Negative 5. Colony formation: After culturing at 30 ° C for 48 hours on nutrient agar medium, round, marginal wavy, convex, slightly shiny, cream-colored colonies formed
6. 10°Cでの生育性：陽性 6. Growth at 10 ° C: Positive
7. 37°Cでの生育性：弱陽性  7. Viability at 37 ° C: weak positive
8. 45°Cでの生育性：陰性  8. Viability at 45 ° C: Negative
9.カタラーゼ：陽性  9. Catalase: positive
10.ォキシダーゼ：陰十生  10. Oxidase: Yinsei
11.酸/ガス産性（グノレコース）：ともに陰性  11. Acid / Gas Productivity (Gnore Course): Both are negative
12.酸化/発酵テスト（グノレコース）：ともに陰性  12. Oxidation / fermentation test (Gnore course): Both negative
13.硝酸塩還元：陰性  13. Nitrate reduction: negative
14.ピラジナミダーゼ：陽性  14. Pyrazinamidase: positive
15.ピロリドニルァリルアミダーゼ：陰性  15. Pyrrolidonyl amidase: negative
16.アルカリフォスファターゼ：陽性  16. Alkaline phosphatase: positive
17. β -ダルクロニダーゼ：陰性  17. β-Dalcronidase: negative
18. β -ガラクトシダーゼ：陰性  18. β-galactosidase: negative
19. a -ダルコシダーゼ：陽性  19. a-Dalcosidase: positive
20. N—ァセチルー β—ダルコサミニダーゼ：陰性  20. N-acetylene β-darcosaminidase: negative
21.エスクリン：陽性  21. Esculin: Positive
22.ゥレアーゼ：陽性  22.urease: positive
23.ゼラチン加水分解：陰性  23. Gelatin hydrolysis: negative
24.ブドウ糖発酵性：陰性  24. Glucose fermentability: negative
25.リボース発酵性：陰性  25. Ribose fermentability: Negative
26.キシロース発酵性：陰性  26. Xylose fermentability: negative
27.マンニトール発酵性：陰性  27. Mannitol fermentability: negative
28.マルトース発酵性：陰性  28. Maltose fermentability: negative
29.乳糖発酵性：陰性  29. Lactose fermentability: Negative
30. 白糖発酵性：陰性  30. Fermentability of sucrose: negative
31.グリコーゲン発酵性：陰性 32.抗酸染色:部分的に陽性 31. Glycogen fermentability: negative 32. Anti-acid staining: partially positive
33.チロシンの溶解性：陽性  33. Tyrosine solubility: Positive
34.フルクトースの酸化：陰性  34. Oxidation of fructose: negative
[0018] ロドコッカス ·エスピー（Rhodococcus s ) KNK0401は、 ¾託番号 FERM BP— 1 0911として、 日本国茨城県つくば巿東 1丁目 1番 1号 (郵便番号 305— 8566)独立 行政法人産業技術総合研究所特許生物寄託センターに寄託されている（原寄託日 2004年 10月 22日の国内寄託株をブダペスト条約に基づく国際寄託へ移管（2007 年 9月 21日））。  [0018] Rhodococcus sp (The original deposit date was transferred to the international deposit under the Budapest Treaty (September 21, 2007)).
[0019] 本願発明の酵素を生産する微生物の培養に用いる培地は、その微生物が増殖し 得るものである限り特に限定されない。例えば、炭素源、窒素源、無機塩類、有機栄 養素等を含有する通常の液体栄養培地が使用され得る。また、本願発明の酵素を生 産する微生物からの該酵素の精製は、常法により行!/、得る。  [0019] The medium used for culturing the microorganism producing the enzyme of the present invention is not particularly limited as long as the microorganism can grow. For example, a normal liquid nutrient medium containing a carbon source, nitrogen source, inorganic salts, organic nutrients and the like can be used. Further, purification of the enzyme from the microorganism producing the enzyme of the present invention can be carried out by a conventional method.
[0020] 本願発明の酵素は、例えば、ロドコッカス'エスピー（Rhodococcus sp.) KNK0401 より以下のように単離され得る。まず、当該菌株を適当な培地で培養し、培養液から 遠心分離により菌体を集める。菌体を、例えば、ダイノミル (Dyno— Mill社製）などで 破砕し、遠心分離にて菌体残渣を除き、無細胞抽出液を得る。この無細胞抽出液に 対して、塩析 (硫酸アンモユウム沈殿、リン酸ナトリウム沈殿など）、溶媒沈殿（アセトン またはエタノールなどによる蛋白質分画沈殿法）、透析、ゲル濾過、イオン交換、逆 相等のカラムクロマトグラフィー、限外濾過等の処理を単独で、または組み合わせて 施すことにより、酵素が精製され得る。エステル加水分解活性は lOOmMリン酸力リウ ム緩衝液（pH7. 0)に基質 p—二トロフエニルアセテート ImMおよび酵素を添加して 、 30°Cで波長 405nmの吸光度の増加を測定することにより行い得る。この反応条件 において、 1分間に l〃molの p—二トロフエニルアセテートを加水分解する活性を lu nitと定義する。  [0020] The enzyme of the present invention can be isolated from Rhodococcus sp. KNK0401 as follows, for example. First, the strain is cultured in an appropriate medium, and the cells are collected from the culture solution by centrifugation. The cells are crushed with, for example, Dynomill (Dyno-Mill), and the cell residue is removed by centrifugation to obtain a cell-free extract. For this cell-free extract, columns such as salting out (ammonium sulfate precipitation, sodium phosphate precipitation, etc.), solvent precipitation (protein fraction precipitation with acetone or ethanol, etc.), dialysis, gel filtration, ion exchange, reverse phase, etc. The enzyme can be purified by performing treatments such as chromatography and ultrafiltration alone or in combination. Ester hydrolysis activity is performed by measuring the increase in absorbance at 405 nm at 30 ° C by adding the substrate p-nitrophenyl acetate ImM and enzyme to lOOmM phosphate phosphate buffer (pH 7.0). obtain. Under these reaction conditions, the activity of hydrolyzing l〃mol of p-nitrotrophyl acetate per minute is defined as lu nit.
[0021] 本願発明において、特定の化合物により酵素が「阻害を受ける」とは、特定の化合 物を当該酵素の反応液中に ImM以下の濃度で添加したときに、無添加の場合の 5 0%あるいはそれ以下に活性が低下することを!/、う。  [0021] In the present invention, an enzyme is "inhibited" by a specific compound means that when a specific compound is added to a reaction solution of the enzyme at a concentration of ImM or less, the enzyme is not added. The activity decreases to% or less!
[0022] 酵素の分子量の測定は、 Superdex 200 HR 10/30 (lOmml. D. X 30cm) (GEヘルスケア バイオサイエンス株式会社製)カラムを用いたゲル濾過分析により 行う。溶離液としては、 0. 2M NaClを含む 10mMリン酸カリウム緩衝液（ρΗ7· 0) を用いる。サブユニット分子量は、還元条件下 (還元剤： 2% (ν/ν) 2—メルカプトェ タノール）で、 10%SDS—ポリアクリルアミドゲルでの電気泳動を行い、そして標準蛋 白質の相対移動度から算出することにより決定される。 [0022] The molecular weight of the enzyme was measured using Superdex 200 HR 10/30 (lOmml. D. X 30cm) Performed by gel filtration analysis using a column (GE Healthcare Biosciences). As eluent, 10 mM potassium phosphate buffer (ρΗ7 · 0) containing 0.2 M NaCl is used. Subunit molecular weight is calculated from the relative mobility of standard proteins by electrophoresis on 10% SDS-polyacrylamide gel under reducing conditions (reducing agent: 2% (ν / ν) 2-mercaptoethanol). To be determined.
[0023] 本願発明のエステル加水分解酵素は、例えば、以下の（1)から（5)の理化学的性 質を有する。 [0023] The ester hydrolase of the present invention has, for example, the following physicochemical properties (1) to (5).
(1)作用：イブプロフェンメチルに作用し、 (R)—イブプロフェンを生成し（S)—イブプ 口フェンメチルを残存させる。  (1) Action: Acts on ibuprofen methyl to produce (R) -ibuprofen and (S) -ibupofenmethyl.
(2)至適 ρΗ : ρΗ8· 0〜9· 0である。  (2) Optimum ρΗ: ρΗ8 · 0 to 9 · 0.
(3)作用至適温度： 60〜70°Cである。  (3) Optimum temperature of action: 60-70 ° C.
(4)阻害剤:銀イオン、水銀イオン、ョード酢酸により阻害され、 PMSFに阻害されな い。  (4) Inhibitor: Inhibited by silver ion, mercury ion, and odoacetic acid, not inhibited by PMSF.
(5)分子量:ゲル濾過分析において約 57, 000、 SDSポリアクリルアミド電気泳動分 析 ίこおレヽて約 37, 000である。  (5) Molecular weight: about 57,000 in gel filtration analysis, about 37,000 in SDS polyacrylamide electrophoresis analysis.
[0024] 本願発明の酵素とほぼ同一の性質を有する酵素は天然酵素であってもよぐまたは 組換え酵素であってもよい。例えば、組換え酵素は、以下に示す方法により決定した 口ドコッカス (Rhodococcus)属に由来する酵素の遺伝子を、適当な宿主に導入するこ とにより得られる。また、導入する当該酵素遺伝子の一部を予め変異させておくことに より、当該酵素のアミノ酸配列中の 1つまたは数個のアミノ酸を置換、欠失、揷入また は付加させた変異酵素を作製することも可能である。このようにして得られる変異酵 素も、それらが、イブプロフェンメチルに作用し (R)—イブプロフェンを生成し（S)—ィ ブプロフェンメチルを残存させる活性を有する限り、本願発明に含まれる。欠失、置 換、揷入または付加するアミノ酸の数は、 40個以下が好ましぐより好ましくは 25個以 下であり、更に好ましくは 10個以下であり、最も好ましくは 5個、 4個、 3個、または 2個 以下である。  [0024] The enzyme having almost the same properties as the enzyme of the present invention may be a natural enzyme or a recombinant enzyme. For example, the recombinant enzyme can be obtained by introducing a gene of an enzyme derived from the genus Rhodococcus determined by the following method into an appropriate host. In addition, by mutating a part of the enzyme gene to be introduced in advance, a mutant enzyme in which one or several amino acids in the amino acid sequence of the enzyme are substituted, deleted, inserted or added is added. It is also possible to produce it. Mutant enzymes thus obtained are also included in the present invention as long as they have the activity of acting on ibuprofen methyl to produce (R) -ibuprofen and leave (S) -ibuprofenmethyl. The number of amino acids to be deleted, replaced, inserted or added is preferably 40 or less, more preferably 25 or less, still more preferably 10 or less, most preferably 5 or 4 , 3 or 2 or less.
[0025] また、配列表の配列番号 2に示すアミノ酸配列と 85%以上の配列同一性を有する ポリペプチドが、イブプロフェンメチルに作用し (R)—イブプロフェンを生成し（S)—ィ ブプロフェンメチルを残存させる活性を有する場合は、これも本発明のポリペプチド に含まれる。配列表の配列番号 2のアミノ酸配列と 85%以上の配列同一性を有する ポリペプチドは本発明のポリペプチドに含まれる力 S、その配列同一性は 90%以上が 好ましぐ 95%以上がより好ましぐ 98%以上が更に好ましぐ 99%以上がより最も好 ましい。 [0025] Further, a polypeptide having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing acts on ibuprofen methyl to produce (R) -ibuprofen (S)- A polypeptide having the activity of leaving buprofen methyl is also included in the polypeptide of the present invention. A polypeptide having 85% or more sequence identity with the amino acid sequence of SEQ ID NO: 2 in the sequence listing is a force S included in the polypeptide of the present invention, and its sequence identity is preferably 90% or more, more preferably 95% or more. More than 98% is more preferable. More than 99% is more preferable.
[0026] アミノ酸配列の配列同一性は、配列表の配列番号 2に示したアミノ酸配列と評価し たレ、アミノ酸配列とを比較し、両方の配列でアミノ酸が一致した位置の数を比較総ァ ミノ酸数で除して、さらに 100を乗じた値で表される。  [0026] The sequence identity of amino acid sequences is determined by comparing the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing with the evaluated amino acid sequence and the number of positions where the amino acid matches in both sequences. Divided by the number of mino acids and then multiplied by 100.
[0027] (エステル加水分解酵素遺伝子のクローニング）  [0027] (Cloning of ester hydrolase gene)
上述のように精製された本願発明の酵素を適当な条件下で変性した後、適当なェ ンドぺプチダーゼで消化することにより、本願発明の酵素を構成するペプチド断片を 得ること力 Sできる。これらのペプチド断片を、例えば、 YMC-Pack SIL-06 (YM C社製)カラムを用いた逆相 HPLCにより精製後、プロテインシークェンサ一に供する ことで、本願発明の酵素の部分アミノ酸配列を決定し得る。そして、得られた部分アミ ノ酸配列情報をもとに PCR (Polymerase Chain Reaction)プライマーを合成し得る。  The enzyme of the present invention purified as described above can be denatured under appropriate conditions and then digested with an appropriate endopeptidase to obtain a peptide fragment constituting the enzyme of the present invention. These peptide fragments are purified by reverse phase HPLC using, for example, a YMC-Pack SIL-06 (manufactured by YMC) column, and then subjected to a protein sequencer, whereby the partial amino acid sequence of the enzyme of the present invention is obtained. Can be determined. A PCR (Polymerase Chain Reaction) primer can be synthesized based on the partial amino acid sequence information obtained.
[0028] 次に、本願発明のエステル加水分解酵素の起源となる微生物より、例えば Murray 等の方法（Nucleic Acids Res., 8:4321-4325 (1980))により、該微生物の染色体 DNA を調製し得る。この染色体 DNAを铸型として、先述の PCRプライマーを用いて PCR を行い、該酵素をコードする DNAの一部（コア配歹 IJ)を増幅し、塩基配列決定し得る 。塩基配列はジデォキシ ·チェイン 'ターミネーシヨン法等により決定でき、例えば、 A BI 3130xlDNA Sequencer (Applied Biosystems社製）等を用いて行うことが できる。  [0028] Next, chromosomal DNA of the microorganism is prepared from the microorganism that is the origin of the ester hydrolase of the present invention by, for example, the method of Murray et al. (Nucleic Acids Res., 8: 4321-4325 (1980)). obtain. Using this chromosomal DNA as a saddle, PCR can be performed using the PCR primers described above to amplify a part of the DNA encoding the enzyme (core arrangement IJ) and determine the nucleotide sequence. The base sequence can be determined by a dideoxy chain termination method or the like, and can be performed using, for example, ABI 3130xlDNA Sequencer (Applied Biosystems).
[0029] 次に、コア配列の周辺領域の塩基配列を明らかにするため、該微生物の染色体 D NAを、コア配列中にその認識配列が存在しない制限酵素により消化し、生成した D NA断片を T4リガーゼにより自己環化させることにより逆 PCR (Nucleic Acids Res., 1 6:8186 (1988))用の铸型 DNAを調製する。次に、コア配列をもとに、コア配列の外側 に向力、う DNA合成の開始点となるプライマーを合成し、逆 PCRによりコア配列の周 辺領域を増幅する、こうして得られた DNAの塩基配列を明らかにすることにより、 目 的酵素の全コード領域の塩基配列を読み取ることができる。 [0029] Next, in order to clarify the base sequence of the peripheral region of the core sequence, the chromosomal DNA of the microorganism is digested with a restriction enzyme whose recognition sequence does not exist in the core sequence, and the generated DNA fragment is A cage DNA for reverse PCR (Nucleic Acids Res., 16: 8186 (1988)) is prepared by self-cyclization with T4 ligase. Next, based on the core sequence, a primer that acts as a starting point for DNA synthesis is synthesized outside the core sequence, and the peripheral region of the core sequence is amplified by inverse PCR. By clarifying the base sequence, The base sequence of the entire coding region of the target enzyme can be read.
[0030] 本発明のポリペプチドをコードする DNAとして、例えば、配列表の配列番号 1に示 した塩基配列からなる DNA、又は、配列表の配列番号 1に示した塩基配列と相補的 な塩基配列を含む DNAと、ストリンジェントな条件下でハイブリダィズし、かつイブプ 口フェンメチルに作用し（R)—イブプロフェンを生成し（S)—イブプロフェンメチルを 残存させる活性を有するポリペプチドをコードする DNA、を挙げること力 Sできる。  [0030] As the DNA encoding the polypeptide of the present invention, for example, DNA consisting of the base sequence shown in SEQ ID NO: 1 in the sequence listing, or a base sequence complementary to the base sequence shown in SEQ ID NO: 1 in the sequence listing And DNA encoding a polypeptide that has the activity of hybridizing under stringent conditions and acting on ibuprofen phenmethyl to produce (R) -ibuprofen and (S) -ibuprofenmethyl to remain. The power to raise S.
[0031] また、「配列表の配列番号 1に示した塩基配列と相補的な塩基配列を含む DNAと 、ストリンジェントな条件下でハイブリダィズし、かつイブプロフェンメチルに作用し（R) イブプロフェンを生成し ） イブプロフェンメチルを残存させる活性を有するポリ ペプチドをコードする DNA」とは、配列表の配列番号 1に示した塩基配列と相補的な 塩基配列からなる DNAをプローブとして、ストリンジェントな条件下にコロニー 'ノヽィ ブリダィゼーシヨン法、プラーク 'ハイブリダィゼーシヨン法、あるいはサザンハイブリダ ィゼーシヨン法等を用いることにより得られる DNAで、かつイブプロフェンメチルに作 用し (R)—イブプロフェンを生成し（S)—イブプロフェンメチルを残存させる活性を有 するポリペプチドをコードする DNAを意味する。  [0031] Further, "DNA containing a base sequence complementary to the base sequence shown in SEQ ID NO: 1 in the sequence listing" is hybridized under stringent conditions and acts on ibuprofen methyl to produce (R) ibuprofen. ) “DNA encoding a polypeptide having the activity of leaving ibuprofen methyl” refers to a colony under stringent conditions using a DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 1 in the Sequence Listing as a probe. DNA obtained by using the 'noisy hybridization method, plaque' hybridization method, or Southern hybridization method, etc. and acting on ibuprofen methyl to produce (R) -ibuprofen. (S) —refers to DNA encoding a polypeptide having the activity of leaving ibuprofen methyl.
[0032] ノヽィプリダイでーシヨンは、 Molecular Cloning, A laboratory manual, second edition  [0032] Noisy Pre-Daisyong is Molecular Cloning, A laboratory manual, second edition
(Cold Spring Harbor Laboratory Press, 1989)等に記載されている方法に準じて行う こと力 Sできる。ここで、「ストリンジェントな条件でハイブリダィズする DNA」とは、例えば 、 コロニーあるいはプラーク由来の DNAを固定化したフィルターを用いて、 0. 7〜1 . 0Mの NaCl存在下、 65°Cでハイブリダィゼーシヨンを行った後、 2倍濃度の SSC溶 液（1倍濃度の SSC溶液の組成は、 150mM塩化ナトリウム、 15mMクェン酸ナトリウ ムよりなる）を用い、 65°Cの条件下でフィルターを洗浄することにより取得できる DNA をあげること力 Sできる。好ましくは 65°Cで 0. 5倍濃度の SSC溶液で洗浄、より好ましく は 65°Cで 0. 2倍濃度の SSC溶液で洗浄、更に好ましくは 65°Cで 0. 1倍濃度の SS C溶液で洗浄することにより取得できる DNAである。  (Cold Spring Harbor Laboratory Press, 1989). Here, “DNA that hybridizes under stringent conditions” means, for example, hybridization at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which colony or plaque-derived DNA is immobilized. After dialysis, filter at 65 ° C using 2x SSC solution (1x SSC solution consists of 150mM sodium chloride, 15mM sodium citrate). It is possible to increase the DNA that can be obtained by washing. Preferably washed with 0.5 times SSC solution at 65 ° C, more preferably washed with 0.2 times SSC solution at 65 ° C, more preferably 0.1 times SSC at 65 ° C This DNA can be obtained by washing with a solution.
[0033] 以上のようにハイブリダィゼーシヨン条件を記載した力 これらの条件に特に制限さ れない。ノ、イブリダィゼーシヨンのストリンジエンシーに影響する要素としては温度や 塩濃度など複数の要素が考えられ、当業者であればこれら要素を適宜選択すること で最適なストリンジエンシーを実現することが可能である。 [0033] Forces that describe hybridization conditions as described above. The conditions are not particularly limited. However, there are several factors that influence the stringency of hybridization, such as temperature and salt concentration, and those skilled in the art should select these factors as appropriate. It is possible to achieve an optimal stringency.
[0034] 上記の条件にてハイブリダィズ可能な DNAとしては、配列番号 1に示される DNA と、配列同一性が 85%以上、好ましくは 90%以上、より好ましくは 95%以上、さらに より好ましくは 98%以上、最も好ましくは 99%以上の DNAをあげることができ、コー ドされるポリペプチド力 イブプロフェンメチルに作用し (R) イブプロフェンを生成し (S) イブプロフェンメチルを残存させる活性を有する限り、上記 DNAに包含される[0034] The DNA that can be hybridized under the above conditions is 85% or more, preferably 90% or more, more preferably 95% or more, and still more preferably 98%, with the DNA represented by SEQ ID NO: 1. As long as it has the activity of acting on the encoded polypeptide force ibuprofen methyl to produce (R) ibuprofen and (S) leave ibuprofen methyl, Included in DNA
〇 Yes
[0035] ここで、「配列同一性（％)」とは、対比される 2つの DNAを最適に整列させ、核酸塩 基 (例えば、 A、 T、 C、 G、 U、または I)が両方の配列で一致した位置の数を比較塩基 総数で除し、そして、この結果に 100を乗じた数値で表される。  [0035] Here, "sequence identity (%)" means that two DNAs to be compared are optimally aligned and both nucleic acid bases (eg, A, T, C, G, U, or I) are both aligned. The number of matching positions in this sequence is divided by the total number of comparison bases, and this result is expressed by multiplying by 100.
[0036] 配列同一性は、例えば、以下の配列分析用ツールを用いて算出し得る： GCG Wise onsin Package (Program Manual ror he Wisconsin Package, Version8, 1994年 9月， enetics Computer Group, 575 Science Drive Medison, Wisconsin, USA 53711; Ric e, P. (1996) Program Manual for EGCG Package, Peter Rice, The Sanger Centre, Hi nxton Hall, Cambridge, CB10 IRQ, England)、及び、 the ExPASy World Wide Web 分子生物字用サーノ 一 (Geneva University Hospital and University or ueneva, en eva, Switzerland)。  [0036] Sequence identity can be calculated, for example, using the following sequence analysis tool: GCG Wise onsin Package (Program Manual ror he Wisconsin Package, Version 8, September 1994, enetics Computer Group, 575 Science Drive Medison , Wisconsin, USA 53711; Rice, P. (1996) Program Manual for EGCG Package, Peter Rice, The Sanger Centre, Hinxton Hall, Cambridge, CB10 IRQ, England), and the ExPASy World Wide Web for molecular biology Geneva University Hospital and University or ueneva, en eva, Switzerland.
[0037] (エステル加水分解酵素を含む組換えプラスミドの作製）  [0037] (Preparation of recombinant plasmid containing ester hydrolase)
本願発明のエステル加水分解酵素遺伝子を宿主微生物内に導入し、そしてその 導入された宿主微生物内で発現させるために用いられるベクター DNAとしては、適 切な宿主微生物内でこのエステル加水分解酵素を発現させ得る限り、任意のベクタ 一 DNAが用いられ得る。このようなベクター DNAとしては、例えば、プラスミドベクタ 一、ファージベクター、コスミドベクターなどが挙げられる。また、他の宿主株との間で 遺伝子交換が可能なシャトルベクターも使用され得る。さらに、このようなベクター DN Aは、作動可能に連結されたプロモーター（lacUV5プロモーター、 trpプロモーター 、 trcプロモーター、 tacプロモーター、 lppプロモーター、 tufBプロモーター、 recAプ 口モーター、 PLプロモーター等）、ェンハンサー配列等の制御因子を有し得る。例え ば、 pUCNT (WO94/03613)等が好適に用いられる。このプラスミド pUCNTは、 1 acプロモーター下流に、 Ndel、 EcoRI部位等の揷入部位を有しているので、好適に 用いられ得る。 As the vector DNA used for introducing the ester hydrolase gene of the present invention into a host microorganism and expressing it in the introduced host microorganism, the ester hydrolase is expressed in an appropriate host microorganism. Any vector can be used as long as it is available. Examples of such vector DNA include plasmid vector 1, phage vector, cosmid vector and the like. A shuttle vector that can exchange genes with other host strains can also be used. Furthermore, such vector DNA is operably linked to promoters (lacUV5 promoter, trp promoter, trc promoter, tac promoter, lpp promoter, tufB promoter, recA promoter motor, PL promoter, etc.), enhancer sequences, etc. Can have the following control factors: For example, pUCNT (WO94 / 03613) is preferably used. This plasmid pUCNT is 1 Since it has an insertion site such as Ndel or EcoRI site downstream of the ac promoter, it can be suitably used.
[0038] (形質転換）  [0038] (Transformation)
得られたエステル加水分解酵素を有する組換えプラスミドは、常法により宿主細胞 に導入され得る。宿主細胞としては、細菌、酵母、糸状菌、植物細胞、動物細胞など が使用され得る。大腸菌の使用が特に好ましい。宿主へのプラスミドの導入は、当業 者に周知の方法、例えばコンビテント（competent)状態にされた宿主細胞と組換えプ ラスミドを混合する工程を包含する方法、ヘルパー ·プラスミドを用いて接合伝達によ り移入する工程を包含する方法などが挙げられる。宿主に導入されたプラスミドは、 ェピゾームとして自立的に複製し得る力、、あるいはその全部または一部が染色体に 組み込まれて染色体とともに複製し得る。  The obtained recombinant plasmid having ester hydrolase can be introduced into a host cell by a conventional method. As host cells, bacteria, yeasts, filamentous fungi, plant cells, animal cells and the like can be used. The use of E. coli is particularly preferred. The introduction of the plasmid into the host can be performed by methods well known to those skilled in the art, for example, a method including a step of mixing a recombinant host cell with a competent host cell, a conjugative transfer using a helper plasmid. For example, a method including a step of transferring by the above method. The plasmid introduced into the host can replicate autonomously as an episome, or all or part of it can be integrated into the chromosome and replicated together with the chromosome.
[0039] (光学活性カルボン酸の取得）  [0039] (Acquisition of optically active carboxylic acid)
光学活性カルボン酸である（S)—イブプロフェンは、例えば以下のようにして取得さ れる。  The optically active carboxylic acid (S) -ibuprofen is obtained, for example, as follows.
[0040] 基質としてはイブプロフェンメチルが用いられ得る。  [0040] As the substrate, ibuprofen methyl may be used.
[0041] 反応は、適当な溶媒中に基質イブプロフェンメチル、および微生物の培養物または その処理物等を添加し、 pH調整下攪拌することにより行い得る。反応は温度 10°C〜 70°C、 pH4〜； 10で行う。また、基質の仕込み濃度は 0. 1 %〜90% (W/V)であるが 、基質を連続的に添加し得る。反応はバッチ方式または連続方式で行い得る。ここで 、微生物の処理物等とは、例えば、粗抽出液、培養菌体、凍結乾燥生物体、アセトン 乾燥生物体、またはそれらの菌体の磨砕物等で、エステル加水分解酵素の触媒活 性が残存している物を意味する。更にそれらは、酵素自体あるいは菌体のまま公知 の手段で固定化されて用いられ得る。固定化は、当業者に周知の方法 (例えば、架 橋法、物理的吸着法、包括法など）で行い得る。反応で生じたイブプロフェンおよび 残存したイブプロフェンメチルは、常法により精製され得る。例えば、イブプロフェンメ チルは、微生物を用いた場合などには必要に応じ遠心分離、濾過などの処理を施し て菌体等の懸濁物を除去し、次いで酢酸ェチル、トルエン等の有機溶媒で抽出し、 硫酸ナトリウム等の脱水剤で脱水し、有機溶媒を減圧条件下で除去し、そして減圧 蒸留またはクロマトグラフィー等（例えば、シリカゲルカラムクロマトグラフィー）の処理 を行うことにより、精製され得る。また、得られた光学活性イブプロフェンメチルは常法 により化学的に加水分解され得る、例えば、適当な溶媒中において、強塩基の条件 化にて攪拌することにより加水分解され得る。 [0041] The reaction can be carried out by adding the substrate ibuprofen methyl, a microorganism culture or a treated product thereof in an appropriate solvent, and stirring under pH adjustment. The reaction is carried out at a temperature of 10 ° C to 70 ° C, pH 4 to 10; In addition, the substrate concentration is 0.1% to 90% (W / V), but the substrate can be added continuously. The reaction can be carried out batchwise or continuously. Here, the treated product of microorganisms is, for example, a crude extract, cultured cells, freeze-dried organisms, acetone-dried organisms, or a ground product of these cells, and the catalytic activity of ester hydrolase. Means the remaining item. Furthermore, they can be used after being immobilized by a known means with the enzyme itself or cells. Immobilization can be performed by methods well known to those skilled in the art (for example, bridge method, physical adsorption method, inclusion method, etc.). The ibuprofen produced in the reaction and the remaining ibuprofen methyl can be purified by conventional methods. For example, ibuprofen methyl is subjected to treatment such as centrifugation and filtration as necessary when microorganisms are used, etc. to remove cell suspensions, and then extracted with an organic solvent such as ethyl acetate or toluene. Dehydrate with a dehydrating agent such as sodium sulfate, remove the organic solvent under reduced pressure, and It can be purified by performing a treatment such as distillation or chromatography (for example, silica gel column chromatography). The obtained optically active ibuprofen methyl can be chemically hydrolyzed by a conventional method. For example, it can be hydrolyzed by stirring under conditions of a strong base in an appropriate solvent.
[0042] イブプロフェンメチルの定量は、ガスクロマトグラフィーにおいて TC— FFAP (ジー エルサイエンス株式会社製）を用い、カラム温度 80°C〜200°Cでクロマトグラフィーを 行い、 FIDにより検出することにより行い得る。 [0042] Quantification of ibuprofen methyl can be performed by gas chromatography using TC-FFAP (manufactured by GL Sciences Inc.), chromatography at a column temperature of 80 ° C to 200 ° C, and detection by FID. .
[0043] (S) イブプロフェンの光学純度の測定は、光学分離カラム CHIRALCEL OJ—[0043] (S) The optical purity of ibuprofen was measured using the optical separation column CHIRALCEL OJ—
H (ダイセル化学工業株式会社製）を用いた HPLCにより行!/、得る。 Obtained by HPLC using H (manufactured by Daicel Chemical Industries, Ltd.).
[0044] 以上のように、本願発明に従って、エステル加水分解酵素の大量生産が可能であ る。さらに、この酵素を利用することにより、（S) イブプロフェンの優れた製造法が提 供される。 [0044] As described above, according to the present invention, mass production of an ester hydrolase is possible. Furthermore, by using this enzyme, an excellent method for producing (S) ibuprofen is provided.
[0045] 以下、実施例より本願発明を詳細に説明する。しかし、本願発明はこれらの実施例 に限定されるものではない。  Hereinafter, the present invention will be described in detail with reference to examples. However, the present invention is not limited to these examples.
[0046] 以下の実施例にお!/、て用いた組換え DNA技術に関する詳細な操作方法などは、 次のテキストに記載されて!/、る。 [0046] Detailed operating methods related to the recombinant DNA technology used in the following examples are described in the following text!
[004 (I) Molecular し lomng 2nd edition (し old spring Haroor Laboratory Press (1989))。 [004 (I) Molecular lomng 2nd edition (Old spring Haroor Laboratory Press (1989)).
[0048] (II) Current Protocols in Molecular Biology (Greene Publishing Associates and Wil ey-Interscience)o [0048] (II) Current Protocols in Molecular Biology (Greene Publishing Associates and Wiley-Interscience) o
実施例  Example
[0049] (実施例 1：エステル加水分解酵素の精製）  [0049] (Example 1: Purification of ester hydrolase)
以下の方法に従って、ロドコッカス'エスピー（Rhodococcus sp.) KNK0401よりイブ プロフェンメチルを不斉的に加水分解して（R)—イブプロフェンを生成し（S)—イブ プロフェンメチルを残存させる活性を有する、エステル加水分解酵素を電気泳動的 に単一に精製した。  It has the activity of asymmetric hydrolysis of ibuprofenmethyl from Rhodococcus sp. KNK0401 to form (R) -ibuprofen and leave (S) -ibuprofenmethyl according to the following method. Then, the ester hydrolase was purified to a single electrophoresis.
[0050] バクト 'トリプトン 16g、バクト 'イーストエキス 10g、塩化ナトリウム 5g (いずれも 1L当 たり）の組成からなる液体培地を 12L調製し、 5L容ジャーファメンターに 3Lずつ分注 し、アデ力ノール（LG— 10) (株式会社 ADEKA社製）を 3滴加え、 120°Cで 20分間 蒸気殺菌をおこなった。 [0050] Prepare 12 L of liquid culture medium consisting of 16 g of Bacto 'trypton, 10 g of Bacto' yeast extract and 5 g of sodium chloride (each per 1 L), and dispense 3 L each into a 5 L jar fermenter. (LG-10) (made by ADEKA Co., Ltd.) 3 drops, 120 minutes at 120 ° C Steam sterilization was performed.
これらの培地に、予め同培地中で前培養しておいたロドコッカス.エスピー（E ^ ^ occus sp.) KNK0401の培養液を 30mlずつ接種し、培養温度 30°C、攪拌数 500rp m、通気量 0. 5L/分の条件で 3日間培養を行った。なお、培養中 pHが 7. 0を下回 る場合は、 5規定の水酸化ナトリウム水溶液で pH7. 0に調整した。この培養液から遠 心分離により菌体を集めた。このようにして、該菌株の湿菌体 470gを得た。この湿菌 体を 2Lの 10mMリン酸カリウム緩衝液（pH7. 0)に懸濁し、 50°Cの恒温槽中で 20分 間攪拌することにより熱処理を行った。次レ、で菌体をダイノミル（Dyno-Mill社製）で 破砕した。この菌体破砕物から遠心分離にて菌体残渣を除き、無細胞抽出液 2100 mlを得た。この無細胞抽出液に硫酸プロタミン 5gを加えて 4°Cでー晚攪拌後、生じ た沈殿を遠心分離により除去した。上清を 10mMリン酸カリウム緩衝液 (pH7. 0)で あらかじめ平衡化した DEAE— TOYOPEARL 650M (東ソ一株式会社製）カラム (400ml)に供し、酵素を吸着させ、同一緩衝液でカラムを洗浄後 OmMから 400mM までの塩化ナトリウム濃度のリニアグラジェントによりエステル加水分解酵素活性画分 を溶出させた。この活性画分に終濃度 1. 5Mとなるように硫酸ナトリウムを添加し、あ らかじめ 1. 5Mの硫酸ナトリウムを含む 10mMリン酸カリウム緩衝液（pH7. 0)で平 衡化した Phenyl— TOYOPEARL 650M (東ソ一株式会社製）カラム（125ml)に 供し、該酵素を吸着させ、同一緩衝液でカラムを洗浄後、 1. 5Mから 0Mまでの硫酸 ナトリウム濃度のリニアグラジェントによりエステル加水分解酵素活性画分を溶出させ た。この活性画分を 10mMリン酸カリウム緩衝液（pH7. 0)に対して 1夜透析を行つ た。この透析液を 10mMリン酸カリウム緩衝液（pH7. 0)であらかじめ平衡化したセ ルロファイン Hap (生化学工業株式会社製)カラム（40ml)に供し、同一緩衝液で洗 浄を行った。洗浄液中のエステル加水分解酵素活性画分を集め、終濃度 0. 25Mと なるように塩化ナトリウムを添加した。この活性画分を 0. 25Mの塩化ナトリウムを含む 10mMリン酸カリウム緩衝液（pH7. 0)であらかじめ平衡化した HiTrap Benzamid ine (GEヘルスケア バイオサイエンス株式会社製）カラム（1ml)に供し、同一緩衝液 で洗浄を行った。洗浄液中のエステル加水分解酵素活性画分を集め精製酵素標品 を得た。 [0052] (実施例 2 :酵素の性質の測定） Inoculate 30 ml of each medium of Rhodococcus sp. KNK0401 previously cultured in the same medium, culture temperature 30 ° C, stirring rate 500 rpm, aeration rate The culture was performed for 3 days under the condition of 0.5 L / min. When the pH dropped below 7.0 during cultivation, the pH was adjusted to 7.0 with a 5N aqueous sodium hydroxide solution. The cells were collected from the culture solution by centrifugation. In this way, 470 g of wet cells of the strain was obtained. The wet cells were suspended in 2 L of 10 mM potassium phosphate buffer (pH 7.0) and heat-treated by stirring for 20 minutes in a 50 ° C constant temperature bath. Next, the cells were crushed with Dynomill (Dyno-Mill). The cell residue was removed from the crushed cell by centrifugation to obtain 2100 ml of a cell-free extract. After adding 5 g of protamine sulfate to this cell-free extract and stirring at 4 ° C, the resulting precipitate was removed by centrifugation. Apply the supernatant to a DEAE— TOYOPEARL 650M column (400 ml) pre-equilibrated with 10 mM potassium phosphate buffer (pH 7.0), adsorb the enzyme, and wash the column with the same buffer. Thereafter, the ester hydrolase active fraction was eluted with a linear gradient of sodium chloride concentration from OmM to 400 mM. To this active fraction, sodium sulfate was added to a final concentration of 1.5 M, and Phenyl— equilibrated with 10 mM potassium phosphate buffer (pH 7.0) containing 1.5 M sodium sulfate in advance. Apply to TOYOPEARL 650M (Tosohichi Co., Ltd.) column (125 ml), adsorb the enzyme, wash the column with the same buffer, and then perform ester hydrolysis with a linear gradient of sodium sulfate concentration from 1.5 M to 0 M The enzyme activity fraction was eluted. This active fraction was dialyzed overnight against 10 mM potassium phosphate buffer (pH 7.0). This dialysate was applied to a Cellulofine Hap (Seikagaku Corporation) column (40 ml) equilibrated in advance with 10 mM potassium phosphate buffer (pH 7.0) and washed with the same buffer. Fractions containing ester hydrolase activity in the washing solution were collected, and sodium chloride was added to a final concentration of 0.25M. This active fraction was applied to a HiTrap Benzamid ine (GE Healthcare Biosciences) column (1 ml) pre-equilibrated with 10 mM potassium phosphate buffer (pH 7.0) containing 0.25 M sodium chloride. Washing with buffer was performed. The fractions of ester hydrolase activity in the washing solution were collected to obtain a purified enzyme preparation. [0052] (Example 2: Measurement of enzyme properties)
実施例 1におレ、て得られたエステル加水分解酵素の酵素学的性質につ!/、て検討し た。  The enzymatic properties of the ester hydrolase obtained in Example 1 were examined.
[0053] エステル加水分解酵素活性の測定は、基本的には、 lOOmMリン酸カリウム緩衝液  [0053] The measurement of ester hydrolase activity is basically performed by measuring lOOmM potassium phosphate buffer.
(pH7. 0)に基質 p—ニトロフエニルアセテート ImMおよび酵素を添加して、 30°Cで 波長 405nmの吸光度の増加を測定することにより行った。なお、作用については、 基質 P—二トロフエニルアセテートの代わりにイブプロフェンを用いて、反応生成物を ガスクロマトグラフィーで同定した。  The substrate p-nitrophenyl acetate ImM and enzyme were added to (pH 7.0), and the increase in absorbance at a wavelength of 405 nm was measured at 30 ° C. Regarding the action, the reaction product was identified by gas chromatography using ibuprofen instead of the substrate P-nitrophenyl acetate.
(1)作用：基質 P—二トロフエニルアセテートの代わりにイブプロフェンメチルに作用さ せたところ、（R)—イブプロフェンを生成し（S)—イブプロフェンメチルを残存させた。  (1) Action: Substrate P When ibuprofenmethyl was used instead of ditrophenyl acetate, (R) -ibuprofen was produced, and (S) -ibuprofenmethyl was left.
(2)至適 pH :リン酸カリウム緩衝液（pH7. 0)に代えて各種 pHの酢酸ナトリウム緩衝 液、リン酸カリウム緩衝液またはトリスー塩酸緩衝液を用いて pH4. 0〜9. 5の範囲で 、上記方法に準じて酵素活性を測定した。その結果、 p—二トロフエニルアセテートに 作用する至適 pHは 8. 0〜9. 0であった。  (2) Optimum pH: pH 4.0 to 9.5 using sodium acetate buffer, potassium phosphate buffer or Tris-HCl buffer instead of potassium phosphate buffer (pH 7.0). Thus, the enzyme activity was measured according to the above method. As a result, the optimum pH acting on p-nitrotrophyl acetate was 8.0 to 9.0.
(3)作用至適温度： 30°Cのみならず、 10°C〜70°Cの温度で 1分間の、 p—二トロフエ ニルアセテートを基質とした場合の本願発明酵素の活性を測定して、至適温度を求 めた。その結果、至適温度は 60〜70°Cであった。  (3) Optimum temperature of action: Measure the activity of the enzyme of the present invention not only at 30 ° C but also at 10 ° C to 70 ° C for 1 minute using p-nitrophenyl acetate as a substrate. The optimum temperature was obtained. As a result, the optimum temperature was 60 to 70 ° C.
(4)阻害剤：上記反応溶液に、表 1に示す濃度の各種金属イオンまたは阻害剤を添 加して、 p—二トロフエニルアセテートを基質として活性を測定した。表 1に示す様に、 本願発明の酵素は銀イオン、水銀イオンおよびョード酢酸によって阻害を受け、 PM SFによって阻害を受けな力、つた。  (4) Inhibitor: Various metal ions or inhibitors having the concentrations shown in Table 1 were added to the above reaction solution, and the activity was measured using p-nitrophenyl acetate as a substrate. As shown in Table 1, the enzyme of the present invention was inhibited by silver ions, mercury ions, and odoacetic acid, and not inhibited by PMSF.
(5)分子量：酵素の分子量の測定は Superdex 200 HR 10/30 (lOmml. D. X 30cm) (GEヘルスケア バイオサイエンス株式会社製）カラムを用い、溶離液とし て 0. 2M塩化ナトリウムを含む 10mMリン酸カリウム緩衝液（pH7. 0)を用いた場合、 約 57, 000であった。酵素のサブユニットの分子量は、還元条件下（還元剤： 2% (V /v) 2—メルカプトエタノール）で、 10%SDS—ポリアクリルアミドゲルで電気泳動し、 そして標準タンパクの相対移動度から算出することにより決定した。その結果、本酵 素のサブユニットの分子量は約 37, 000であった。 [0054] [表 1] (5) Molecular weight: The molecular weight of the enzyme is measured using a Superdex 200 HR 10/30 (lOmml. D. X 30cm) column (manufactured by GE Healthcare Biosciences Inc.) and contains 0.2M sodium chloride as the eluent. When 10 mM potassium phosphate buffer (pH 7.0) was used, it was about 57,000. The molecular weight of the subunit of the enzyme is calculated from the relative mobility of the standard protein after electrophoresis on 10% SDS-polyacrylamide gel under reducing conditions (reducing agent: 2% (V / v) 2-mercaptoethanol). Was determined by As a result, the molecular weight of the subunit of this enzyme was about 37,000. [0054] [Table 1]
Figure imgf000016_0001
Figure imgf000016_0001
[0055] (実施例 3：エステル加水分解酵素のクローニング） [Example 3: Cloning of ester hydrolase]
(染色体 DNAの取得）  (Acquisition of chromosomal DNA)
ロドコッカス.エスピー（Rhodococcus sp.) KNK0401の培養 ί夜力、ら、 Murray等の 方法（Nucleic Acids Res., 8:4321-4325 (1980))に従って染色体 DNAを抽出した。  Cultivation of Rhodococcus sp. KNK0401 Chromosomal DNA was extracted in accordance with the method of Nakuryoku et al., Murray et al. (Nucleic Acids Res., 8: 4321-4325 (1980)).
[0056] (エステル加水分解酵素遺伝子の PCR法によるクローニング）  [0056] (Cloning of ester hydrolase gene by PCR)
実施例 1のようにして得られた精製エステル加水分解酵素を 8M尿素存在下で変 性させた後、ァクロモパクター由来のリシルエンドぺプチダーゼ（和光純薬工業株式 会社製）で消化し、得られたペプチド断片の配列をエドマン法により決定した。このァ ミノ酸配列から予想される DNA配列を考慮し、 PCRプライマー 2種 5'— CCRTGR TTRTANCCYTCCCA- 3' (プライマー 1：配列表配列番号 3)、 5'— GARGCNG TNAGYGTNGAYGG - 3' (プライマー 2：配列表配列番号 4)を合成した。  The purified ester hydrolase obtained as in Example 1 was denatured in the presence of 8M urea and then digested with acrymopacter-derived lysyl endopeptidase (manufactured by Wako Pure Chemical Industries, Ltd.). The sequence of the fragment was determined by the Edman method. Considering the DNA sequence expected from this amino acid sequence, two kinds of PCR primers 5'—CCRTGR TTRTANCCYTCCCA-3 ′ (primer 1: SEQ ID NO: 3), 5′—GARGCNG TNAGYGTNGAYGG-3 ′ (primer 2: Sequence listing SEQ ID NO: 4) was synthesized.
[0057] プライマー 2種（プライマー 1及びプライマー 2)各 50pmol、染色体 DNA240ng、 d NTP各 10nmol、 ExTaq (タカラバイオ株式会社製） 1. 3Uを含む ExTaq用緩衝液 50 1を調製し、熱変性（97°C、 30秒）、アニーリング（50°C、 1分）、伸長反応（72°C 、 1分）を 30サイクル行い、 4°Cまで冷却後、ァガロースゲノレ電気泳動により増幅 DN Aを確認した。  [0057] 2 types of primers (Primer 1 and Primer 2) 50 pmol each, chromosomal DNA 240 ng, d NTP 10 nmol each, ExTaq (manufactured by Takara Bio Inc.) 1. Prepare ExTaq buffer solution 50 1 containing 3 U, and heat denaturation ( 97 ° C, 30 seconds), annealing (50 ° C, 1 minute), extension reaction (72 ° C, 1 minute) for 30 cycles. After cooling to 4 ° C, amplified DNA was confirmed by agarose genomic electrophoresis. .
[0058] (PCR法により増幅した DNA断片のサブクローニング） 増幅 DNAを pT7Blue Vector (Novagen社製）にサブクローユングし、その塩基 配列を決定した。以後この配列を「コア配歹 IJ」と記す。 [0058] (Subcloning of DNA fragment amplified by PCR method) The amplified DNA was subcloned into pT7Blue Vector (Novagen), and its nucleotide sequence was determined. This sequence is hereinafter referred to as “core arrangement IJ”.
[0059] (逆 PCR法によるコア配列周辺配列のクローニング） [0059] (Cloning around the core sequence by reverse PCR)
コア配列の 5'—側に近い部分の相補配列 5'— TTCGCGGAATCGTCGTATC CGGATTC— 3' (プライマー 3：配列表配列番号 5)及び 3'—側に近い部分の配列 5 ' -ACGAGTTGCTTCGCAACCTGGACATC - 3' (プライマー 4：配列表配列 番号 6)を作製した。  5'—complementary sequence close to the 5 'side of the core sequence 5'— TTCGCGGAATCGTCGTATC CGGATTC— 3' (primer 3: SEQ ID NO: 5) 4: Sequence Listing Sequence No. 6) was prepared.
[0060] 逆 PCRの铸型 して、まずロドコッカス.エスピー (Rhodococcus sp. ) KNK040 1の染色体 DNAを制限酵素 Apalにより消化し、消化物を T4DNAリガーゼを用いて 自己閉環した。この自己閉環物 230ng、プライマー 2種 (プライマー 3及びプライマー 4)各 50pmol、 dNTP各 10nmol、 ExTaq (タカラバイオ株式会社製） 1. 3Uを含む ExTaq用緩衝液 50 1を調製し、熱変性（97°C、 30秒）、アニーリング（70°C、 1分） 、伸長反応（72°C、 5分）を 30サイクル行い、 4°Cまで冷却後、ァガロースゲル電気泳 動により増幅 DNAを確認した。  [0060] As a reverse PCR variant, first, the chromosomal DNA of Rhodococcus sp. KNK0401 was digested with the restriction enzyme Apal, and the digest was self-cycled using T4 DNA ligase. 230 ng of this self-closing ring, 2 types of primers (primer 3 and primer 4) 50 pmol each, dNTP 10 nmol each, ExTaq (manufactured by Takara Bio Inc.) 1. Prepare ExTaq buffer solution 50 1 containing 3 U and heat denaturation (97 30 ° C, 30 seconds), annealing (70 ° C, 1 minute), extension reaction (72 ° C, 5 minutes) were performed for 30 cycles. After cooling to 4 ° C, amplified DNA was confirmed by agarose gel electrophoresis.
[0061] 増幅 DNAを pT7Blue Vector (Novagen社製）にサブクローユングし、その塩基 配列を決定した。この結果とコア配列の結果より、エステル加水分解酵素をコードす る DNAの全塩基配列を決定した。全塩基配列及び該 DNAがコードする推定アミノ 酸配列を配列表の配列番号 2に示した。  [0061] The amplified DNA was subcloned into pT7Blue Vector (Novagen), and its nucleotide sequence was determined. From this result and the result of the core sequence, the entire nucleotide sequence of DNA encoding the ester hydrolase was determined. The entire base sequence and the deduced amino acid sequence encoded by the DNA are shown in SEQ ID NO: 2 in the sequence listing.
[0062] (実施例 4：エステル加水分解酵素遺伝子を含む組換えベクターの作製）  (Example 4: Production of recombinant vector containing ester hydrolase gene)
大腸菌においてエステル加水分解酵素を発現させるために、形質転換に用いる組 換えベクターを作製した。まず、エステル加水分解酵素の構造遺伝子の開始コドン 部分に Ndel部位を付加し、かつ終始コドンの直後に終止コドン（TAA)と EcoRI部 位を付加した二本鎖 DNAを以下の方法により取得した。実施例 3で決定した塩基配 列に基づき、エステル加水分解酵素の構造遺伝子の開始コドン部分に Ndel部位を 付加したプライマー： 5'— GTGCATATGTCTATTCGTGAAGCCGTC— 3' (プ ライマー 5：配列表配列番号 7)と、エステル加水分解酵素の構造遺伝子の終始コド ンの直後に終止コドン（TAA)と EcoRI部位を付加したプライマー： 5'— GAGGAA TTCTTACTAACCGAGGCTCGAGATGA— 3' (プライマー 6：配列表配列番 号 8)とを合成した。 In order to express an ester hydrolase in E. coli, a recombinant vector used for transformation was prepared. First, double-stranded DNA in which an Ndel site was added to the start codon portion of the structural gene of ester hydrolase and a stop codon (TAA) and an EcoRI site were added immediately after the stop codon was obtained by the following method. Based on the nucleotide sequence determined in Example 3, a primer in which an Ndel site was added to the start codon portion of the structural gene for ester hydrolase: 5′—GTGCATATGTCTATTCGTGAAGCCGTC—3 ′ (Primer 5: SEQ ID NO: 7) , Primer with a stop codon (TAA) and EcoRI site added immediately after the end codon of the structural gene of ester hydrolase: 5'—GAGGAA TTCTTACTAACCGAGGCTCGAGATGA— 3 ′ (Primer 6: Sequence Listing SEQ ID NO: No. 8) was synthesized.
[0063] プライマー 2種（プライマー 5及びプライマー 6)各 50pmol、ロドコッカス'エスピー（β hodococcus sp.) KNK0401の染色体 DNA15ng、 dNTP各 10nmol、 ExTaq (タカ ラバイオ株式会社製） 1. 3Uを含む ExTaq用緩衝液 50 1を調製し、熱変性（97°C、 30秒）、アニーリング（60°C、 1分）、伸長反応（72°C、 5分）を 30サイクル行い、 4°Cま で冷却後、ァガロースゲル電気泳動により増幅 DNAを確認した。この増幅断片を Nd el及び EcoRIで消化し、プラスミド pUCNT (WO94/03613)の lacプロモーターの 下流の Ndel、 EcoRI部位に揷入することにより、組換えベクター pNTHRを得た。  [0063] 2 primers (primer 5 and primer 6) 50 pmol each, Rhodococcus sp. KNK0401 chromosomal DNA 15 ng, dNTP 10 nmol each, ExTaq (manufactured by Takara Bio Inc.) 1. ExTaq buffer containing 3 U Prepare 501, heat denaturation (97 ° C, 30 seconds), annealing (60 ° C, 1 minute), extension reaction (72 ° C, 5 minutes) for 30 cycles, and after cooling to 4 ° C The amplified DNA was confirmed by agarose gel electrophoresis. This amplified fragment was digested with Ndel and EcoRI and inserted into the Ndel and EcoRI sites downstream of the lac promoter of the plasmid pUCNT (WO94 / 03613) to obtain a recombinant vector pNTHR.
[0064] (実施例 5：組換え大腸菌の作製）  [Example 5: Production of recombinant Escherichia coli]
実施例 4で得た組換えベクター pNTHRを DNA Ligation Kit Ver. 2. 1 I液 (タカラバイオ株式会社製）と混合し 16°Cで 30分間保温した。得られた反応液を大腸 菌 HB101コンビテントセル (タカラバイオ株式会社製）に添加し、 30分間氷上で保温 、 42°Cで 45秒保温、その後氷上で 2分間冷却することにより形質転換し、組換え大 腸菌 HB101 (pNTHR)を得た。こうして得られた形質転換体である大腸菌 HB101 ( pNTHR)は、受託番号 FERM P— 20271として、平成 16年（2004年） 10月 22曰 付けで、 日本国茨城県つくば巿東 1丁目 1番 1号 (郵便番号 305— 8566)独立行政 法人産業技術総合研究所特許生物寄託センターに寄託されている。  The recombinant vector pNTHR obtained in Example 4 was mixed with DNA Ligation Kit Ver. 2.1 I solution (manufactured by Takara Bio Inc.) and incubated at 16 ° C. for 30 minutes. The resulting reaction solution was added to Escherichia coli HB101 combined cell (manufactured by Takara Bio Inc.), incubated on ice for 30 minutes, incubated at 42 ° C for 45 seconds, and then cooled on ice for 2 minutes, Recombinant E. coli HB101 (pNTHR) was obtained. Escherichia coli HB101 (pNTHR), a transformant obtained in this way, has the accession number FERM P-20271, dated October 22, 2004, Tsukuba Sakai Higashi 1-chome, 1-chome, Ibaraki, Japan 1 No. (Postal code: 305-8566) Deposited at the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary.
[0065] (実施例 6：組換え大腸菌におけるエステル加水分解酵素の発現）  [Example 6: Expression of ester hydrolase in recombinant Escherichia coli]
実施例 5で得た組換え大腸菌 HB101 (pNTHR)を 120 μ g/mlのアンピシリンを 含む 2 XYT培地（バタト'トリプトン 1 · 6% (w/v)、バタト 'イーストエキス 1 · 0% (w/ v)、 NaClO. 5% (w/v)、 pH7. 0)で培養し、得られた培養液を超音波破砕するこ とにより無細胞抽出液を得た。この無細胞抽出液のエステル加水分解活性を実施例 2に記載の方法で測定した。  Recombinant Escherichia coli HB101 (pNTHR) obtained in Example 5 was mixed with 2 XYT medium containing 120 μg / ml ampicillin (batato'tryptone 1 · 6% (w / v), butato'yeast extract 1 · 0% (w / v), NaClO. 5% (w / v), pH 7.0), and the resulting culture broth was sonicated to obtain a cell-free extract. The ester hydrolysis activity of this cell-free extract was measured by the method described in Example 2.
[0066] 表 2に示す様に、大腸菌 HB101 (pNTHR)では、ベクタープラスミドのみの形質転 換体である大腸菌 HB101 (pUCNT)と比較して明らかなエステル加水分解活性の 増加がみられた。  [0066] As shown in Table 2, E. coli HB101 (pNTHR) showed a clear increase in ester hydrolysis activity as compared to E. coli HB101 (pUCNT), which is a transformant of only the vector plasmid.
[0067] [表 2] 菌株名 エステラーゼ比活性（UZmg)[0067] [Table 2] Strain name Esterase specific activity (UZmg)
HB 1 01 (pUCNT) 0 03 HB 1 01 (pUCNT) 0 03
HB 1 01 (pNTHR) 1 . 1 3  HB 1 01 (pNTHR) 1.1 3
[0068] (実施例 7：エステル加水分解酵素遺伝子を導入した組換え大腸菌による（S)—ィ
Figure imgf000019_0001
[Example 7: (S) -i by recombinant Escherichia coli into which an ester hydrolase gene has been introduced
Figure imgf000019_0001
実施例 5で得た組換え大腸菌 HB101 (pNTHR)を、 500ml容坂ロフラスコ中で滅 菌した 50mlの 2 X YT培地に接種し、 37°Cで 48時間振とう培養した。得られた培養 液を PH7. 0に調整し超音波破砕することにより無細胞抽出液を得た。無細胞抽出 液 lmlにラセミ体のイブプロフェンメチル 10mgを添加して、 30°Cで 12時間振とうした 。変換反応ののち反応液を硫安飽和にし、酢酸ェチルを加えて抽出を行い、残存す The recombinant Escherichia coli HB101 (pNTHR) obtained in Example 5 was inoculated into 50 ml of 2X YT medium sterilized in a 500 ml volumetric flask and cultured with shaking at 37 ° C for 48 hours. The resulting culture solution to give a P H7. Cell-free extract by adjusting to sonication at 0. 10 mg of racemic ibuprofen methyl was added to 1 ml of the cell-free extract and shaken at 30 ° C. for 12 hours. After the conversion reaction, the reaction solution is saturated with ammonium sulfate, extracted with ethyl acetate, and remains.
：ジーエルサイエンス株式会社製 TC一 FFAP、検出： FID、カラム温度： 80— 200°C )で分析した。また、得られたイブプロフェンの光学純度は、高速液体クロマトグラフィ 一（カラム：ダイセル化学工業株式会社製 CHIRALCEL 〇J— H、溶離液：へキサ ン/イソプロパノール = 99/1、流速： 1. Oml/min,検出： 254nm、カラム温度：室 温、溶出時間： R体 30分、 S体 25分）により分析した。その結果、イブプロフェンが 3. 3mg生成し、その光学純度は（R)体 58. l%e. e.であり、イブプロフェンメチルは 4. 8mg残存した。 : TC FFAP manufactured by GL Sciences Inc., detection: FID, column temperature: 80-200 ° C). In addition, the optical purity of the obtained ibuprofen is high performance liquid chromatography (column: CHIRALCEL 〇J—H, manufactured by Daicel Chemical Industries, Ltd., eluent: hexane / isopropanol = 99/1, flow rate: 1. Oml / min. , Detection: 254 nm, column temperature: room temperature, elution time: R-form 30 minutes, S-form 25 minutes). As a result, 3.3 mg of ibuprofen was produced, its optical purity was 58. l% e.e., And 4.8 mg of ibuprofen methyl remained.
[0069] (実施例 8：ロドコッカス.エスピー（Rhodococcus sp.) KNK0401による（S)—イブプ 口フェンメチルの合成）  [0069] (Example 8: Synthesis of (S) -Ibupu-Fufenmethyl by Rhodococcus sp. KNK0401)
実施例 1に記載の方法で得たロドコッカス ·エスピー（Rhodococcus sp.) KNK0401 の培養液 1Lから遠心分離によって菌体を取得し、 l OOmMリン酸カリウム緩衝液 (p H8. 0) 250mlにラセミ体のイブプロフェンメチノレ 2. 5gを添カロして、 40°Cで 25日寺間 攪拌した。変換反応ののち反応液を硫安飽和にし、酢酸ェチルを加えて抽出を行い 、残存するイブプロフェンメチルおよび生成したイブプロフェンを実施例 7記載の方法 で分析した。その結果、イブプロフェンが 750mg生成していた。  The cells were obtained from 1 L of the Rhodococcus sp. KNK0401 culture solution obtained by the method described in Example 1 by centrifugation, and racemic into 250 ml of OOmM potassium phosphate buffer (pH 8.0). 2.5g of ibuprofen methinore was added and stirred for 25 days at 40 ° C. After the conversion reaction, the reaction solution was saturated with ammonium sulfate, extracted with ethyl acetate, and the remaining ibuprofen methyl and the produced ibuprofen were analyzed by the method described in Example 7. As a result, 750 mg of ibuprofen was produced.

Claims

請求の範囲 [1] 以下の（1)から（3)のいずれかに記載のポリペプチドであるエステル加水分解酵素 (1) 配列表の配列番号 2に記載のアミノ酸配列からなるポリペプチド； (2) 配列表の配列番号 2に記載のアミノ酸配列において 1または数個のアミノ酸が 欠失、置換、揷入または付加されたアミノ酸配列を有し、かつイブプロフェンメチルを 不斉的に加水分解して (R)—イブプロフェンを生成し（S)—イブプロフェンメチルを 残存させる活性を有するポリペプチド； (3) 配列表の配列番号 2に記載のアミノ酸配列と 85%以上の配列同一性を有し、 かつイブプロフェンメチルを不斉的に加水分解して (R)—イブプロフェンを生成し（S )一イブプロフェンメチルを残存させる活性を有するポリペプチド。 [2] 以下の（1)から（5)の理化学的性質を有するエステル加水分解酵素： Claims [1] An ester hydrolase which is a polypeptide according to any one of (1) to (3) below (1) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing; ) It has an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, and ibuprofen methyl is asymmetrically hydrolyzed ( R) —a polypeptide having the activity of producing ibuprofen and leaving (S) —ibuprofen methyl; (3) having at least 85% sequence identity with the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing, and ibuprofen A polypeptide having the activity of asymmetrically hydrolyzing methyl to produce (R) -ibuprofen (S) and remaining ibuprofenmethyl. [2] Ester hydrolases having the following physicochemical properties (1) to (5):
(1)作用：イブプロフェンメチルに作用し、 (R)—イブプロフェンを生成し（S)—イブプ 口フェンメチルを残存させる。  (1) Action: Acts on ibuprofen methyl to produce (R) -ibuprofen and (S) -ibupofenmethyl.
(2)至適 ρΗ : ρΗ8· 0〜9· 0である。  (2) Optimum ρΗ: ρΗ8 · 0 to 9 · 0.
(3)作用至適温度： 60〜70°Cである。  (3) Optimum temperature of action: 60-70 ° C.
(4)阻害剤:銀イオン、水銀イオン、ョード酢酸により阻害され、 PMSFに阻害されな い。  (4) Inhibitor: Inhibited by silver ion, mercury ion, and odoacetic acid, not inhibited by PMSF.
(5)分子量:ゲル濾過分析において約 57, 000、 SDSポリアクリルアミド電気泳動分 析 ίこおレヽて約 37, 000である。  (5) Molecular weight: about 57,000 in gel filtration analysis, about 37,000 in SDS polyacrylamide electrophoresis analysis.
[3] 上記エステル加水分解酵素が、ロドコッカス（Rhodococcus)属に属する微生物から 得られるエステル加水分解酵素である、請求項 1または 2に記載のエステル加水分解 酵素。  [3] The ester hydrolase according to claim 1 or 2, wherein the ester hydrolase is an ester hydrolase obtained from a microorganism belonging to the genus Rhodococcus.
[4] 前記エステル加水分解酵素が、ロドコッカス'エスピー (Rhodococcus sp.) KNK04 [4] The ester hydrolase is Rhodococcus sp. KNK04
01 (FERM BP— 10911)から得られるエステル加水分解酵素である、請求項 3に 記載のエステル加水分解酵素。 The ester hydrolase according to claim 3, which is an ester hydrolase obtained from 01 (FERM BP-10911).
[5] 請求項 1〜4のいずれかに記載のエステル加水分解酵素をコードする DNA。  [5] DNA encoding the ester hydrolase according to any one of claims 1 to 4.
[6] 以下の（1)から（4)の!/、ずれかに記載の塩基配列からなる DNA: (1) 配列表の配列番号 1に記載の塩基配列； [6] DNA comprising the base sequence described in any of the following (1) to (4)! /: (1) the nucleotide sequence set forth in SEQ ID NO: 1 in the sequence listing;
(2) 配列表の配列番号 2に記載のアミノ酸配列からなるポリペプチドをコードする塩 基配列；  (2) a base sequence encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 in the Sequence Listing;
(3) 配列表の配列番号 1に記載の塩基配列と相補的な塩基配列とストリンジェント な条件下でハイブリダィズし、かつイブプロフェンメチルを不斉的に加水分解して (R) イブプロフェンを生成し ） イブプロフェンメチルを残存させる活性を有するポリ ペプチドをコードする塩基配列；  (3) Hybridize under stringent conditions with a base sequence complementary to the base sequence shown in SEQ ID NO: 1 in the sequence listing and asymmetrically hydrolyze ibuprofen methyl to produce (R) ibuprofen) A base sequence encoding a polypeptide having an activity of leaving ibuprofen methyl;
(4) 配列表の配列番号 1に記載の塩基配列と 85%以上の配列同一性を有し、かつ イブプロフェンメチルを不斉的に加水分解して (R)—イブプロフェンを生成し（S)—ィ ブプロフェンメチルを残存させる活性を有するポリペプチドをコードする塩基配列。  (4) It has 85% or more sequence identity with the nucleotide sequence set forth in SEQ ID NO: 1 in the Sequence Listing, and asymmetrically hydrolyzes ibuprofen methyl to produce (R) —ibuprofen (S) — A base sequence encoding a polypeptide having an activity of leaving ibuprofen methyl.
[7] 請求項 5または 6に記載の DNAを有するベクター。  [7] A vector having the DNA of claim 5 or 6.
[8] 請求項 7に記載のベクターにより形質転換された形質転換細胞。 [8] A transformed cell transformed with the vector according to claim 7.
[9] 前記形質転換細胞が大腸菌である、請求項 8に記載の形質転換細胞。 9. The transformed cell according to claim 8, wherein the transformed cell is Escherichia coli.
[10] 請求項 1〜4のいずれかに記載のエステル加水分解酵素および/または請求項 8 〜9のいずれかに記載の形質転換細胞をエステル化合物に作用させ、生成する光 学活性カルボン酸化合物および/または残存する光学活性エステル化合物を採取 することを特徴とする、光学活性カルボン酸化合物および/または光学活性エステ ル化合物の製造方法。 [10] A photoactive carboxylic acid compound produced by reacting the ester hydrolase according to any one of claims 1 to 4 and / or the transformed cell according to any one of claims 8 to 9 with an ester compound. And / or collecting the remaining optically active ester compound, a method for producing an optically active carboxylic acid compound and / or an optically active ester compound.
[11] 前記生成する光学活性カルボン酸化合物が（R) イブプロフェンであり、前記残存 する光学活性エステル化合物が（S) イブプロフェンメチルである、請求項 10に記 載の光学活性カルボン酸化合物および/または光学活性エステル化合物の製造方 法。  [11] The optically active carboxylic acid compound and / or the optically active carboxylic acid compound according to claim 10, wherein the generated optically active carboxylic acid compound is (R) ibuprofen and the remaining optically active ester compound is (S) ibuprofenmethyl. A method for producing an optically active ester compound.
[12] ロドコッカス ·エスピー（Rhodococcus sp.) KNK0401 (FERM BP— 10911)の菌 体または培養物あるいはその処理物をエステル化合物に作用させ、生成した光学活 性カルボン酸化合物および/または残存する光学活性エステル化合物を採取するこ とを特徴とする、請求項 10または 11に記載の光学活性カルボン酸化合物および/ または光学活性エステル化合物の製造方法。  [12] Rhodococcus sp. KNK0401 (FERM BP—10911) cells or cultures or their treated products are allowed to act on ester compounds to produce optically active carboxylic acid compounds and / or remaining optical activity. The method for producing an optically active carboxylic acid compound and / or an optically active ester compound according to claim 10 or 11, wherein an ester compound is collected.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110592049A (en) * 2019-09-29 2019-12-20 北京工商大学 Aspergillus niger ester hydrolase AnCu3, encoding gene and application thereof in DEHP hydrolysis

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03224496A (en) * 1989-12-21 1991-10-03 Asahi Chem Ind Co Ltd Production of optically active alpha-substituted organic acid

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03224496A (en) * 1989-12-21 1991-10-03 Asahi Chem Ind Co Ltd Production of optically active alpha-substituted organic acid

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
CHUNG Y.M. ET AL.: "Partial purification and characterization of thermostable esterase from the hyperthermophilic archaeon Sulfolobus solfataricus", BIOTECHNOLOGY AND BIOPROCESS ENGINEERING, vol. 5, no. 1, 2000, pages 53 - 56, XP003022309 *
DATABASE SWISSPROT [online] XU Y. ET AL.: "Carboxyeasterase", XP003022311, Database accession no. (Q5MAF6) *
DROGE M.J. ET AL.: "Comparison and functional characterization of three homologous intracellular carboxyesterases of Bacillus subtilis", JOURNAL OF MOLECULAR CATALYSIS B ENZYMATIC, vol. 32, no. 5-6, 2005, pages 261 - 270, XP004752016 *
LEE W.H. ET AL.: "Enzymatic resolution of racemic ibuprofen esters: effects of organic cosolvents and temperature", J. FERMENT. BIOENG., vol. 80, no. 6, 1995, pages 613 - 615, XP009022668 *
MADHAV M.V. ET AL.: "Study on the enzymatic hydrolysis of racemic methyl ibuprofen ester", J. CHEM. TECHNOL. BIOTECHNOL., vol. 76, no. 9, 2001, pages 941 - 948, XP001092368 *
RHODOCOCCUS SP. CDT3 *
SNELL D. ET AL.: "Enantioselective hydrolysis of racemic ibuprofen amide to S-(plus)-ibuprofen by RHodococcus AJ270", ENZYME AND MICROBIAL TECHNOLOGY, vol. 24, no. 3-4, 1999, pages 160 - 163, XP003022310 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110592049A (en) * 2019-09-29 2019-12-20 北京工商大学 Aspergillus niger ester hydrolase AnCu3, encoding gene and application thereof in DEHP hydrolysis

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