Classifications

• • 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
• • 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)
• • 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)
• • 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
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
  • Y02P20/00—Technologies relating to chemical industry
  • Y02P20/50—Improvements relating to the production of bulk chemicals
  • Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts

Definitions

• the present invention relates to a novel hydrolase protein that can be used for the production of (1S, 2S) -1-alkoxycarbonyl-2-vinylcyclopropanecarboxylic acid, and the use thereof.
• Non-Patent Document 1 describes a method for hydrolyzing dimethyl 2-vinylcyclopropane-1,1-dicarboxylic acid with an enzyme to obtain (1S, 2S) -1-methoxycarbonyl-2-vinylcyclopropanecarboxylic acid.
• the optical selectivity of the enzyme is insufficient, and the obtained (1S, 2S) -1-methoxycarbonyl-2-vinylcyclopropanecarboxylic acid has an optical purity of 90% ee, which is an intermediate of a pharmaceutical product. It was unsuitable as an industrial production method.
• Bacillus subtilis is ATCC23857
• the amino acid sequence of paranitrobenzyl esterase derived from the Bacillus ⁇ ⁇ subtilis ATCC23857 strain (hereinafter sometimes referred to as “PNBE23857”). Is registered in GenBank Accession No. ZP_03593235.
• the present invention is useful as an intermediate for the production of a therapeutic agent for hepatitis C by hydrolyzing a dialkyl ⁇ 2-vinylcyclopropane-1,1-dicarboxylic acid with an enzyme.
• paranitrobenzyl hydrolase protein derived from Bacillus subtilis has good selectivity and diethyl 2-vinylcyclopropane-1,1- It was found that (1S, 2S) -1-ethoxycarbonyl-2-vinylcyclopropanecarboxylic acid can be efficiently obtained by hydrolyzing dicarboxylic acid.
• a hydrolase comprising the amino acid sequence shown in any of SEQ ID NOs: 2 to 5 and having an activity of catalyzing the reaction shown in formula (1) with higher selectivity than the protein consisting of the amino acid sequence shown in SEQ ID NO: 1. protein.
• amino acid sequence shown in any one of SEQ ID NOs: 1 to 5 amino acid numbers 70, 106, 107, 108, 219, 270, 271, 272, 273, 273, 274, 275
• the amino acid sequence of Nos. 276 and 313 has an amino acid sequence in which one or more amino acids are substituted with amino acids having a lower side chain than the wild-type amino acid, and the reaction shown in Formula (1) is performed.
• amino acid sequence shown in any one of SEQ ID NOs: 1 to 5 the amino acid numbers 188, 190, 193, 215, 216, 217, 314, 358, 362, and 363
• One or more of the amino acids consists of an amino acid sequence substituted with an amino acid having a bulky side chain compared to the wild-type amino acid, and the reaction shown in Formula (1) is converted from the amino acid sequence shown in SEQ ID NO: 1.
• a hydrolase protein having an activity of catalyzing with higher selectivity than the protein.
• amino acid numbers 70, 106, 107, 108, 219, 270, 271, 272, 273, 273, 274, 275 One or more of amino acids No. 276, No. 313 is substituted with an amino acid whose side chain is less bulky than wild-type amino acids, and amino acid Nos. 188, 190, 193,
• amino acids Nos. 215, 216, 217, 314, 358, 362, and 363 are substituted with amino acids having a higher bulk side chain than wild-type amino acids.
• a hydrolase protein comprising an amino acid sequence and having an activity of catalyzing the reaction represented by the formula (1) with higher selectivity than a protein comprising the amino acid sequence represented by SEQ ID NO: 1.
• the leucine of amino acid number 270 is substituted with any amino acid of serine, glutamine, glutamic acid, or alanine, according to [5] or [6] Hydrolase protein.
• the leucine at amino acid number 70 is aspartic acid
• the leucine at 270 number is any one of glutamine, glutamic acid, or alanine
• the 273th leucine is at arginine
• a hydrolase protein comprising an amino acid sequence in which the 313th leucine is substituted with methionine, and having an activity of catalyzing the reaction represented by formula (1) with higher selectivity than the protein comprising the amino acid sequence represented by SEQ ID NO: 1.
• a hydrolase protein according to any one of [1] to [12] is allowed to act on a dialkyl 2-vinylcyclopropane-1,1-dicarboxylic acid to produce (1S, 2S) -1-alkoxycarbonyl.
• the hydrolase protein of the present invention when hydrolyzing dialkyl 2-vinylcyclopropane-1,1-dicarboxylic acid to produce 1-alkoxycarbonyl-2-vinylcyclopropanecarboxylic acid, (1S, 2S) 1-alkoxycarbonyl-2-vinylcyclopropanecarboxylic acid has high selectivity that it can be produced preferentially. That is, according to the hydrolase protein of the present invention, (1S, 2S) -1-alkoxycarbonyl-2-vinylcyclopropanecarboxylic acid, which is useful as an intermediate for producing a therapeutic agent for hepatitis C, is industrially put into practical use. And can be manufactured more efficiently than conventional methods.
• the method for producing (1S, 2S) -1-alkoxycarbonyl-2-vinylcyclopropanecarboxylic acid using the hydrolase protein of the present invention can be used for the production of a therapeutic agent for hepatitis C and its intermediate. it can.
• the compound produced by the production method of the present invention can be used as a raw material or an intermediate in the production of a therapeutic agent for hepatitis C.
• hydrolase protein of the present invention comprises the amino acid sequence shown in any one of SEQ ID NOs: 2 to 5, or an amino acid sequence obtained by mutating the amino acid sequence, and a reaction represented by the formula (1) Is characterized in that it has an activity of catalyzing with higher selectivity than a protein consisting of the amino acid sequence shown in SEQ ID NO: 1.
• SEQ ID NO: 1 is an amino acid sequence of paranitrobenzyl esterase (PNBE23857, GenBank Accession No. ZP_03593235) derived from Bacillus subtilis ATCC23857 strain.
• Et represents an ethyl group.
• selectivity means optical selectivity and means a property of producing an optically active compound by preferentially producing a certain isomer. Specifically, it means the property of preferentially producing (1S, 2S) -1-alkoxycarbonyl-2-vinylcyclopropanecarboxylic acid by hydrolyzing dialkyl 2-vinylcyclopropane-1,1-dicarboxylic acid. To do. Dialkyl 2-vinylcyclopropane-1,1-dicarboxylic acid is required for hydrolase protein to hydrolyze (1S, 2S) -1-alkoxycarbonyl-2-vinylcyclopropanecarboxylic acid The following points are mentioned as selectivity.
• dialkyl 2-vinylcyclopropane-1,1-dicarboxylic acid has two types of stereoisomers, (2R) and (2S), but there are special conditions that cause steric inversion at the 2-position. Unless otherwise, only the (2S) isomer can be the starting material for the desired (1S, 2S) -1-alkoxycarbonyl-2-vinylcyclopropanecarboxylic acid.
• the hydrolase protein is pro-R among the two alkoxycarbonyl groups bonded to the 1-position prochiral carbon.
• Pro-R is a notation that distinguishes two Xs on CX 2 YZ, and the outline is as follows. Priorities are determined for each substituent on C according to the CIP rule. Then, assume that one of the two Xs has a higher priority than the other. The priority relationship with Y and Z is not changed. Based on provisional priorities, the central notation chirality is determined to be R or S using RS notation.
• the priority X at that time is designated as pro-R
• pro-S the priority X at that time
• This selectivity can be compared using the ratio of (1S, 2S) and (1R, 2S) isomers of 1-alkoxycarbonyl-2-vinylcyclopropanecarboxylic acid produced by hydrolysis as an index, and (1S, 2S )
• the yield of (1S, 2S) -1-alkoxycarbonyl-2-vinylcyclopropanecarboxylic acid is improved as the ratio of the amount of (1R, 2S) produced is lower than the amount produced. Is advantageous.
• the hydrolase protein is a pro-R alkoxycarbonyl group out of two alkoxycarbonyl groups bonded to the 1-position prochiral carbon of dialkyl (2R) -2-vinylcyclopropane-1,1-dicarboxylic acid.
• any selectivity can be compared by the enantiomeric excess (% ee) of the (1S, 2S) form of the 1-alkoxycarbonyl-2-vinylcyclopropanecarboxylic acid produced by hydrolysis to the (1R, 2R) form.
• the hydrolase protein is pro-R among the two alkoxycarbonyl groups bonded to the 1-position prochiral carbon of dialkyl (2R) -2-vinylcyclopropane-1,1-dicarboxylic acid.
• Hydroxyloxycarbonyl group can be hydrolyzed to produce (1S, 2R) -1-alkoxycarbonyl-2-vinylcyclopropanecarboxylic acid corresponding to the desired (1S, 2S) diastereomer.
• This selectivity can be compared by the ratio of the generated amount of (1S, 2S) isomer and (1S, 2R) isomer of 1-alkoxycarbonyl-2-vinylcyclopropanecarboxylic acid to be generated.
• Those having a lower production amount of (1S, 2R) body relative to the production amount of 2S) body are less likely to adversely affect the subsequent production process and the physiological activity of the manufactured pharmaceutical, and are advantageous in industrialization.
• the selectivity in the present invention is an index of “1R2S / 1S2S ratio”, “1S2S optical purity (% ee)”, or “1S2R / 1S2S ratio” in 1-alkoxycarbonyl-2-vinylcyclopropanecarboxylic acid. It is what. That is, “the activity of catalyzing the reaction shown in Formula (1) with higher selectivity than the protein consisting of the amino acid sequence shown in SEQ ID NO: 1” of the present invention is the product 1-ethoxycarbonyl-2-vinylcyclohexane.
• the “1R2S / 1S2S ratio”, “1S2S optical purity (% ee)”, or “1S2R / 1S2S ratio” in propanecarboxylic acid can be determined as an index.
• Preferred embodiments of the hydrolase protein of the present invention include the following.
• Reaction consisting of an amino acid sequence in which one or more of the amino acids of Nos. 276 and 313 is substituted with an amino acid having a lower side chain bulk than the wild-type amino acid, and represented by the above formula (1)
• a hydrolase protein having an activity of catalyzing with higher selectivity than a protein comprising (C) In the amino acid sequence shown in any one of SEQ ID NOs: 1 to 5, amino acid numbers 70, 106, 107, 108, 219, 270, 271, 272, 273, 274, 275 One or more of amino acids No. 276, No. 313 is substituted with an amino acid whose side chain is less bulky than wild-type amino acids, and amino acid Nos. 188, 190, 193, One or more of amino acids Nos. 215, 216, 217, 314, 358, 362, and 363 are substituted with amino acids having a higher bulk side chain than wild-type amino acids.
• a hydrolase protein comprising an amino acid sequence and having an activity of catalyzing the reaction represented by the above formula (1) with higher selectivity than a protein comprising the amino acid sequence represented by SEQ ID NO: 1.
• D The hydrolase protein of (a) or (c) above, wherein at least one amino acid of amino acids Nos. 70, 270, 273, and 313 is substituted.
• a hydrolase protein comprising an amino acid sequence and having an activity of catalyzing the reaction represented by the above formula (1) with higher selectivity than a protein comprising the amino acid sequence represented by SEQ ID NO: 1.
• the hydrolase protein (d) is particularly preferable.
• “Bulk” in “an amino acid having a lower side chain bulk than a wild-type amino acid” or “an amino acid having a higher side chain bulk than a wild-type amino acid” specifically refers to “J. Theoret. Biol. , 1968, 21, 170, the value shown as Bulkiness in Table 3 can be used as an index. Specifically, the Bulkiness value of each amino acid is as follows.
• substitution with an amino acid whose side chain is lower in bulk than the wild-type amino acid means that the amino acid has been substituted with an amino acid having a smaller Bulkiness value than the wild-type amino acid.
• substitution with an amino acid having a bulky side chain compared to the wild-type amino acid means that the amino acid has been substituted with an amino acid having a greater Bulkiness value than the wild-type amino acid.
• the hydrolase protein of the present invention includes one or more of leucine or isoleucine having amino acid numbers 70, 270, 273, or 313 in the amino acid sequence shown in any one of SEQ ID NOs: 1 to 5. However, those substituted with an amino acid having a smaller Bulkiness value than these are preferred.
• hydrolase proteins include the following.
• E The hydrolase protein of (d) above, wherein in the amino acid sequence shown in any one of SEQ ID NOs: 1 to 5, at least leucine of amino acid number 70 is aspartic acid, asparagine, serine, threonine, or glycine A hydrolase protein in which any amino acid is substituted.
• F The hydrolase protein of (d) or (e) above, wherein in the amino acid sequence shown in SEQ ID NO: 3 or 4, the leucine at amino acid number 270 is one of serine, glutamine, glutamic acid, or alanine A hydrolase protein that is substituted with an amino acid.
• (G) The hydrolase protein of (d) or (e) above, wherein in the amino acid sequence shown in SEQ ID NO: 1, 2, or 5, isoleucine of amino acid number 270 is serine, glutamine, glutamic acid, Alternatively, a hydrolase protein substituted with any amino acid of alanine.
• (H) The hydrolase protein of any one of (d) to (g) above, wherein in the amino acid sequence shown in any one of SEQ ID NOs: 1 to 5, leucine of amino acid number 273 is arginine or histidine A hydrolase protein substituted with any amino acid.
• leucine at amino acid number 70 is aspartic acid
• leucine at 270 is any amino acid of glutamine, glutamic acid, or alanine
• leucine at 273 is arginine.
• the hydrolase protein of the present invention further includes an amino acid sequence having 90% or more homology with the amino acid sequence of the hydrolase protein of the present invention described above, or the amino acid sequence of the hydrolase protein of the present invention described above. It consists of an amino acid sequence having 1 to several amino acid substitutions, deletions and / or additions, and catalyzes the reaction shown in formula (1) with higher selectivity than the protein consisting of the amino acid sequence shown in SEQ ID NO: 1. Those having the activity of
• amino acid sequence having 90% or more homology with the above-mentioned amino acid sequence of the hydrolase protein of the present invention means the amino acid of the hydrolase protein of the present invention. It means that the homology or identity with the sequence is 90% or more, preferably 93% or more, more preferably 95% or more, particularly preferably 98% or more.
• substitution, deletion and / or addition of one to several amino acids means, for example, about 1 to 40, preferably about 1 to 20 More preferably, it means about 1 to 10, more preferably about 1 to 5.
• the origin of the hydrolase protein of the present invention is not particularly limited, and may be a natural enzyme derived from a microorganism or a genetically modified protein.
• the natural enzyme can be preferably obtained from Bacillus subtilis. That is, the hydrolase protein of the present invention includes, for example, Bacillus subtilis NBRC3026, Bacillus ⁇ subtilis 3 NBRC3108, Bacillus ⁇ subtilis NBRC3027, and Bacillus ⁇ subtilis 13 NBRC3027, and Bacillus ⁇ subtilis NBRC3027.
• Specific purification methods include, for example, salting out with ammonium sulfate (ammonium sulfate) or sodium sulfate, centrifugation, dialysis, ultrafiltration, adsorption chromatography, ion exchange chromatography, hydrophobic chromatography, reverse phase chromatography. Processing operations such as chromatography, gel filtration, gel permeation chromatography, affinity chromatography, electrophoresis, zymography, and combinations thereof.
• ammonium sulfate ammonium sulfate
• sodium sulfate sodium sulfate
• centrifugation dialysis
• ultrafiltration ultrafiltration
• adsorption chromatography ion exchange chromatography
• hydrophobic chromatography hydrophobic chromatography
• reverse phase chromatography reverse phase chromatography.
• Processing operations such as chromatography, gel filtration, gel permeation chromatography, affinity chromatography, electrophoresis, zymography, and combinations thereof.
• the hydrolase protein of the present invention can be obtained as a recombinant protein by cloning the gene of the hydrolase protein of the present invention according to a known method, introducing the gene into a suitable host and expressing it.
• a probe or primer specific to the gene of the hydrolase protein of the present invention is designed based on the information on the base sequence of the gene encoding the hydrolase protein of the present invention, and the probe or primer is used to DNA libraries (e.g., genomic libraries or cDNA such as Bacillus subtilis NBRC3026, Bacillus subtilis NBRC3108, Bacillus subtilis NBRC3027, and Bacillus subtilis NBRC3013)
• the DNA encoding the hydrolase protein of the present invention is isolated or amplified from the rally, etc., put into a vector by gene recombination technology, introduced into a host cell, and expressed therein, thereby allowing the hydrolysis of the present invention. Enzyme protein can be obtained.
• DNA of the present invention DNA encoding the hydrolase protein of the present invention described above is provided.
• Specific examples of the DNA of the present invention include DNA comprising the following base sequences.
• substitution of one to several bases in the base sequence described in SEQ ID NOs: 7 to 10 lack of A base sequence having a loss and / or an addition, which encodes a hydrolase protein having an activity of catalyzing the reaction shown in Formula (1) with higher selectivity than the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 Base sequence:
• DNA encoding the hydrolase protein of the present invention includes Bacillus subtilis NBRC3026, Bacillus ⁇ subtilis NBRC3108, Bacillus ⁇ subtilis NBRC3027, and Bacillus ⁇ subtilis 13 NBRC3027 and Bacillus ⁇ subtilis (Bacillus RC RC30 It is a cloned DNA or a mutation introduced into the DNA.
• Bacillus RC RC30 It is a cloned DNA or a mutation introduced into the DNA.
• the base sequence is determined according to the present invention, it is possible to synthesize based on this base sequence.
• DNA hybridization of a microorganism such as Bacillus subtilis is performed by hybridization using an oligonucleotide prepared based on this base sequence as a probe, or by PCR using an oligonucleotide prepared based on the sequence as a primer. It can also be isolated from a library (eg, genomic DNA library or cDNA library).
• the DNA of the hydrolase protein of the present invention is not limited to those isolated from natural microorganisms, but also known DNA synthesis methods such as the methods described in US Pat. No. 6,472,184 and US Pat. No. 5,750,380. It may be synthesized using.
• DNA capable of hybridizing under stringent conditions refers to DNA having the nucleotide sequence set forth in SEQ ID NOs: 7 to 10 or a complementary sequence thereof and 80% or more, preferably 90% by BLAST analysis. % Or more, more preferably 95% or more of DNA containing a base sequence having homology.
• hybridization under stringent conditions is a reaction in a normal hybridization buffer at a temperature of 40 to 70 ° C., preferably 60 to 65 ° C., and a salt concentration of 15 mM to 300 mM, preferably The washing can be performed in a washing solution of 15 mM to 60 mM or the like.
• “1 to several” in the “base sequence having substitution, deletion and / or addition of 1 to several bases” means, for example, about 1 to 30, preferably 1 Means about 20, more preferably about 1 to 10, more preferably about 1 to 5.
• substitution, deletion, and / or addition of amino acids that do not substantially harm the enzyme activity can be easily selected by those skilled in the art.
• DNA encoding the hydrolase protein of the present invention having amino acid substitutions, deletions, and / or additions that do not substantially affect enzyme activity should be obtained from, for example, natural mutants or variants. Can do.
• DNA having a base sequence having 1 to several base substitutions, deletions and / or additions can be prepared by using a mutagen or site-specific mutation. It can be obtained by a usual mutation operation such as a method. These can be easily performed with a commercially available kit such as PrimeSTAR Mutagenesis Basal Kit (Takara Bio) or Quick Change Site Directed Muta Genesis Kit (Stratagene).
• the present invention further provides a recombinant vector having the DNA of the present invention.
• the DNA of the present invention is usually added to the vector together with a promoter suitable for the host microorganism so that the 5 ′ end of the coding region of the DNA of the present invention is linked downstream of this promoter. insert.
• the DNA of the present invention may be inserted into an expression vector containing a promoter.
• the expression vector is not particularly limited as long as it can replicate and proliferate in the host microorganism, and examples thereof include plasmid vectors, shuttle vectors, and phage vectors.
• Specific plasmid vectors include pBR322, pUC18, pHSG298, pUC118, pSTV28, pTWV228, pHY300PLK (the above plasmid vectors can be purchased from, for example, Takara Bio Inc.), pET expression vector series (manufactured by Novagen), and the like. Can do. Examples of E.
• coli-coryneform bacterium shuttle vectors include plasmid pCRY30 described in JP-A-3-210184; plasmids pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE and pCRY3KX described in JP-A-2-276575; Plasmids pCRY2 and pCRY3 described in Kaihei 1-191686; pAM330 described in JP-A-58-67679; pHM1519 described in JP-A-58-77895; described in JP-A-58-192900 PAJ655, pAJ611 and pAJ1844; pCG1 described in JP-A-57-134500; pCG2 described in JP-A-58-35197; pCG4 and pCG1 described in JP-A-57-183799 Etc., or it may be derivatives thereof.
• the phage vector include ( ⁇ FixI
• a promoter for expressing the DNA encoding the hydrolase protein of the present invention a promoter possessed by the host microorganism can be generally used, but is not limited thereto, and the promoter of the hydrolase protein DNA of the present invention is not limited thereto. Any promoter may be used as long as it is a prokaryotic base sequence for initiating transcription. Specifically, a lactose operon promoter, a tryptophan operon promoter, a lambda phage-derived PL promoter, a tryptophan lactose hybrid (tac) promoter [H. A. Bose et al., Proc. Natl. Acad. Sci. U.
• inducible promoters can be used for the purpose of improving the expression efficiency.
• gene expression can be induced by adding lactose or isopropyl- ⁇ -D-thiogalactoside (IPTG).
• a transformant obtained by introducing the DNA or recombinant vector of the present invention into a host cell.
• the host into which the DNA or recombinant vector of the present invention is introduced is not particularly limited, but dialkyl 2-vinylcyclopropane-1,1-dicarboxylic acid is converted to (1S, 2S) -1-alkoxycarbonyl-2- Those having activity of hydrolyzing other than vinylcyclopropanecarboxylic acid are not suitably used. Further, those having an activity of changing the produced (1S, 2S) -1-alkoxycarbonyl-2-vinylcyclopropanecarboxylic acid into another compound are also not preferable.
• the host that can be used in the present invention is brought into contact with a dialkyl 2-vinylcyclopropane-1,1-dicarboxylic acid or the desired product (1S, 2S) -1-alkoxycarbonyl-2-vinylcyclopropanecarboxylic acid. As a result, it can be selected by analyzing the resulting compound.
• Specific examples of host microorganisms that can be used in the present invention include Escherichia bacteria (for example, Escherichia coli), Actinomycetes bacteria, Bacillus bacteria, Serratia.
• (Serratia) bacteria Pseudomonas bacteria, Corynebacterium bacteria, Brevibacterium bacteria, Rhodococcus bacteria, Lactobacillus bacteria, Streptomyces ( Streptomyces bacteria, Thermus bacteria, Streptococcus bacteria and the like can also be used as hosts for introducing the DNA or recombinant vector of the present invention.
• a competent cell method [Journalourof Molecular Biology, logyVol.53, p.159 (1970)], pulse wave energization method [J. Indust.Microbiol., Vol.5, p.159 (1990)], etc., transduction methods using phages [E. Ohtsubo, Genetics, Vol.64, p.189 (1970)], conjugation transfer method [J. G. C. Ottow , Ann. Rev. Microbiol., Vol. 29, p. 80 (1975)], cell fusion method [MH Gabor, J. Bacteriol., Vol. 137, p. 1346 (1979)] and the like can be used. From these methods, a method suitable for the host microorganism may be appropriately selected.
• homologous recombination technology or transposon or insertion sequence for directly introducing the DNA encoding the hydrolase protein of the present invention linked to a promoter into the chromosome of the host microorganism It can also be expressed by a technique introduced using the above. Therefore, the transformant of the present invention is not limited as long as the enzyme of the present invention is expressed, and the method of gene introduction is not limited.
• the transformant obtained as described above is cultured, and the hydrolase protein of the present invention is obtained from the culture. Can be collected.
• the transformant can be cultured in a normal nutrient medium containing a carbon source, nitrogen source, inorganic salt, various vitamins, etc.
• a carbon source include sugars such as glucose, sucrose, fructose and maltose, ethanol Alcohols such as methanol, citric acid, malic acid, succinic acid, maleic acid, organic acids such as fumaric acid, molasses, etc. are used.
• the nitrogen source for example, ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, urea or the like is used alone or in combination.
• inorganic salts that can be used include potassium monohydrogen phosphate, potassium dihydrogen phosphate, and magnesium sulfate.
• nutrients such as various vitamins such as peptone, meat extract, yeast extract, corn steep liquor, casamino acid, and biotin can be added to the medium.
• Cultivation is usually carried out under aerobic conditions such as aeration stirring and shaking.
• the culture temperature is not particularly limited as long as the host microorganism can grow, and the pH during the culture is not particularly limited as long as the host microorganism can grow.
• the pH adjustment during the culture can be performed by adding an acid or an alkali.
• the enzyme can be collected from the culture by a known collection method using the enzyme activity as an index.
• the enzyme does not necessarily need to be purified to homogeneity, and may be purified to a degree of purification according to the application.
• the crudely purified fraction or purified enzyme used in the present invention not only the cells isolated from the culture solution in which the transformant was cultured, but also the culture solution and the cells are crushed by means of ultrasonic waves, crushing or the like.
• the resulting crushed product, the extract containing the hydrolase protein of the present invention obtained by extracting the crushed product with water, and the like, and further subjected to treatment such as ammonium sulfate salting out and column chromatography.
• the obtained crude enzyme preparation or purified enzyme preparation of the hydrolase protein of the present invention may be used.
• carrier can also be used.
• Immobilization of these cells, disrupted cells, extract or purified enzyme is carried out by immobilizing the cells on an appropriate carrier such as acrylamide monomer, alginic acid, or carrageenan in accordance with a known and commonly used method. It can be performed by the method of making it. For example, when the cells are immobilized on a carrier, they are recovered from the culture or washed with an appropriate buffer, for example, a phosphate buffer (pH 6 to 10) of about 0.02 M to 0.2 M. Can be used.
• an appropriate buffer for example, a phosphate buffer (pH 6 to 10) of about 0.02 M to 0.2 M.
• Examples of the optionally substituted alkyl group having 1 to 10 carbon atoms represented by R include a substituted or unsubstituted linear, branched or cyclic alkyl group having 1 to 10 carbon atoms, and more preferable. Includes a substituted or unsubstituted linear, branched or cyclic alkyl group having 1 to 6 carbon atoms.
• a normal hexyl group, a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group and the like are preferable, and an alkyl group having 1 to 4 carbon atoms is particularly preferable.
• the optionally substituted aralkyl group having 7 to 20 carbon atoms represented by R is preferably a substituted or unsubstituted aralkyl group having 7 to 12 carbon atoms.
• benzyl group 4-methylbenzyl group, 4-methoxybenzyl group, 4-chlorobenzyl group, 4-bromobenzyl group, 2-phenylethyl group, 1-phenylethyl group, 3-phenylpropyl group, etc.
• benzyl group 4-methylbenzyl group, 4-methoxybenzyl group, 4-chlorobenzyl group, 4-bromobenzyl group, 2-phenylethyl group, 1-phenylethyl group, 3-phenylpropyl group, etc.
• benzyl group 4-methylbenzyl group, 4-methoxybenzyl group, 4-chlorobenzyl group, 4-bromobenzyl group, 2-phenylethyl group, 1-phenylethyl group, 3-phenyl
• R examples of the optionally substituted aryl group having 6 to 12 carbon atoms represented by R include a phenyl group, 1-naphthyl group, 2-naphthyl group, o-methylphenyl group, m-methylphenyl group, p -Methylphenyl group, o-methoxyphenyl group, m-methoxyphenyl group, p-methoxyphenyl group, 2,3-dimethylphenyl group, 2,4-dimethylphenyl group, 2,5-dimethylphenyl group, 2,6 -Dimethylphenyl group, 3,4-dimethylphenyl group, 3,5-dimethylphenyl group, 2,3,5-trimethylphenyl group, 2,3,6-trimethylphenyl group, 2,4,6-trimethylphenyl group And o-nitrophenyl group, m-nitrophenyl group, p-nitrophenyl group and the like are preferable.
• R is preferably
• the hydrolase protein of the present invention When the hydrolase protein of the present invention is allowed to act on dialkyl 2-vinylcyclopropane-1,1-dicarboxylic acid, the hydrolase protein of the present invention, which has been purified or roughly purified, is produced.
• (1S) by allowing a microorganism (eg, a transformant having a DNA encoding the hydrolase protein of the present invention) or a treated product thereof to act on a dialkyl 2-vinylcyclopropane-1,1-dicarboxylic acid.
• 2S -1-alkoxycarbonyl-2-vinylcyclopropanecarboxylic acid.
• the reaction method is not particularly limited, and a dialkyl ⁇ 2-vinylcyclopropane-1,1-dicarboxylic acid serving as a substrate is added to a liquid containing the hydrolase protein of the present invention, and an appropriate temperature (for example, 10 ° C. to 40 ° C.). Or about pressure (eg about atmospheric pressure). Thereby, (1S, 2S) -1-alkoxycarbonyl-2-vinylcyclopropanecarboxylic acid can be produced.
• the method for fractionating the desired (1S, 2S) -1-alkoxycarbonyl-2-vinylcyclopropanecarboxylic acid from the reaction mixture is not particularly limited, and any method for separation or purification known to those skilled in the art may be used. it can. For example, it can be carried out by solvent extraction, crystallization, resin adsorption, column chromatography, etc., but is not limited thereto.
• the hydrolase protein of the present invention is a method for producing (1S, 2S) -1-ethoxycarbonyl-2-vinylcyclopropanecarboxylic acid by hydrolyzing diethyl 2-vinylcyclopropane-1,1-dicarboxylic acid In particular, it can be suitably used.
• the dialkyl 2-vinylcyclopropane-1,1-dicarboxylic acid of the present invention can be produced by the reaction of the following formula (3). That is, it can be produced by reacting the raw material compound of the formula (3) with malonic acid esters in the presence of an alkali metal alkoxide or an alkali metal hydride.
• R 3 represents an optionally substituted arylsulfonyl group having 6 to 12 carbon atoms, an alkylsulfonyl group having 1 to 10 carbon atoms, and an aralkylsulfonyl group having 7 to 20 carbon atoms.
• Examples include benzenesulfonyl group, p-toluenesulfonyl group, 1-naphthalenesulfonyl group, 2-naphthalenesulfonyl group, methanesulfonyl group, ethanesulfonyl group, propanesulfonyl group, trifluoromethanesulfonyl group, and benzylsulfonyl group.
• R 3 is preferably a methanesulfonyl group, a benzenesulfonyl group or a p-toluenesulfonyl group, particularly preferably a p-toluenesulfonyl group.
• R is the same as defined above.
• the starting compound of the formula (3) can be produced according to a known method, for example, a method described in Frederic Dolle et al., Bioorg. Med. Chem. 2006, 14, 1115-1125 and the like. Moreover, it can manufacture by reaction of the following formula
• 1,4-butenediol is reacted with a compound represented by R 3 X and then crystallized, or a commercially available raw material compound is hydrolyzed with an acid or a base to produce 1,4-butenediol. It can be produced by obtaining butenediol and further reacting with a compound represented by R 3 X, followed by crystallization.
• X 1 represents a hydrogen atom or R 1
• X 2 represents a hydrogen atom or R 2
• R 1 and R 2 each independently have 2 to 11 carbon atoms that may be substituted
• X 1 is R 1 and X 2 is R 2 , more preferably R 1 and R 2 are an acetyl group, an ethylcarbonyl group, a tert-butylcarbonyl group, and a benzoyl group, and more preferably an acetyl group It is.
• (1S, 2S) -1-ethoxycarbonyl-2-vinylcyclopropanecarboxylic acid obtained in the present invention is useful for the production of various HCV NS3 protease inhibitors and the like that are under development as therapeutic agents for hepatitis C.
• An intermediate (1R, 2S) / (1S, 2R) -1-amino-1-ethoxycarbonyl-2-vinylcyclopropane and a salt thereof can be produced.
• Example 1 Cloning of paranitrobenzyl esterase gene (pnbA; SEQ ID NOs: 6 to 10) (1) Cloning of gene Bacillus subtilis ATCC23857, Bacillus subtilis NBRC3026, Bacillus subtilis (Bacillus subtilis) ) DNeasy Blood & Tissue Kit from cells obtained by culturing NBRC3108, Bacillus subtilis NBRC3027, and Bacillus subtilis NBRC3013 overnight in a liquid medium designated by each strain preservation institution. Chromosomal DNA was prepared using (Qiagen).
• pnbA23857 SEQ ID NO: 6
• PNBE23857 GenBank Accession No. ZP_03593235, SEQ ID NO: 1
• Primers pnbA F and pnbA R for amplifying the full length of the esterase gene were designed and synthesized.
• the respective base sequences are shown in SEQ ID NOs: 11 (pnbAF) and 12 (pnbA R) in the sequence listing.
• PCR polymerase chain reaction
• PCR was performed using KOD-plus-Ver.2 (manufactured by Toyobo Co., Ltd.) according to the conditions of the attached instruction manual. Regarding the temperature condition, after holding at 94 ° C. for 2 minutes, (94 ° C., 15 seconds; 58 ° C., 30 seconds; 68 ° C., 4 minutes) was repeated 30 cycles, and the temperature was held at 68 ° C. for 5 minutes to finish.
• pnbA3026 For the plasmids ppnbA3026, ppnbA3108, ppnbA3027, and ppnbA3013, the inserted DNA sequence was analyzed, and it was confirmed that each gene contained a 1467 bp ORF. These genes were named pnbA3026, pnbA3108, pnbA3027, and pnbA3013, respectively. Each sequence was as shown by SEQ ID NO: 7 (pnbA3026), 8 (pnbA3108), 9 (pnbA3027) and 10 (pnbA3013). The amino acid sequences encoded by the respective DNA sequences were named PNBE3026, PNBE3108, PNBE3027, and PNBE3013.
• Each sequence was as shown in SEQ ID NO: 2 (PNBE3026), 3 (PNBE3108), 4 (PNBE3027) and 5 (PNBE3013).
• Each amino acid sequence showed a sequence identity of 97%, 91%, 90%, and 98% with respect to the known amino acid sequence of PNBE23857 (SEQ ID NO: 1), respectively.
• Example 2 Comparison of Selectivity of Novel Paranitrobenzyl Esterase Using the five plasmids obtained in Example 1, Escherichia coli JM109 (manufactured by Takara Bio Inc.) was transformed according to a conventional method. Each of the obtained recombinant Escherichia coli was cultured by shaking at 30 ° C. in a liquid LB medium containing 20 mg / l kanamycin and 0.2 mM IPTG, and collected at 20 hours of culture.
• VCPDE 100 mM phosphoric acid containing 5 g / l racemic 1,1-diethoxycarbonyl-2-vinylcyclopropane
• VCPDE 5 g / l racemic 1,1-diethoxycarbonyl-2-vinylcyclopropane
• VCPME 1-ethoxycarbonyl-2-vinylcyclopropanecarboxylic acid
• the three enzymes PNBE3026, PNBE3108, and PNBE3027 showed higher values in the “1S2S optical purity” than the enzyme PNBE23857 with a known sequence.
• PNBE3013 showed a lower value in the “1S2R / 1S2S ratio” than the enzyme PNBE23857 with a known sequence.
• Synthesis Example VCPDE used in Example 1 was synthesized as follows. A 2 L 4-neck flask was charged with 93.4 g (583 mmol) of malonic acid diethyl ester and 1000 mL of toluene, and 223 mL (569 mmol) of a 20% sodium ethoxide ethanol solution was added as a base. After stirring at room temperature for 1.5 hours, 59.4 g of trans-1,4-dibromo-2-butene (278 mmol, Aldrich reagent) was added. After stirring at room temperature for 2 hours, 109 mL (278 mmol) of a 20% sodium ethoxide ethanol solution was further added.
• Example 3 Three-dimensional structure modeling of the hydrolase protein of the present invention (SEQ ID NO: 4)
• the amino acid sequence of PNBE3027 was subjected to a BLAST search against Protein Data Bank (PDB), and the following three types of crystal structures were referenced. Selected as.
• the notation A56V indicates that the 56th amino acid residue alanine (A) is substituted with valine (V).
• the S-form and R-form of the substrate VCPDE are generated in the direction of (1S, 2S) -VCPME and the direction of (1R, 2R) -VCPME. It was created by coordinating to the initial structure and minimizing energy. When performing energy minimization, the following distance constraint conditions were set in consideration of the hydrolysis reaction mechanism of esterase.
• Example 4 Estimation of amino acid residues involved in substrate recognition in paranitrobenzyl esterase First, in each of the “1S2S model” and “1R2R model” obtained in Example 3, the carbon at the 2-position of the vinyl group of VCPDE All amino acid residues that were partially or wholly within 8 cm from the atom were extracted. Use Discovery Studio Visualizer v2.5.5.9350 to calculate the distance. Among the extracted amino acid residues, other than the hydrogen atom closest to the carbon atom at the 2-position of the vinyl group of VCPDE of each model The distance from each atom was defined as the distance from each amino acid residue.
• Example 5 Modification of paranitrobenzyl esterase gene by mutagenesis Using the plasmid ppnbA3027 obtained in Example 1 as a template, a primer (L70FW) shown in SEQ ID NO: 13 and a primer (L70RV) shown in SEQ ID NO: 14 were used. Then, a random mutation was introduced into the base encoding leucine with amino acid number 70 by QuikChange Multi Site-Directed Mutagenesis Kit (Stratagene). About 200 colonies obtained were each cultured with shaking at 30 ° C. in a liquid LB medium containing 20 mg / l kanamycin and 0.2 mM IPTG, and collected at 20 hours of culture.
• reaction was performed in 100 mM potassium phosphate buffer containing 5 g / l or 30 g / l racemic VCPDE and 5% dimethyl sulfoxide at 30 ° C and pH 7 for 21 hours. I let you. Further, in the same manner as described above, a primer (L313FW) shown in SEQ ID NO: 15 and a primer (L313RV) shown in SEQ ID NO: 16 are used to randomly generate a base encoding the leucine residue of amino acid number 313. About 200 colonies were reacted in the same manner as described above.
• SEQ ID Nos: 1 to 20 in the present specification are as follows.
• SEQ ID No. 1 Amino acid sequence of Bacillus subtilis ATCC23857-derived paranitrobenzyl esterase (PNBE23857, GenBank Accession No.
• SEQ ID NO: 2 Amino acid sequence PNBE3026 of a hydrolase protein derived from Bacillus subtilis NBRC3026
• SEQ ID NO: 3 Amino acid sequence PNBE3108 of the hydrolase protein derived from Bacillus subtilis NBRC3108
• SEQ ID NO: 4 Amino acid sequence PNBE3027 of the hydrolase protein derived from Bacillus subtilis NBRC3027
• SEQ ID NO: 5 Amino acid sequence PNBE3013 of the hydrolase protein derived from Bacillus subtilis NBRC3013
• SEQ ID NO: 1 SEQ ID NO: 7: Base sequence of a DNA encoding a hydrolase protein derived from Bacillus subtilis NBRC3026 pnbA3026 SEQ ID NO: 8: Base sequence of DNA encoding a hydrolase protein derived from Bacillus subtilis NBRC3108 pnbA3108 SEQ ID NO: 9: Base sequence of DNA pnbA3027 encoding a hydrolase protein derived from Bacillus subtilis NBRC3027 SEQ ID NO: 10: Base sequence of DNA encoding the hydrolase protein derived from Bacillus subtilis NBRC3013 pnbA3013 SEQ ID NO: 11: Primer pnbA F for amplifying the full length of the paranitrobenzyl esterase gene SEQ ID NO: 12: Primer pnbA R for amplifying the full length of paranitrobenzyl esterase gene Sequence number 13: Primer (L70FW) Sequence number 14: Primer (L)

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