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WO2008047819A1 - Nouvelle ester hydrolase, gène codant pour ladite enzyme et utilisation - Google Patents

Nouvelle ester hydrolase, gène codant pour ladite enzyme et utilisation 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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English (en)
Japanese (ja)
Inventor
Teruaki Takesue
Noriyuki Kizaki
Yoshihiko Yasohara
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Kaneka Corporation
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Publication date
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Publication of WO2008047819A1 publication Critical patent/WO2008047819A1/fr

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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

L'invention concerne : un polypeptide présentant une activité d'hydrolyse des esters, ledit polypeptide étant isolé à partir d'un microorganisme appartenant au genre Rhodococcus ; de l'ADN codant pour ledit polypeptide ; un transformant capable de produire ledit polypeptide ; et un procédé permettant de produire un acide carboxylique optiquement actif ou un ester optiquement actif par réaction dudit polypeptide ou transformant avec un ester. Il est ainsi possible de produire avec un bon rendement un composé optiquement actif (ibuprofène optiquement actif, par exemple) utile en tant qu'agent pharmaceutique ou bien un intermédiaire d'agent pharmaceutique.
PCT/JP2007/070225 2006-10-18 2007-10-17 Nouvelle ester hydrolase, gène codant pour ladite enzyme et utilisation WO2008047819A1 (fr)

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Cited By (1)

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CN110592049A (zh) * 2019-09-29 2019-12-20 北京工商大学 一种黑曲霉酯水解酶AnCu3、编码基因及其在水解DEHP中的应用

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JPH03224496A (ja) * 1989-12-21 1991-10-03 Asahi Chem Ind Co Ltd 光学活性なα―置換有機酸を製造する方法

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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 *
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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 (zh) * 2019-09-29 2019-12-20 北京工商大学 一种黑曲霉酯水解酶AnCu3、编码基因及其在水解DEHP中的应用

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