WO2012142921A1 - 生物催化不对称还原制备(r)-邻氯扁桃酸甲酯的方法 - Google Patents

生物催化不对称还原制备(r)-邻氯扁桃酸甲酯的方法 Download PDF

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WO2012142921A1
WO2012142921A1 PCT/CN2012/073812 CN2012073812W WO2012142921A1 WO 2012142921 A1 WO2012142921 A1 WO 2012142921A1 CN 2012073812 W CN2012073812 W CN 2012073812W WO 2012142921 A1 WO2012142921 A1 WO 2012142921A1
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recombinant
sequence
base sequence
amino acid
reductase
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French (fr)
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许建和
倪燕
潘江
李春秀
马宏敏
郑高伟
郁惠蕾
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华东理工大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (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
    • C12P41/002—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by oxidation/reduction reactions

Definitions

  • the invention belongs to the technical field of bioengineering, and particularly relates to a method for preparing (R)-o-chloromandelic acid methyl ester by biocatalytic asymmetric reduction, and a recombinant carrier and a genetic engineering bacteria used. Background technique
  • Clopidogrel chemical name 03 ⁇ 4- ⁇ -(2-chlorophenyl)-6,7-dihydrothieno[3,2-c]pyridine-5(4H)-acetic acid methyl ester, a type Platelet aggregation inhibitor, developed by the French company Sanofi-Aventis in 1986, clinically used its sulfate, trade name Plavix® (Plavix:), mainly used to treat arterial porridge Cardiovascular and cerebrovascular diseases such as sclerosis. In 2009, the drug's global sales reached US$10 billion, second only to the lipid-lowering drug atorvastatin, making it the second-best selling drug in the global pharmaceutical market.
  • (R)-o-chloromandelic acid and its methyl ester are important chiral blocks for the synthesis of clopidogrel, and the method for synthesizing clopidogrel by sulfonation and nucleophilic substitution of (R)-o-chloromandelic acid methyl ester
  • the yield is high and the product is essentially free of racemization. Therefore, the study of chiral synthesis of (R)-o-chloromandelic acid methyl ester has broad application prospects.
  • the synthetic route of (R)-o-chloromandelic acid and its methyl ester mainly includes the following three: (1) Starting from racemic o-chloromandelic acid or its ester, using diastereomeric salt crystal resolution or enzyme A hydrolysis-promoting method results in a methyl form of o-chloromandelic acid in a single configuration.
  • the commercial enzyme CAL-A Novozym 735 can hydrolyze the methyl or ethyl ethanomandelate in the aqueous phase, the substrate concentration is 1% (v/v), and the product ee value is 99. %the above.
  • CAL-B (Novozym 435) The product ee value was 95.9% when the o-chloromandelic acid methyl ester was separated, and the product ee value was greater than 99% when the o-chloromandelic acid ethyl ester was separated.
  • the product yield is 98%, the ee value is 90%, and (R)-o-chloromandelic acid is obtained after acid hydrolysis. After further recrystallization, the product ee value can reach above 99%.
  • Glieder et al. (Awgew Chem. Int. Ed., 2003, 42: 4815-4818) used (P m ⁇ a ygifo/ ⁇ ) cyanohydrin hydrolase as a catalyst to obtain (R)-o-chlorol from o-chlorobenzaldehyde. Mandelonitrile, product ee value of 96.5%. After the enzyme is cross-linked and immobilized, the catalyst can be reused in more than 10 batches (Org. Lett., 2005, 7: 327-329). Although the method has high yield and good selectivity, the use of highly toxic hydrocyanic acid increases the difficulty and danger of the reaction operation.
  • the biocatalytic asymmetric reduction reaction conditions are very mild and the environmental pollution is small, making it one of the most promising green production methods.
  • the substrate was added in an amount of 10 g/L.
  • the product ee value was mostly above 98%, and the highest yield of the product was 88.7%. . Guo et al. (/. Chem. Technol.
  • reductase catalyzes the asymmetric reduction reaction, it usually needs to be carried out in the presence of a coenzyme, by reductase and coenzyme.
  • Co-expression of regenerative enzymes may solve the problem of coenzyme regeneration. Japanese Ema et al. Synth.
  • the technical problem to be solved by the present invention is to solve the serious defect that the (R)-o-chloromandelic acid methyl ester needs to additionally add a coenzyme to the existing biological reduction method, and provide a double enzyme using a reductive reductase and a coenzyme regenerating enzyme.
  • the genetically engineered whole cells are used as catalysts to catalyze the asymmetric reduction of methyl o-chlorobenzoylformate to prepare optically pure (R)-o-chloromandelic acid methyl ester, and the recombinant vector and recombinant bacteria used therein.
  • the method does not require the addition of the expensive coenzyme NADP+, which greatly reduces the production cost, and has high production efficiency, high optical purity of the product, mild reaction conditions, environmental friendliness, easy operation and easy amplification.
  • a first aspect of the invention provides a method for the preparation of (R)-o-chloromandelic acid methyl ester by biocatalytic asymmetric reduction, comprising co-expression of recombinant reductase and recombination in the presence of glucose at pH 6-8 Glucose dehydrogenase genetically engineered bacteria wet cells or lyophilized cells thereof are used as catalysts, and biotransformation reaction is carried out using methyl o-chlorobenzoylformate as a substrate, wherein the recombinant reductase is a recombinant aldehyde ketone reductase (aldo-keto reductase, AKR).
  • the recombinant aldosterone reductase is derived from Bacillus subtilis ( ⁇ zd// ⁇ subtilis), and the amino acid sequence is as shown in SEQ. ID NO: 2 in the sequence listing; or is maintained by the aldehyde ketone.
  • a variant amino acid sequence obtained by inserting, deleting or replacing at least one amino acid in the amino acid sequence shown by SEQ. ID NO: 2 in the Sequence Listing, on the premise of enzymatic activity.
  • the recombinant glucose dehydrogenase (GDH) may be any existing glucose dehydrogenase of any source, as long as it can be expressed in the genetic engineering bacteria in which it is located and the regeneration of the coenzyme can be achieved.
  • the recombinant glucose dehydrogenase is derived from Bacillus subtilis ( ⁇ zd// ⁇ TM fo), and more preferably the amino acid sequence is dehydrogenated by recombinant glucose as shown in SEQ. ID NO: 3 in the Sequence Listing.
  • An amino acid sequence obtained by inserting, deleting or replacing at least one amino acid in the amino acid sequence shown by SEQ. ID NO: 3 in the Sequence Listing, while maintaining the catalytic activity of the glucose dehydrogenase; .
  • the genetically engineered bacteria may be various microorganisms conventional in the art as long as it can satisfy the recombinant aldosterone reductase and the recombinant glucose dehydrogenase capable of efficiently expressing the present invention.
  • the genetically engineered bacteria simultaneously express aldosterone reductase and glucose dehydrogenase, and can achieve efficient regeneration of intracellular coenzyme.
  • the genetically engineered bacteria is recombinant Escherichia coli, more preferably recombinant Escherichia coli) BL21 (DE3
  • the genetically engineered bacteria of the present invention can be prepared according to a conventional method in the art, and generally will contain The recombinant aldehyde ketone reductase of the present invention and the recombinant vector of the recombinant glucose dehydrogenase may be introduced into a host cell.
  • the reaction is carried out in a water-organic two-phase system, and the mass to volume ratio (g/L) of the substrate methyl ortho-chlorobenzoylformate to the buffer is from 50 to 1,000, preferably 100-. 600. Since the substrate o-chlorobenzoic acid methyl ester is hardly soluble in water, it can directly form a two-phase system with the buffer; the substrate ortho-chlorobenzoylformate can also be dissolved in other organic solvents, and the buffer It can constitute a two-phase system.
  • the selected organic solvent may be toluene, n-octanol, butyl acetate, ethyl butyrate, ethyl hexanoate, ethyl octanoate, dibutyl phthalate, etc., preferably ethyl octanoate and phthalic acid Butyl ester.
  • the amount of the genetically engineered bacteria of the present invention is a catalytically effective amount, and the substrate can be transformed to 99% or more, and the preferred amount of the freeze-dried cells is 10 to 50 g/L.
  • the amount of glucose and the mass of the substrate are from 1.0 to 2.0, more preferably from 1.0 to 1.5.
  • the effect of the invention can be achieved without the addition of the coenzyme NADP + in the biotransformation reaction.
  • Coenzyme NADP + can also be added to achieve extremely excellent results.
  • the amount of NADP + used is preferably not more than 1.0 mmol/L.
  • the pH of the reaction solution is 6-8, and it is controlled by using a phosphate buffer.
  • the phosphate buffer is preferably a phosphate-potassium phosphate or a sodium phosphate-sodium phosphate buffer.
  • the concentration of the phosphate buffer is preferably 0.05-0.1 mol/L, and the concentration refers to the total concentration of the conjugate acid base in the buffer solution.
  • an alkali solution such as sodium carbonate, potassium carbonate, sodium hydrogencarbonate, potassium hydrogencarbonate, sodium hydroxide, potassium hydroxide or aqueous ammonia may be added dropwise to maintain the pH of the reaction solution constant in the range of pH 6-8. .
  • an alkali solution such as sodium carbonate, potassium carbonate, sodium hydrogencarbonate, potassium hydrogencarbonate, sodium hydroxide, potassium hydroxide or aqueous ammonia may be added dropwise to maintain the pH of the reaction solution constant in the range of pH 6-8.
  • the temperature of the biotransformation reaction is preferably from 15 to 40 ° C, preferably from 20 to 25 ° C.
  • the time of the biotransformation reaction is completely based on the reaction, and is usually from 1 to 24 hours.
  • (R)-o-chloromandelic acid methyl ester can be extracted from the reaction liquid by a conventional method in the art.
  • the reaction solution is directly extracted with a water-insoluble organic solvent, or the cells are removed by filtration using a microfiltration membrane, and the reaction solution is extracted with a water-insoluble organic solvent.
  • the water-insoluble organic solvent to be used is a conventional chemical solvent, and as long as it has a high solubility for the product (R)-o-chloromandelic acid methyl ester, ethyl acetate or butyl acetate is preferred.
  • the pore size of the microfiltration membrane used should be no more than 0.2 ⁇ .
  • a second aspect of the present invention provides a recombinant vector comprising a base sequence encoding an aldehyde ketone reductase and a glucose dehydrogenase; or an aldehyde ketone reductase or a glucose dehydrogenase which is encoded by the base sequence
  • the base sequence of the mutation obtained by inserting, deleting or replacing at least one base in the base sequence on the premise of catalytic activity.
  • the recombinant aldosterone reductase is derived from Bacillus subtilis ( ⁇ zd// ⁇ subtilis), and the amino acid sequence is as shown in SEQ. ID NO: 2 in the sequence listing; or is maintained by the aldehyde ketone. Under the premise of enzymatic activity, by insertion, deletion or substitution as shown in the sequence listing SEQ.ID NO: A variant amino acid sequence resulting from at least one amino acid in the amino acid sequence.
  • the recombinant glucose dehydrogenase may be any existing glucose dehydrogenase of any kind, as long as it can be expressed in the genetic engineering bacteria in which it is located and the regeneration of the coenzyme can be achieved.
  • the amino acid sequence is such as the recombinant glucose dehydrogenase shown by SEQ. ID NO: 3 in the Sequence Listing; or by insertion, deletion or substitution, while maintaining the catalytic activity of the glucose dehydrogenase A variant amino acid sequence obtained by ligating at least one amino acid in the amino acid sequence shown by SEQ. ID NO: 3.
  • the base sequence encoding the aldosterone reductase may be conventional or may be optimized for the efficient expression of a recombinant aldosterone reductase in a host cell according to the codonence of the codon.
  • Preferred is an aldehyde ketone reductase gene derived from Bacillus subtilis, more preferably a base sequence represented by positions 1 to 843 of SEQ ID NO: 1 of the Sequence Listing; or a sequence encoded by the base sequence A mutated base sequence obtained by inserting, deleting or replacing at least one base in the base sequence on the premise of the catalytic activity of the aldosterone reductase.
  • the nucleotide sequence encoding the glucose dehydrogenase may be conventional or may be optimized for efficient expression of recombinant glucose dehydrogenase in a host cell according to codons.
  • the glucose dehydrogenase gene derived from Bacillus subtilis more preferably the base sequence shown in 859 to 1644 of SEQ ID NO: 1 of the sequence listing, or contained in the sequence retained by the base sequence A mutated base sequence obtained by inserting, deleting or replacing at least one base in the base sequence on the premise of the catalytic activity of the glucose dehydrogenase.
  • the recombinant vector may be various vectors conventional in the art, such as a commercially available plasmid, cosmid, phage or viral vector, etc., preferably plasmid pET28a.
  • the recombinant vector of the present invention can be produced according to a conventional method in the art.
  • the recombinant vector of the present invention is produced by the following method: Primer is designed according to the aldehyde ketoreductase ytbE gene of Bacillus subtilis (d// ⁇ fo) 168 which has been reported in Genbank (the primer Preferred as the upstream primer: CGCGGATCCATGACAACACATTTACAAGCAAAAG; downstream bow I: CCGGTCGAGTTAAAAATCAAAGTTGTCCGGATCo ), using the whole genome of Bacillus subtilis as a template for PCR amplification to obtain a gene fragment encoding aldehyde ketone reductase, and recovering the target fragment amplified by PCR.
  • the obtained gene fragment encoding aldehyde ketone reductase was digested and ligated into pET28a plasmid to construct a recombinant plasmid pET28a-AKR.
  • the recombinant plasmid pET28a-AKR was digested with Xhol and dephosphorylated; primers were designed according to the reported Bacillus subtilis ( ⁇ 7/168 glucose dehydrogenase gene (GDH) sequence in Genbank, in which the primer 5' was introduced into pET28a- On the AKR, the 15 bp sequence at both ends of the nick after single restriction enzyme digestion (the primer is preferably as the upstream primer: TGGTGGTGGTGGTGCTTAACCGCGGCCTGCCTGGAA; the downstream arch I substance: the whole genome of the bacterium is used as a template for PCR amplification of the GDH gene fragment, and the PCR amplification is performed.
  • GDH glucose dehydrogenase gene
  • the target gene was recombined into the single-cut pET28a-AKR by gene homologous recombination using the clone EZ kit to form a recombinant plasmid containing both the aldosterone reductase gene and the glucose dehydrogenase gene sequence.
  • pET28a-AKR-GDH the target gene was recombined into the single-cut pET28a-AKR by gene homologous recombination using the clone EZ kit to form a recombinant plasmid containing both the aldosterone reductase gene and the glucose dehydrogenase gene sequence.
  • a third aspect of the invention provides a genetically engineered bacterium comprising the recombinant vector of the invention as described above.
  • the genetically engineered bacteria can simultaneously express aldosterone reductase and glucose dehydrogenase to achieve efficient regeneration of intracellular coenzymes. It can be prepared by transforming the recombinant vector of the present invention into a host microorganism.
  • the host microorganism may be various host microorganisms conventional in the art, as long as the recombinant plasmid can stably replicate itself, and the reductase gene of the present invention carried can be efficiently expressed.
  • the present invention is preferably Escherichia coli), more preferably E. coli BL21 (DE3).
  • the above recombinant vector pET28a-AKR-GDH is transformed into co/BL21 (DE3) to obtain a preferred genetically engineered strain of the present invention, namely E. coli BL21 (DE3) / pET28a-AKR-GDH.
  • a fourth aspect of the present invention provides a method for fermenting a genetically engineered bacteria as described above, comprising inoculating a genetically engineered Escherichia coli as described above to a kanamycin-containing compound (the kanamycin concentration is 10 to 20 ( ⁇ G/ml, preferably 50 g/ml) is cultured in LB medium, and when the optical density OD 6 of the culture solution reaches 0.5-0.7 (preferably 0.6), the final concentration is 0.1-1 mmol/L (preferably 0.5). Mmmol/L) Isopropyl- ⁇ -D-thiogalactopyranoside (IPTG) was continued for 8-16 hours.
  • the fermented liquid is centrifuged to obtain a wet cell of the recombinant strain, and then freeze-dried to obtain a freeze-dried cell of the recombinant strain.
  • the positive progress of the present invention is as follows:
  • the genetically engineered bacteria co-expressing aldehyde ketone reductase and glucose dehydrogenation of the present invention can efficiently and highly selectively prepare methyl (R)-o-chloromandelate.
  • the genetically engineered whole cells of the present invention can catalyze high concentration of o-chlorobenzoylformic acid without additional coenzyme addition.
  • the complete conversion of the methyl ester to a single configuration of (R)-o-chloromandelic acid methyl ester has high catalytic efficiency and strong stereoselectivity.
  • 50 g/L lyophilized cells can catalyze the complete conversion of 400 g/L methyl chlorobenzoylformate to optically pure (R)-o-chloromandelic acid methyl ester.
  • 30 g/L lyophilized cells can completely convert 600 g/L methyl o-chlorobenzoylformate to produce optically pure (R)-o-chloromandelic acid methyl ester.
  • the method of the present invention is the most efficient method for producing (R)-o-chloromanganate by biological reduction.
  • reaction system does not require the addition of expensive coenzymes, the production cost is greatly reduced, the reaction conditions are mild, environmentally friendly, and easy to operate, and it is excellent in synthesizing the chiral intermediate of the best-selling drug clopidogrel. Industrial application prospects. DRAWINGS
  • Figure 1 shows the electropherogram of the PCR amplification product of the aldosterone reductase gene.
  • Figure 2 is an amplification map of the glucose dehydrogenase gene. Among them, 1, the amplification product of glucose dehydrogenase gene; 2, DNA Marker (Marker IV, Beijing Tiangen Biochemical Technology Co., Ltd.). Figure 3. Single enzyme digestion analysis of plasmid pET28a-AKR. Among them, 1, pET28a-AKR single digestion product; 2, DNA Marker (Marker IV, Beijing Tiangen Biochemical Technology Co., Ltd.).
  • Figure 4 is a schematic diagram showing the construction of the recombinant plasmid pET28a-AKR-GDH. detailed description
  • the expression plasmid pET28a was purchased from Shanghai Novagen Corporation.
  • E. coli DH5a and E. coli BL21 (DE3) competent cells were purchased from Beijing Tiangen Biochemical Technology Co., Ltd.
  • the Clone EZ Recombinant Cloning Kit was purchased from GenScript.
  • Embodiments 1 to 4 The process of Embodiments 1 to 4 is shown in Figure 4.
  • the PCR primers were designed according to the gene sequence of the reductase of Bacillus subtilis ( ⁇ ad//i « subtilis 168) included in Genbank (Gene ID 937984) as follows:
  • Upstream bow I substance CGCGGATCCATGACAACACATTTACAAGCAAAAG; downstream bow I substance: CCGGTCGAGTTAAAAATCAAAGTTGTCCGGATC.
  • the underlined part of the upstream primer is the Bamm cleavage site
  • the underlined part of the downstream primer is the Xhoi cleavage site.
  • PCR amplification was performed using genomic DNA of Bacillus subtilis ( ⁇ ad//i « subtilis) 168 (purchased from the Osage Genetics Center of Venezuela, USA).
  • the PCR system was: 2xTaq PCR MasterMix 15 ⁇ , 1 ⁇ (0.3 ⁇ /L) of the upstream and downstream primers, 1 ⁇ (0.1 g) and dd3 ⁇ 40 12 ⁇ 1 of the DNA template.
  • the PCR amplification steps were: (1) 95 ° C, pre-denaturation for 5 min; (2) 94 ° C, denaturation for 45 s; (3) annealing at 60 ° C for 1 min; (4) extension at 72 ° C for 1 min; (2) ⁇ (4) Repeat 35 times; (5) Continue to extend at 72 °C for 10 min and cool to 4 °C.
  • the PCR product was purified by agarose gel electrophoresis, and the target band in the 700-900 bp interval (Fig. 1), which is the aldehyde-ketal reductase gene, was recovered using an agarose gel DNA recovery kit.
  • the target band of the aldehyde ketoreductase gene recovered in Example 1 was digested with restriction endonuclease BamHl and Xhoi for 12 h at 37 ° C, and purified by agarose gel electrophoresis using agarose gel DNA recovery reagent. The box recycles the target segment.
  • the recombinant plasmid pET28a-AKR was obtained by ligating the target fragment under the action of T4 DNA ligase with the plasmid pET28a which was also digested with BamHl and Xhoi at 4 °C overnight.
  • Upstream primer TGGTGGTGGTGGTGCTTAACCGCGGCCTGCCTGGAA; Downstream primer: ACTTTGATTTTTAACAAGGAGATATACATATGTATCC.
  • the genomic DNA of Bacillus subtilis ( ⁇ ad// ⁇ subtilis) ⁇ 6S was used as a template for PCR amplification.
  • the PCR system was: 2xTaq PCR MasterMix 15 ⁇ , 1 ⁇ (0.3 ⁇ /L) for the upstream and downstream primers, 1 ⁇ (0.1 g) and dd3 ⁇ 40 12 ⁇ 1 for the DNA template.
  • the PCR amplification steps were: (1) 95 ° C, pre-denaturation for 5 min; (2) 94 ° C, denaturation for 45 s; (3) annealing at 57 ° C for 1 min; (4) extension at 72 ° C for 1 min; (2) ⁇ (4) Repeat 35 times; (5) Continue to extend at 72 °C for 10 min and cool to 4 °C.
  • the PCR product was purified by agarose gel electrophoresis, and the target band of 700-900 bp interval (Fig. 2), which is the glucose dehydrogenase gene amplification product, was recovered by using the agarose gel DNA recovery kit.
  • the plasmid pET28a-AKR obtained in Example 2 was digested with restriction endonuclease Xhol for 12 h at 37 ° C, purified by agarose gel electrophoresis, and the target fragment was recovered by agarose gel DNA recovery kit (Fig. 3). ).
  • the linearized plasmid pET28a-AKR was homologously recombined with the target band of the amplification product of the glucose dehydrogenase gene recovered in Example 3.
  • the reaction system is: linearized vector 6 ⁇ l, amplification product of glucose dehydrogenase gene 8 ⁇ , !OxCloneEZ buffer 2 ⁇ 1, CloneEZ 2 ⁇ and (1 (1 ⁇ 2 0 2 ⁇ 1.
  • the mixture was placed at 25 ° C for 30 minutes, kept on ice for 5 minutes, and then immediately transformed into E. coli DH5a competent cells, coated with 50 g / ml kanamycin
  • the LB plate was cultured until the growth of the single bacteria, and the obtained plasmid was the recombinant plasmid pET28a-AKR-GDH.
  • the full length of the gene was 1644 bp, and the base sequence was as shown in SEQ ID No: 1 in the sequence listing. From the 1st to the 843th position, the coding sequence of the aldosterone reductase gene, and the coding sequence of the glucose dehydrogenase gene from the 859th to the 1644th.
  • the recombinant plasmid obtained in Example 4 was retransformed into E. coli BL21 (DE3) competent cells, and the transformant was applied to an LB plate containing kanamycin, and cultured at 37 ° C overnight to obtain a positive recombinant Escherichia coli.
  • the recombinant Escherichia coli obtained above was inoculated into LB medium containing kanamycin, and cultured overnight at 37 ° C with shaking, and 1% (v ⁇ inoculum was added to 100 ml of LB medium (peptone 10 g/L). , 500 g flask of yeast extract 5 g / L, NaCl 10 g / L, pH 7.0), shaken at 37 ° C, 180 rpm shaker, when the OD 6 of the culture solution. When 0.6 is reached, the final concentration is added.
  • Example 5 0.3 g of the lyophilized cells of the recombinant Escherichia coli obtained in Example 5 were suspended in 10 ml of sodium phosphate-sodium phosphate buffer (100 mmol/L, pH 6.5), and 6 g of the substrate methyl o-chlorobenzoylformate was added, 9 g glucose and 10 mol NADP + . The reaction was carried out at 30 ° C under magnetic stirring for 22 h. After the completion of the reaction, the mixture was extracted with ethyl acetate and extracted three times. The combined extracts were dried over anhydrous sodium sulfate and evaporated and evaporated.
  • Example 8-16 Asymmetric Reduction Reaction of Recombinant Bacterial Cells at Different Temperatures
  • Example 17-23 Asymmetric Reduction Catalyzed by Recombinant Bacteria in Organic Solvent-Water Two-Phase System 0.5 g of the lyophilized cells of the recombinant Escherichia coli obtained in Example 5 were suspended in 10 ml of sodium phosphate-sodium phosphate buffer (100 mmol/L, pH 6.5), and 10 ml of different organic solvents and 4 g of the substrate o-chlorobenzene were added. Methyl formylformate, 5.4 g of glucose. The reaction was stirred at 22 ° C for 22 h, and 1 mol/L Na 2 C0 3 was added to maintain the pH of the reaction solution at about 6.5. The reaction conversion rate was analyzed by sampling, and the results are shown in Table 2.
  • Example 24 Expanded preparation of (R)-o-chloromandelic acid methyl ester 200 g of fresh recombinant E. coli cells obtained in Example 5 were suspended in 1000 ml of tap water, and 500 g of substrate o-chlorobenz was added. Methyl formate, 540 g of glucose. The reaction was mechanically stirred at 200 rpm at 20 ° C, concentrated ammonia was added, and the reaction pH was maintained at about 6.5. The reaction conversion rate was analyzed by intermittent sampling. After the conversion, the cells were removed by filtration through a microfiltration membrane with a pore size of 0.2 ⁇ m, and the remaining reaction solution was removed.

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Description

生物催化不对称还原制备 (R)-邻氯扁桃酸甲酯的方法 技术领域
本发明属于生物工程技术领域，特别涉及一种生物催化不对称还原制备 (R)-邻氯扁桃酸甲酯的方法及所用的重组载体和基因工程菌。 背景技术
氯吡格雷 (Clopidogrel) , 化学名 0¾-α-(2-氯苯基 )-6,7-二氢噻吩并 [3,2-c]吡 啶 -5(4H)-乙酸甲酯， 系一种血小板凝集抑制剂， 由法国赛诺菲一安万特公司 (Sanofi-Aventis)公司于 1986 年研究开发成功， 临床用其硫酸盐， 商品名 Plavix® (波立维：)， 主要用于治疗动脉粥样硬化等心脑血管疾病。 2009年该 药品全球销售额达一百亿美元， 仅次于降血脂药物阿伐他汀， 成为全球药品 市场中排名第二位的畅销药物。 （R)-邻氯扁桃酸及其甲酯是合成氯吡格雷的 重要手性砌块， （R)-邻氯扁桃酸甲酯经磺酸酯化和亲核取代合成氯吡格雷的 方法， 反应产率高， 产物基本无消旋化。 因此， 研究 (R)-邻氯扁桃酸甲酯的 手性合成具有广阔的应用前景。
目前为止， （R)-邻氯扁桃酸及其甲酯的合成路线主要包括以下三条： (1) 从消旋邻氯扁桃酸或其酯出发， 采用非对映体盐结晶拆分法或酶促 水解拆分法得到单一构型的邻氯扁桃酸甲酯。 如专利 WO2007078176A1 中 报道了商品酶 CAL-A (Novozym 735)可以在水相中水解拆分邻氯扁桃酸甲酯 或乙酯，底物浓度 1 % (v/v) ,产物 ee值均达 99%以上。 CAL-B (Novozym 435) 拆分邻氯扁桃酸甲酯时的产物 ee值为 95.9% , 拆分邻氯扁桃酸乙酯时产物 ee值大于 99%。 CAL-A亦可以在非水相中拆分邻氯扁桃酸甲酯得到光学纯 (R)-邻氯扁桃酸甲酯(>99% ee) , 产物得率 41% , E=34.7 ( Mol. Catal. B: «^y . , 2007, 45: 34-38)。 但这种合成方法的理论收率仅 50% , —定程度上 造成了资源的浪费和环境的污染。 (2) 利用羟腈化酶催化邻氯苯甲醛与氢氰酸不对称合成 (R)-邻氯扁桃腈， 再经酸水解生成 (R)-邻氯扁桃酸。 如 van Langen等 (Org. Process Res. Dev. , 2003, 7: 828-831)利用商品化的羟腈化酶较低温度下 (0-20°C)合成 (R)-邻氯扁 桃腈， 产物得率 98% , ee值 90% , 酸水解后得到 (R)-邻氯扁桃酸， 进一步重 结晶后产物 ee值可达 99%以上。 Glieder等 (Awgew Chem. Int. Ed. , 2003, 42: 4815-4818)利用扁桃 (P m^ a ygifo/^)羟腈水解酶为催化剂， 从邻氯苯甲醛 出发得到 (R)-邻氯扁桃腈， 产物 ee值为 96.5%。 将该酶交联固定化后， 催化 剂可以重复使用 10个批次以上 (Org. Lett., 2005, 7: 327-329)。 该方法虽然产 物得率高， 选择性较好， 但由于需用到剧毒的氢氰酸， 加大了反应操作难度 和危险性。
(3) 直接不对称还原邻氯苯甲酰甲酸或其甲酯得到 (R)-邻氯扁桃酸或其 甲酯， 该方法理论上可以实现 100%的产率， 使原料得到充分利用。 Yin等 (/. Organometal. Chem. , 2009, 694: 2092-2095)利用金属钌（Ru)催化剂通过不对 称氢化法合成 (R)-邻氯扁桃酸甲酯， 但该反应产物 ee值只有 92%， 且反应条 件相对苛刻， 产物中容易残留有毒的重金属。 相比而言， 生物催化的不对称 还原法反应条件非常温和， 环境污染小， 使其成为最具潜力的绿色生产方法 之一。 如专利 EP13 16613A2 中筛选了几十种微生物， 用以不对称还原邻氯 苯甲酰甲酸， 底物加入量 10 g/L， 产物 ee值大多在 98%以上， 产物的最高 得率为 88.7%。 Guo等 (/. Chem. Technol. Biotechnol , 2009, 84: 1787— 1792)筛
Figure imgf000003_0001
ellipsoideus) GIM2.105 , 可以不对称还 原邻氯苯甲酰甲酸生成光学纯 (R)-邻氯扁桃酸， 但底物浓度仅为 30 mM。 Jeong等 (Biotechnol. Lett., 2010, 32: 1529 - 1531)利用面包酵母催化邻氯苯甲 酰甲酸甲酯的不对称还原， 底物浓度 16.7 g/L， 转化率 100% , ee值 96.1%。 利用高表达还原酶的重组细胞作为催化剂可以克服野生微生物催化时反应 的时空产率和立体选择性较低的障碍， 提高产率和立体选择性。 由于还原酶 催化不对称还原反应通常需要在辅酶存在下才能进行，通过将还原酶和辅酶 再生酶类 (如葡萄糖脱氢酶:)进行共表达有可能可以解决辅酶再生问题。 日本 的 Ema等 Synth. Catal, 2008, 350: 2039-2044)通过将羰基还原酶 Gre2 与葡萄糖脱氢酶共表达，利用重组大肠杆菌作为催化剂不对称还原 1 M邻氯 苯甲酰甲酸甲酯合成 (R)-邻氯扁桃酸甲酯， 反应的转化率和对映选择性均可 达 99%以上，但是该方法并没有解决辅酶再生的问题，还是需额外添加 1 g/L 的昂贵辅酶 NADP+ , 显著增大了生产的成本， 且反应需要在 20°C下进行， 一定程度上也增加了能耗。 发明内容
因此， 本发明要解决的技术问题就是针对现有的生物还原法制备 (R)-邻 氯扁桃酸甲酯需额外添加辅酶的严重缺陷， 提供一种利用重组还原酶和辅酶 再生酶双酶的基因工程菌整细胞为催化剂来催化邻氯苯甲酰甲酸甲酯不对 称还原制备光学纯 (R)-邻氯扁桃酸甲酯的方法， 以及其中所用的重组载体和 重组菌。 该方法不需要额外添加价格昂贵的辅酶 NADP+ , 极大地降低了生 产的成本， 并且反应的生产效率高， 产物的光学纯度高， 反应条件温和， 环 境友好， 操作简便， 易于放大。
本发明通过下述技术方案解决上述问题：
本发明的第一方面提供一种生物催化不对称还原制备 (R)-邻氯扁桃酸甲 酯的方法， 包括在 pH 6-8中， 在葡萄糖的存在下， 以共表达重组还原酶和 重组葡萄糖脱氢酶的基因工程菌湿菌体或其冻干细胞为催化剂， 以邻氯苯甲 酰甲酸甲酯为底物， 进行生物转化反应， 其中， 所述的重组还原酶是重组醛 酮还原酶 (aldo-keto reductase， AKR)。
本发明中，所述的重组醛酮还原酶来源于枯草芽孢杆菌 (^zd//^ subtilis) , 氨基酸序列如序列表中 SEQ.ID NO: 2所示； 或者是在保持由该醛酮还原酶 催化活性的前提下， 由插入、 缺失或替换如序列表中 SEQ.ID NO: 2所示的 氨基酸序列中至少一个氨基酸而得到的变异的氨基酸序列。 本发明中， 所述的重组葡萄糖脱氢酶 （GDH ) 可以是现有的任何来源的 葡萄糖脱氢酶，只要能够在所在的基因工程菌中表达且可以实现辅酶的再生即 可。较佳的，所述的重组葡萄糖脱氢酶来源于枯草芽孢杆菌 (^zd//^™ fo)， 更佳的是氨基酸序列如序列表中 SEQ.ID NO : 3所示的重组葡萄糖脱氢酶； 或者是在保持由该葡萄糖脱氢酶催化活性的前提下， 由插入、 缺失或替换如 序列表中 SEQ.ID NO: 3所示的氨基酸序列中至少一个氨基酸而得到的变异 的氨基酸序列。
本发明中， 所述的基因工程菌可为本领域常规的各种微生物， 只要能满足 能够有效表达本发明的重组醛酮还原酶和和重组葡萄糖脱氢酶即可。 该基因 工程菌同时表达醛酮还原酶和葡萄糖脱氢酶， 能够实现细胞内辅酶的高效再 生。 较佳的， 所述的基因工程菌是重组大肠杆菌， 更佳的为重组大肠埃希氏 菌 coli) BL21(DE3 本发明的基因工程菌可以按照本领域的常规方法制备 而得， 一般将含有本发明的重组醛酮还原酶和和重组葡萄糖脱氢酶的重组载 体导入宿主细胞即可。
本发明中， 反应在水-有机两相体系中进行， 所述底物邻氯苯甲酰甲酸甲 酯与缓冲液的质量体积比 (g/L)为 50-1000 , 较佳的为 100-600。 由于底物邻 氯苯甲酰甲酸甲酯难溶于水， 其与缓冲液可以直接构成两相体系； 也可以将 底物邻氯苯甲酰甲酸甲酯溶解于其他有机溶剂中， 与缓冲液可以构成两相体 系。所选的有机溶剂可以是甲苯、 正辛醇、 乙酸丁酯、 丁酸乙酯、 己酸乙酯、 辛酸乙酯、 邻苯二甲酸二丁酯等， 优选辛酸乙酯和邻苯二甲酸二丁酯。 本发 明的基因工程菌的用量为催化有效量， 能使底物转化达到 99%以上即可， 较 佳的所述冻干细胞的用量为 10~50 g/L。 所述葡萄糖用量与底物质量之比较 佳的为 1.0-2.0 , 更佳的为 1.0-1.5。
本发明中，在所述的生物转化反应中不加入辅酶 NADP+就能够达到发明 效果。 还可以加入辅酶 NADP+， 以达到极其优良的效果。 所述的 NADP+用 量较佳的不超过 1.0 mmol/L。 本发明中， 反应液的 pH为 6-8， 通过使用磷酸盐缓冲液进行控制。 所 述的磷酸盐缓冲液较佳的如磷酸 -磷酸钾或磷酸-磷酸钠缓冲液。 磷酸盐缓冲 液的浓度较佳的为 0.05-0.1 mol/L , 所述的浓度是指缓冲溶液中共轭酸碱的 总浓度。 反应过程中也可以滴加碱液， 如碳酸钠、 碳酸钾、 碳酸氢钠、 碳酸 氢钾、氢氧化钠、氢氧化钾、氨水等的水溶液以维持反应液 pH恒定在 pH 6-8 的范围。 在扩大制备中， 也可以不添加缓冲液， 直接通过滴加碱液控制反应 液 pH的恒定。
本发明中，所述的生物转化反应的温度较佳的为 15~40°C，优选为 20-25 °C。 所述的生物转化反应的时间以反应完全为准， 一般为 1-24小时。 不对 称还原反应结束后， 可按本领域常规方法从反应液中提取 (R)-邻氯扁桃酸甲 酯。 使用水不溶性有机溶剂直接对反应液进行萃取， 或使用微滤膜过滤除去 细胞后， 用水不溶性有机溶剂对反应清液进行萃取。所用的水不溶性有机溶 剂为常规化学溶剂， 只要对产品 (R)-邻氯扁桃酸甲酯有较高溶解度的均可以 选用，优先选用乙酸乙酯或乙酸丁酯。所用的微滤膜的孔径应不大于 0.2 μηι。 合并萃取液， 干燥后采用旋转蒸发的方法除去溶剂， 即得到 (R)-邻氯扁桃酸 甲酯粗产品， 进而采用常规的减压蒸馏方法进行提纯， 在一定的真空度条件 下， 收集与 (R)-邻氯扁桃酸甲酯沸点相对应温度下的馏出组分， 即可获得高 纯度的 (R)-邻氯扁桃酸甲酯。产品的化学纯度高于 98% ,光学纯度高于 99%。 本发明的第二方面提供一种重组载体， 其含有编码醛酮还原酶和编码葡 萄糖脱氢酶的碱基序列； 或者含有在保持由该碱基序列编码的醛酮还原酶或 者葡萄糖脱氢酶的催化活性的前提下， 由插入、 缺失或替换该碱基序列中至 少一个碱基而得到的变异的碱基序列。
本发明中，所述的重组醛酮还原酶来源于枯草芽孢杆菌 (^zd//^ subtilis) , 氨基酸序列如序列表中 SEQ.ID NO: 2所示； 或者是在保持由该醛酮还原酶 催化活性的前提下， 由插入、 缺失或替换如序列表中 SEQ.ID NO: 2所示的 氨基酸序列中至少一个氨基酸而得到的变异的氨基酸序列。
本发明中， 所述的重组葡萄糖脱氢酶可以是现有的任何来源的葡萄糖脱 氢酶， 只要能够在所在的基因工程菌中表达且可以实现辅酶的再生即可。较佳 的，
Figure imgf000007_0001
, 更佳的 是氨基酸序列如序列表中 SEQ.ID NO: 3所示的重组葡萄糖脱氢酶； 或者是 在保持由该葡萄糖脱氢酶催化活性的前提下， 由插入、 缺失或替换如序列表 中 SEQ.ID NO : 3所示的氨基酸序列中至少一个氨基酸而得到的变异的氨基 酸序列。
本发明中， 所述的编码醛酮还原酶的碱基序列可以是常规， 也可以是按 照密码子的间并性， 进行了优化适合在宿主细胞中有效表达重组醛酮还原 酶。较佳的是来源于枯草芽孢杆菌的醛酮还原酶基因， 更佳的是序列表 SEQ ID NO : 1的第 1至 843位所示的碱基序列；或者含有在保持由该碱基序列编 码的醛酮还原酶的催化活性的前提下， 由插入、 缺失或替换该碱基序列中至 少一个碱基而得到的变异的碱基序列。
本发明中， 所述的编码葡萄糖脱氢酶的碱基序列同样的可以是常规， 也 可以是按照密码子的间并性，进行了优化适合在宿主细胞中有效表达重组葡 萄糖脱氢酶。 较佳的是来源于枯草芽孢杆菌的葡萄糖脱氢酶基因， 更佳的是 序列表 SEQ ID NO : 1的第 859至 1644位所示的碱基序列， 或者含有在保持 由该碱基序列编码的葡萄糖脱氢酶的催化活性的前提下， 由插入、 缺失或替 换该碱基序列中至少一个碱基而得到的变异的碱基序列。
本发明中，所述的重组载体的可为本领域常规的各种载体，如市售的质粒、 粘粒、 噬菌体或病毒载体等， 优选质粒 pET28a。 本发明的重组载体可以按照 本领域的常规方法制备得到。本发明的一较佳实施例是通过下述方法制得本发 明的重组载体： 根据 Genbank中已报道的枯草芽孢杆菌 ( d// ^ fo) 168 的醛酮还原酶 ytbE 基因设计引物（该引物较佳的如上游引物： CGCGGATCCATGACAACACATTTACAAGCAAAAG ； 下 游 弓 I 物 ： CCGGTCGAGTTAAAAATCAAAGTTGTCCGGATCo )， 以枯草芽孢杆菌全 基因组为模板进行 PCR扩增得到编码醛酮还原酶的基因片段， 将 PCR扩增 出来的目的片段回收。 将所得到的编码醛酮还原酶（AKR) 的基因片段酶切 后连接到 pET28a 质粒上， 构建得到重组质粒 pET28a-AKR。 将重组质粒 pET28a-AKR进行 Xhol单酶切并去磷酸化； 根据 Genbank中已报道的枯草芽 孢杆菌 ( ϋ7/ 168的葡萄糖脱氢酶基因（GDH )序列设计引物， 其 中引物 5'端引入 pET28a-AKR上经过单酶切后切口两端 15 bp的序列 （该引 物 较 佳 的 如 上 游 引 物 ： TGGTGGTGGTGGTGCTTAACCGCGGCCTGCCTGGAA； 下游弓 I物 ： 菌全基因组为模板进行 PCR扩增 GDH基因片段， 将 PCR扩增出来的目的 片段回收后，利用 clone EZ试剂盒通过基因同源重组的方法将目的基因重组 到单酶切后的 pET28a-AKR上，形成同时含有醛酮还原酶基因和葡萄糖脱氢 酶基因序列的重组质粒 pET28a-AKR-GDH。
本发明的第三方面提供一种基因工程菌， 其包含如上所述的本发明的重 组载体。 该基因工程菌能够同时表达醛酮还原酶和葡萄糖脱氢酶， 实现细胞 内辅酶的高效再生。 其可通过将本发明的重组载体转化至宿主微生物中制得。 所述的宿主微生物可为本领域常规的各种宿主微生物，只要能满足重组质粒可 稳定地自行复制， 且所携带的本发明的还原酶基因可被有效表达即可。本发明 优选大肠杆菌 coli) , 更优选 E. coli BL21(DE3)。 将前述重组载体 pET28a-AKR-GDH转化至 co/ BL21(DE3)中， 即可得本发明优选的基因工 程菌株， 即 E. coli BL21 (DE3)/ pET28a-AKR-GDH。
本发明的第四方面提供一种发酵培养如上所述的基因工程菌的方法，包 括将如上所述的基因工程大肠杆菌接种至含卡那霉素 （卡那霉素浓度为 10~20(^g/ml，优选 50 g/ml )的 LB培养基中培养，当培养液的光密度 OD6。。 达到 0.5-0.7 (优选 0.6)时， 加入终浓度为 0.1-1 mmol/L (优选 0.5 mmol/L)的 异丙基 -β-D-硫代吡喃半乳糖苷 (IPTG) , 继续诱导 8-16小时。 将发酵液离 心， 即得重组菌的湿菌体， 再经冷冻干燥即得重组菌的冻干细胞。
在符合本领域常识的基础上， 上述各优选条件， 可任意组合， 即得本发 明各较佳实例。
本发明所用的原料或试剂除特别说明之外， 均市售可得。
本发明的积极进步效果在于： 本发明的共表达醛酮还原酶和葡萄糖脱氢 的基因工程菌可以高效高选择性地制备 (R)-邻氯扁桃酸甲酯。 与现有报道的 生物还原制备 (R)-邻氯扁桃酸甲酯的方法相比， 本发明的基因工程菌整细胞 可以在不额外添加辅酶的情况下，催化高浓度邻氯苯甲酰甲酸甲酯的完全转 化成单一构型的 (R)-邻氯扁桃酸甲酯， 催化效率高、 立体选择性强。 在不加 入辅酶的情况下， 50 g/L冻干细胞可以催化 400 g/L邻氯苯甲酰甲酸甲酯完 全转化生成光学纯的 (R)-邻氯扁桃酸甲酯。 若在加入 1 mmol/L辅酶的情况 下， 30 g/L冻干细胞可以完全转化 600 g/L邻氯苯甲酰甲酸甲酯生成光学纯 的 (R)-邻氯扁桃酸甲酯。本发明的方法是目前为止生物还原法制备 (R)-邻氯扁 桃酸甲酯生产效率最高的方法。 由于反应体系中不需要额外添加价格昂贵的 辅酶， 极大地降低了生产的成本， 且反应条件温和， 对环境友好， 操作简便， 在合成畅销药物氯吡格雷的手性中间体方面具有很好的工业应用前景。 附图说明
以下结合附图说明本发明的特征和有益效果。
图 1为醛酮还原酶基因的 PCR扩增产物电泳图谱。 其中， 1、 醛酮还原 酶基因的 PCR扩增产物； 2、 Marker ( Marker II， 北京天根生化科技有限公 司） 。
图 2为葡萄糖脱氢酶基因的扩增图谱。 其中， 1、 葡萄糖脱氢酶基因的 扩增产物； 2、 DNA Marker (Marker IV， 北京天根生化科技有限公司） 。 图 3质粒 pET28a-AKR的单酶切分析图谱。 其中， 1、 pET28a-AKR单 酶切产物； 2、 DNA Marker ( Marker IV， 北京天根生化科技有限公司） 。
图 4为重组质粒 pET28a-AKR-GDH的构建示意图。 具体实施方式
下面用实施例来进一步说明本发明， 但本发明并不受其限制。 下列实施 例中未注明具体条件的实验方法， 通常按照常规条件， 或按照制造厂商所建 议的条件。
下列实施例中材料的来源为：
表达质粒 pET28a购自上海 Novagen公司。
E. coli DH5a和 E. coli BL21 (DE3)感受态细胞， 2xTaq PCR MasterMix , 琼脂糖凝胶 DNA回收试剂盒购自北京天根生化科技有限公司。
Clone EZ重组克隆试剂盒购自 GenScript公司。
实施例 1~4过程如图 4所示。
实施例 1 醛酮还原酶基因的克隆
根据 Genbank中已收录的枯草芽孢杆菌 (^ad//i« subtilis 168)的还原酶 的基因序列 （Gene ID 937984)， 设计 PCR引物如下：
上游弓 I物： CGCGGATCCATGACAACACATTTACAAGCAAAAG； 下游弓 I物： CCGGTCGAGTTAAAAATCAAAGTTGTCCGGATC。
其中， 上游引物下划线部分为 Bamm酶切位点， 下游引物下划线部分 为 Xhoi酶切位点。 以枯草芽孢杆菌 (^ad//i« subtilis) 168 (从美国俄亥俄州州 立大学杆状菌遗传库存中心， BGSC购买：)的基因组 DNA为模板， 进行 PCR 扩增。 PCR体系为： 2xTaq PCR MasterMix 15 μΐ ,上游引物和下游引物各 1 μΐ (0.3 μηιοΙ/L) , DNA模板 1 μΐ (0.1 g)和 dd¾0 12 μ1。 PCR扩增步骤为： （1) 95°C， 预变性 5 min; (2) 94°C， 变性 45 s; (3) 60°C退火 1 min; (4) 72°C延 伸 1 min; 步骤 (2)~(4)重复 35次； (5) 72°C继续延伸 10 min, 冷却至 4°C。 PCR产物经琼脂糖凝胶电泳纯化， 利用琼脂糖凝胶 DNA 回收试剂盒回收 700~900 bp区间的目标条带 (图 1)， 即醛酮还原酶基因。
实施例 2 重组质粒 pET28a-AKR的制备
将实施例 1 回收所得的醛酮还原酶基因目标条带在 37°C用限制性内切 酶 BamHl和 Xhoi双酶切 12 h， 经琼脂糖凝胶电泳纯化， 利用琼脂糖凝胶 DNA回收试剂盒回收目标片段。 将目标片段在 T4 DNA连接酶的作用下， 与同样经 BamHl和 Xhoi酶切后的质粒 pET28a，在 4°C下连接过夜得到重组 质粒 pET28a-AKR。
实施例 3 葡萄糖脱氢酶基因的克隆
根据 Genbank中已收录的枯草芽孢杆菌 (^ad//i« subtilis) \ 6S的葡萄糖脱 氢酶基因序列 （Gene ID 938261 )， 设计特异性引物：
上游引物： TGGTGGTGGTGGTGCTTAACCGCGGCCTGCCTGGAA； 下游引物： ACTTTGATTTTTAACAAGGAGATATACATATGTATCC。 以枯草芽孢杆菌 (^ad//^ subtilis) \ 6S的基因组 DNA为模板， 进行 PCR 扩增。 PCR体系为： 2xTaq PCR MasterMix 15 μΐ ,上游引物和下游引物各 1 μΐ (0.3 μηιοΙ/L) , DNA模板 1 μΐ (0.1 g)和 dd¾0 12 μ1。 PCR扩增步骤为： （1) 95°C， 预变性 5 min; (2) 94°C， 变性 45 s; (3) 57°C退火 1 min; (4) 72°C延 伸 1 min; 步骤 (2)~(4)重复 35次； (5) 72°C继续延伸 10 min, 冷却至 4°C。 PCR产物经琼脂糖凝胶电泳纯化， 利用琼脂糖凝胶 DNA 回收试剂盒回收 700~900 bp区间的目标条带 （图 2 ) ， 即葡萄糖脱氢酶基因扩增产物。
实施例 4 重组质粒 pET28a-AKR-GDH的制备
将实施例 2中所得质粒 pET28a-AKR在 37°C用限制性内切酶 Xhol单酶 切 12 h， 经琼脂糖凝胶电泳纯化， 利用琼脂糖凝胶 DNA回收试剂盒回收目 标片段 （图 3 )。 将线性化质粒 pET28a-AKR与实施例 3回收所得葡萄糖脱 氢酶基因的扩增产物目标条带进行同源重组。反应体系为：线性化载体 6 μ1， 葡萄糖脱氢酶基因的扩增产物 8 μΐ , !OxCloneEZ 缓冲液 2 μ1， CloneEZ酶 2 μΐ和（1(1Η20 2 μ1。 将混合物在 25°C放置 30分钟， 冰上保持 5分钟， 然后立 即转化 E. coli DH5a感受态细胞， 涂在含 50 g/ml卡那霉素的 LB平板上培 养至长出单菌落后，抽提所得质粒即为重组质粒 pET28a-AKR-GDH，经 DNA 测序， 基因全长 1644bp， 碱基序列如序列表中 SEQ ID No : 1所示。 其中， 从第 1〜第 843位是醛酮还原酶基因的编码序列，从第 859〜第 1644位是葡萄 糖脱氢酶基因的编码序列。
实施例 5 重组菌的制备及培养
将实施例 4所得重组质粒重新转化至 E. coli BL21 (DE3)感受态细胞中， 转化液涂布到含有卡那霉素的 LB平板上， 37°C倒置培养过夜， 即获得阳性 重组大肠杆菌 E. coli BL21 (DE3)/ pET28a-AKR-GDH。
将上述所得的重组大肠杆菌接种至含卡那霉素的 LB培养基中， 37°C振 荡培养过夜，按 1%(νΑ 的接种量接入装有 100 ml LB培养基 (蛋白胨 10 g/L， 酵母膏 5 g/L, NaCl 10 g/L, pH 7.0)的 500 ml三角瓶中， 置 37°C、 180 rpm 摇床培养， 当培养液的 OD6。。达到 0.6时，加入终浓度为 0.5 mmol/L的 IPTG 作为诱导剂， 25°C诱导 12 h后， 将培养液离心， 收集细胞， 并用生理盐水 洗涤两次， 将所得的静息细胞冷冻干燥得冻干细胞。
实施例 6 重组菌催化邻氯苯甲酰甲酸甲酯的不对称还原
取 0.3 g实施例 5所得的重组大肠杆菌的冻干细胞悬浮于 10 ml磷酸-磷 酸钠缓冲液 (100 mmol/L， pH 6.5)中， 加入 6 g底物邻氯苯甲酰甲酸甲酯， 9 g 葡萄糖和 10 mol NADP+。 在 30°C， 磁力搅拌下反应 22h。 反应结束后用乙 酸乙酯进行萃取， 萃取三次， 合并萃取液， 加无水硫酸钠干燥过夜， 减压蒸 馏除去乙酸乙酯并真空干燥， 即得 (R)-邻氯扁桃酸甲酯。 用气相色谱 (手性毛 细管柱 CP-Chirasil-DEX CB， 载气氮气， 进样口温度 280°C， 检测器温度 280°C，柱温 180°C)和液相色谱 (手性 OD-H柱，流动相：正己垸 /异丙醇 =97/3， 流速 1 ml/min，检测器波长 254 nm)分析测定底物转化率和还原产物的 ee值。 核磁共振分析产物纯度， 旋光仪测定比旋光度。 结果如下： 转化率 100% ; 分离得率 92%;66值>99.9%； [ag: -187.1 (c 1.0, CHCl^ HNMR (CDC13, 500 Hz): 3.76 (s, 4H), 5.58 (s, 1H), 7.25-7.29 (m, 2H), 7.38-7.41 (m, 2H)。
实施例 7 重组菌催化邻氯苯甲酰甲酸甲酯的不对称还原
取 5 g实施例 5所得的重组大肠杆菌的冻干细胞悬浮于 100 ml磷酸-磷 酸钠缓冲液 (100 mmol/L， pH 6.5)中， 加入 40 g底物邻氯苯甲酰甲酸甲酯， 60 g葡萄糖。 在 30°C， 机械搅拌 (400rpm)下反应 5 h。 反应结束后用乙酸乙 酯进行萃取， 萃取三次， 合并萃取液， 加无水硫酸钠干燥过夜， 旋转蒸发除 去溶剂， 获得 37.2 g(R)-邻氯扁桃酸甲酯， 66值>99.9%。
实施例 8-16 不同温度下重组菌整细胞催化的不对称还原反应
取 0.5 g实施例 5所得的重组大肠杆菌的冻干细胞悬浮于 10 ml磷酸-磷 酸钠缓冲液 (100mmol/L， pH6.5)中， 加入 4 g底物邻氯苯甲酰甲酸甲酯， 5.4 g葡萄糖。 在不同温度下， 磁力搅拌， 流加 1 mol/L Na2CO 反应液 pH维 持在 6.5左右。 间歇取样， 分析反应转化率， 直至转化终止， 结果见表 1 表 1 不同温度下重组菌催化不对称还原
底物与水相的质
实施例 反应温度 （°C) 反应时间 (h) 转化率 （％) 量体积比 (gl 1)
8 40 50 1 >99
9 30 100 1.5 >99
10 30 200 2 93
11 25 200 4 >99
12 25 400 10 96
13 20 400 12 >99
14 20 500 13 >99
15 20 600 18 95
16 15 1000 48 72
实施例 17-23 有机溶剂-水两相体系中重组菌催化的不对称还原反应 取 0.5 g实施例 5所得的重组大肠杆菌的冻干细胞悬浮于 10 ml磷酸-磷 酸钠缓冲液 (100 mmol/L , pH 6.5)中， 加入 10 ml不同的有机溶剂和 4 g底物 邻氯苯甲酰甲酸甲酯， 5.4 g葡萄糖。在 25°C，磁力搅拌反应 22 h，流加 1 mol/L Na2C03使反应液 pH维持在 6.5左右。 取样分析反应转化率， 结果见表 2。
表 2 有机溶剂 -水两相体系中重组菌催化的不对称还原 实施例 有机溶剂 转化率 （％)
17 辛酸乙酯 > 99
18 邻苯二甲酸二丁酯 97
19 己酸乙酯 83
20 甲苯 77
21 丁酸乙酯 71
22 正辛醇 66
23 乙酸丁酯 52 实施例 24 (R)-邻氯扁桃酸甲酯的扩大制备 取 200 g实施例 5所得的新鲜重组大肠杆菌细胞悬浮于 1000 ml自来水 中， 加入 500 g底物邻氯苯甲酰甲酸甲酯， 540 g葡萄糖。 在 20°C， 200 rpm 机械搅拌反应， 流加浓氨水， 控制反应 pH维持在 6.5左右， 间歇取样分析 反应转化率， 转化完毕后， 采用 0.2 μηι孔径的微滤膜过滤除去细胞， 剩余 反应液用等体积乙酸丁酯萃取 2次， 萃取液加入无水硫酸钠干燥后旋转蒸发 除去溶剂， 进而减压蒸馏， 在 200~350 Pa真空度下， 收集 126°C ~135 °C的馏 分， 获得 (R)-邻氯扁桃酸甲酯 462 g， 66值 > 99.5%， 比旋光度 [α] 5 = - 187°。

Claims

权利要求
1、 一种生物催化不对称还原制备 (R)-邻氯扁桃酸甲酯的方法， 包括在 pH 6-8中，在葡萄糖的存在下， 以共表达重组还原酶和重组葡萄糖脱氢酶的 基因工程菌湿菌体或其冻干细胞为催化剂， 以邻氯苯甲酰甲酸甲酯为底物， 进行生物转化反应， 其特征在于， 所述的重组还原酶是重组醛酮还原酶。
2、 如权利要求 1所述的方法， 其特征在于， 所述的重组醛酮还原酶来源 于枯草芽孢杆菌 (^ad//^ ™ /ώ)， 氨基酸序列如序列表中 SEQ.ID NO : 2所 示； 或者是在保持由该醛酮还原酶催化活性的前提下， 由插入、 缺失或替换 如序列表中 SEQ.ID NO : 2所示的氨基酸序列中至少一个氨基酸而得到的变 异的氨基酸序列。
3、 如权利要求 1或 2所述的方法， 其特征在于， 还加入辅酶 NADP+， 所述的 NADP+用量不超过 1.0 mmol/L。
4、一种重组载体， 其特征在于， 含有编码醛酮还原酶和编码葡萄糖脱氢 酶的碱基序列； 或者含有在保持由该碱基序列编码的醛酮还原酶或者葡萄糖 脱氢酶的催化活性的前提下， 由插入、 缺失或替换该碱基序列中至少一个碱 基而得到的变异的碱基序列。
5、 如权利要求 4所述的重组载体， 其特征在于，
所述的编码醛酮还原酶的碱基序列，
( 1 ) 是编码氨基酸序列如序列表中 SEQ.ID NO : 2所示的重组醛酮还原 酶的碱基序列； 或者是编码在保持由该醛酮还原酶催化活性的前提下， 由插 入、 缺失或替换如序列表中 SEQ.ID NO: 2所示的氨基酸序列中至少一个氨 基酸而得到的变异的氨基酸序列的碱基序列； 或者
( 2 )是序列表 SEQ ID NO : 1的第 1至 843位所示的碱基序列； 或者是 在保持由该碱基序列编码的醛酮还原酶的催化活性的前提下， 由插入、 缺失 或替换该碱基序列中至少一个碱基而得到的变异的碱基序列；
所述的编码葡萄糖脱氢酶的碱基序列，
( 1 ) 是编码氨基酸序列如序列表中 SEQ.ID NO: 3所示的重组葡萄糖脱 氢酶的碱基序列； 或者是编码在保持由该葡萄糖脱氢酶催化活性的前提下， 由插入、 缺失或替换如序列表中 SEQ.ID NO : 3所示的氨基酸序列中至少一 个氨基酸而得到的变异的氨基酸序列的碱基序列；
( 2 ) 是序列表 SEQ ID NO : 1的第 859至 1644位所示的碱基序列； 或 者是在保持由该碱基序列编码的葡萄糖脱氢酶的催化活性的前提下， 由插 入、 缺失或替换该碱基序列中至少一个碱基而得到的变异的碱基序列。
6、 如权利要求 4或 5所述的重组载体， 其特征在于， 所述的重组载体是 质粒 pET28a
7、 一种基因工程菌， 其特征在于， 包含如权利要求 4~6任一项所述的重 组载体。
8、 如权利要求 7所述的基因工程菌， 其特征在于， 所述的基因工程菌为 大肠奸菌。
9、 一种发酵培养如权利要求 8所述的基因工程菌的方法， 其特征在于， 包括将如权利要求 8所述的大肠杆菌接种至含卡那霉素的 LB培养基中培养， 当培养液的光密度 OD6。。达到 0.5-0.7时， 加入终浓度为 0.1-1 mmol/L的异 丙基 -β-D-硫代吡喃半乳糖苷， 继续诱导 8-16小时。
10、 如权利要求 7或 8所述的基因工程菌在生物催化不对称还原邻氯苯 甲酰甲酸甲酯制备 (R)-邻氯扁桃酸甲酯中的应用。
11、 如权利要求 10所述的应用， 其特征在于： 反应体系为有机-水两相 体系， 其中有机相可以是完全由底物邻氯苯甲酰甲酸甲酯构成， 或者是将邻 氯苯甲酰甲酸甲酯溶解于其他有机溶剂中。
12、 如权利要求 1 1所述的反应在水 -底物两相体系中的应用， 其特征在 于： 底物与水相的质量体积比 (g/L)为 50-1000， 优选为 100-600。
13、 如权利要求 1 1所述的应用， 其特征在于： 所述的有机溶剂可以是 甲苯、 正辛醇、 乙酸丁酯、 丁酸乙酯、 己酸乙酯、 辛酸乙酯、 邻苯二甲酸二 丁酯， 优选辛酸乙酯和邻苯二甲酸二丁酯。
14、 如权利要求 10和 1 1所述的应用， 其特征在于， 反应 pH为 6-8 ; 反应温度为 15-35°C， 优选 20-25°C。
15、 如权利要求 10-14所述的应用， 其特征在于， 反应结束后， 用水不 溶性有机溶剂直接对反应液进行萃取， 或使用微滤膜过滤除去细胞后， 用水 不溶性有机溶剂对反应清液进行萃取， 蒸发萃取液除去溶剂， 即得到 (R)-邻 氯扁桃酸甲酯粗产品， 进而采用常规的减压蒸馏方法进行提纯， 获得高纯度 的 (R)-邻氯扁桃酸甲酯。 所述的水不溶性有机溶剂可以有很多种， 优先选用 乙酸乙酯或乙酸丁酯。
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