Enzymatic synthesis method of melogabalin intermediate and related alcohol dehydrogenase

Abstract

The invention discloses an enzymatic synthesis method of a melogabalin intermediate and related alcohol dehydrogenase, racemic 3-ethyl bicyclo [3.2. 0] hept-3-ene-6-ketone is used as a substrate, a genetically engineered bacterium for producing alcohol dehydrogenase is used as an enzyme catalyst, a genetically engineered bacterium for producing formate dehydrogenase, ammonium formate and coenzyme are matched, an enzymatic reaction is carried out under a stirring condition, and the melogabalin intermediate is obtained. And carrying out purification and separation on a reaction product to obtain the melogabalin intermediate (1R, 5S)-3-ethyl bicyclo [3.2. 0] hept-3-ene-6-ketone. The method has the advantages of simple reaction, few steps, mild reaction, less pollution and the like, and has great industrial application potential and commercial value.

Description

Technical Field

[0001] This invention relates to the field of biosynthesis technology, and in particular to an enzymatic method for the synthesis of melogabalin intermediate and related alcohol dehydrogenases. Background Technology

[0002] Alcohol dehydrogenases (ADH, EC 1.1.1.1) are a class of enzymes that catalyze the reduction of ketones and aldehydes to their corresponding alcohols. They are widely distributed in the cells of various organisms and are among the most important redox catalysts in living organisms. In recent years, with a deeper understanding of the catalytic mechanism of these enzymes, ADHs have been widely used in organic synthesis to produce chiral alcohols or aldehydes and other fine chemicals, offering advantages such as high stereoselectivity and environmental friendliness.

[0003] Merogabalin besylate is a novel analgesic belonging to the gabapentin class of drugs, developed by Daiichi Sankyo Co., Ltd. It was initially approved in Japan in 2019 for the treatment of peripheral neuropathic pain (including diabetic peripheral neuropathy and postherpetic neuralgia), and was approved for marketing in China in 2024. Its key intermediate—(1R,5S)-3-ethylbicyclo[3.2.0]heptane-3-en-6-one (also known as merogabalin DB01)—is primarily used in the synthesis of merogabalin. This molecule has multiple chiral centers, and its stereoselective construction is both a key focus and a challenge in the preparation process.

[0004] In 2008, Daiichi Sankyo Co., Ltd. reported a synthetic route for melogabalone in patent CN101878193A. This method uses ethyl 3-oxohexanoate as a starting material, reacting it with allyl bromide to prepare ethyl 4-ethyl-3-hydroxyhept-6-enoate, which is then reduced and hydrolyzed to obtain 4-ethyl-3-hydroxyhept-6-enoic acid. This acid is dissolved in acetic anhydride, potassium acetate is added, and the mixture is stirred at room temperature for 100 minutes, then refluxed for 3.5 hours to cyclize and obtain racemic 3-ethylbicyclo[3.2.0]hept-3-en-6-one. Subsequently, the racemic mixture reacted sequentially with tert-butyl dimethoxyphosphoryl acetate and nitromethane, introducing ester side chains and nitro groups, respectively, to obtain (±)-[(1R,5S,6S)-3-ethyl-6-(nitromethyl)bicyclo[3.2.0]hept-3-en-6-yl]tert-butyl acetate. This was then resolved using a Chiralpak IC chiral column to obtain a single configuration product, which was finally obtained through reduction, Boc protection, and hydrochloric acid hydrolysis. In 2010, patent CN102356061A optimized this route, mainly improving the chiral resolution strategy: D-mandelic acid was used for resolution in the unprotected [6-aminomethyl-3-ethylbicyclo[3.2.0]hept-3-en-6-yl]tert-butyl acetate stage. However, the above chemical synthesis route still suffers from lengthy steps and the need for chiral resolution reagents, resulting in high costs and poor environmental friendliness. In 2013, Daiichi Sankyo Co., Ltd. proposed an enzymatic synthesis process in patent CN104245951A, attempting to achieve optical resolution through biocatalysis at an early stage. This method uses racemic 3-ethylbicyclo[3.2.0]hept-3-en-6-one as a substrate, converting it into an alcohol of the non-target configuration and the desired configuration (1R,5S)-3-ethylbicyclo[3.2.0]hept-3-en-6-one under the action of reductase and coenzyme. However, the highest substrate concentration achieved by this enzymatic process is only 60 g / L, which is still far from industrial application. Summary of the Invention

[0005] The purpose of this invention is to provide an enzymatic synthesis method for melogabalin intermediates, which has good application prospects in the industrial production of (1R,5S)-3-ethylbicyclo[3.2.0]hepten-3-en-6-one; and solves the defects of existing preparation methods for melogabalin intermediates, such as low substrate concentration, poor stereoselectivity, high cost, and environmental pollution.

[0006] The present invention also provides an alcohol dehydrogenase for the enzymatic synthesis of melogabalin intermediates, which has good catalytic activity.

[0007] The technical solution adopted by this invention to solve its technical problem is: An enzymatic synthesis method for melogabalone intermediates is disclosed, using racemic 3-ethylbicyclo[3.2.0]hepten-3-en-6-one as a substrate, a genetically engineered bacterium producing alcohol dehydrogenase as an enzyme catalyst, and a genetically engineered bacterium producing formate dehydrogenase, ammonium formate, and a coenzyme. The enzyme-catalyzed reaction is carried out under stirring conditions, and the reaction product is purified and separated to obtain melogabalone intermediate (1R,5S)-3-ethylbicyclo[3.2.0]hepten-3-en-6-one. The proportions of raw materials in a 1L reaction system are as follows: substrate 50g-300g, wet cells of genetically engineered bacteria producing alcohol dehydrogenase 10-60g, wet cells of genetically engineered bacteria producing formate dehydrogenase 20-50g, ammonium formate 400-700mM, coenzyme 0.5-1.0 mM, and buffer solution balance. This invention utilizes formate dehydrogenase for NADPH regeneration cycling, significantly reducing production costs.

[0008] The alcohol dehydrogenase described in this invention can also be a culture of the aforementioned engineered alcohol dehydrogenase bacteria, or it can be bacterial cells obtained by centrifuging the culture medium or its processed products. Processed products refer to extracts obtained from bacterial cells, lysates, or isolated products obtained by separating and / or purifying extracts, or immobilized products obtained by immobilizing extracts or processed products. In this invention, the water content of the wet bacterial cells is 70-90 wt%.

[0009] Preferably, the enzyme-catalyzed reaction is carried out at a temperature of 5–50°C for 2–24 hours.

[0010] Preferably, the coenzyme is sodium NADP.

[0011] Preferably, the buffer solution is a phosphate buffer solution with a concentration of 0.05–0.10 M and a pH value of 6–8.

[0012] Preferably, the genetically engineered bacteria producing alcohol dehydrogenase is a recombinant Escherichia coli producing alcohol dehydrogenase, and the genetically engineered bacteria producing formate dehydrogenase is a recombinant Escherichia coli producing formate dehydrogenase.

[0013] Preferably, the alcohol dehydrogenase is ADH4, and the amino acid sequence of ADH4 is shown in SEQ ID NO:8. Enzymes whose amino acid sequences have been substituted, deleted, or have added several amino acids and which have the function of catalyzing racemic 3-ethylbicyclo[3.2.0]hept-3-en-6-one are also within the scope of protection of this invention. In the art, substitution with amino acids with similar or comparable properties generally does not change the function of the protein. Similarly, adding one or more amino acids to the C-terminus or N-terminus generally does not change the function of the protein.

[0014] Preferably, the alcohol dehydrogenase is a mutant of ADH4, and the mutant of ADH4 is selected from one or more of the following: The mutant ADH-4-S94Y is formed by mutating S to Y at position 94 of the ADH4 amino acid sequence. The mutant ADH-4-V141F is formed by mutating V to F at position 141 of the ADH4 amino acid sequence. The mutant ADH-4-A185W is formed by mutating the 185th amino acid sequence of ADH4 from A to W. The mutant ADH-4-S94Y / V141F / A185W is formed by mutating the S at position 94 of the ADH4 amino acid sequence to Y, the V at position 141 to F, and the A at position 185 to W. The mutant ADH-4-S94Y / V141F / A185W / I16M is formed by mutating the S at position 94 of the ADH4 amino acid sequence to Y, the V at position 141 to F, the A at position 185 to W, and the I at position 16 to M. The mutant ADH-4-S94Y / V141F / A185W / I16M / Y153S is formed by mutating the S at position 94 of the ADH4 amino acid sequence to Y, the V at position 141 to F, the A at position 185 to W, the I at position 16 to M, and the Y at position 153 to S. The mutant ADH-4-S94Y / V141F / A185W / I16M / Y153S / A194R is formed by mutating the following amino acid sequence of ADH4: S at position 94 to Y, V at position 141 to F, A at position 185 to W, I at position 16 to M, Y at position 153 to S, and A at position 194 to R.

[0015] Compared to the parental ADH-4, the catalytic activities of the above mutants were increased by 1.71, 1.42, 1.68, 3.85, 6.16, 9.24, and 15.71 times, respectively. Conserved substitutions at other amino acid sites, additions or deletions of one or more amino acids, amino-terminal truncation, and carboxyl-terminal truncation of the above alcohol dehydrogenases are also included within the scope of this invention. This invention has discovered the above series of alcohol dehydrogenase mutants, which can efficiently catalyze racemic 3-ethylbicyclo[3.2.0]hept-3-en-6-one, providing a good method and catalyst for the industrial production of the melogabalin chiral intermediate (1R,5S)-3-ethylbicyclo[3.2.0]hept-3-en-6-one.

[0016] An alcohol dehydrogenase for the enzymatic synthesis of melogabalin intermediates, wherein the alcohol dehydrogenase is a mutant of ADH4, and the mutant of ADH4 is selected from one or more of the following: The mutant ADH-4-S94Y is formed by mutating S to Y at position 94 of the ADH4 amino acid sequence; the amino acid sequence is shown in SEQ ID NO.21. The mutant ADH-4-V141F is formed by mutating V to F at position 141 of the ADH4 amino acid sequence; the amino acid sequence is shown in SEQ ID NO.22. The mutant ADH-4-A185W is formed by mutating the A at position 185 of the ADH4 amino acid sequence to W; the amino acid sequence is shown in SEQ ID NO.23. The mutant ADH-4-S94Y / V141F / A185W is formed by mutating S to Y, V to F, and A to W at position 94 of the ADH4 amino acid sequence; the amino acid sequence is shown in SEQ ID NO.24. The mutant ADH-4-S94Y / V141F / A185W / I16M is formed by mutating the S at position 94 of the ADH4 amino acid sequence to Y, the V at position 141 to F, the A at position 185 to W, and the I at position 16 to M; the amino acid sequence is shown in SEQ ID NO.25. The mutant ADH-4-S94Y / V141F / A185W / I16M / Y153S is formed by mutating the S at position 94 of the ADH4 amino acid sequence to Y, the V at position 141 to F, the A at position 185 to W, the I at position 16 to M, and the Y at position 153 to S; the amino acid sequence is shown in SEQ ID NO.26. The mutant ADH-4-S94Y / V141F / A185W / I16M / Y153S / A194R is formed by mutating the following amino acid sequences in ADH4: S at position 94 to Y, V at position 141 to F, A at position 185 to W, I at position 16 to M, Y at position 153 to S, and A at position 194 to R. The amino acid sequence is shown in SEQ ID NO.27.

[0017] A recombinant vector containing the encoding gene for the alcohol dehydrogenase. The recombinant vector refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, animal cell viruses, retroviruses, or other vectors well-known in the art. Vectors used in this invention include, but are not limited to, the pET-28a vector expressed in *Escherichia coli*. Any vector can be used to construct a recombinant expression vector, provided it can stably replicate and exist in the host cell.

[0018] Preferably, the recombinant vector is obtained by inserting the alcohol dehydrogenase DNA fragment into the multiple cloning site of the pET-28a vector to obtain the recombinant plasmid pET-28a-ADH. Optionally, the cloning site is an NdeI restriction site and a SalI restriction site.

[0019] A recombinant host cell containing the recombinant vector. The recombinant host cell refers to a prokaryotic cell, such as an archaea cell, a bacterial cell, or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell, preferably *Escherichia coli* BL21.

[0020] The beneficial effects of this invention are: it has the advantages of simple reaction, few steps, mild reaction, and low pollution, and has great potential for industrial application and commercial value. Attached Figure Description

[0021] Figure 1 This is the HPLC chromatogram of racemic 3-ethylbicyclo[3.2.0]hept-3-en-6-one; Figure 2 This is an HPLC chromatogram of (1R,5S)-3-ethylbicyclo[3.2.0]hept-3-en-6-one. Detailed Implementation

[0022] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0023] In this invention, unless otherwise specified, all raw materials and equipment used are commercially available or commonly used in the field. The methods described in the following embodiments are conventional methods in the field, unless otherwise specified.

[0024] The catalytic process flow of this invention is as follows: .

[0025] In the preparation of the alcohol dehydrogenase of the present invention, the host microorganism for protein expression can be Escherichia coli. Escherichia coli BL21(DE3) can be used as the recombinant plasmid, and pET28 plasmid can be selected. The molecular biology operations involved in the procedure are all well-known and routine experimental procedures in the biological field, including gene acquisition (PCR), splicing of plasmid and target gene (i.e., vector construction), introduction of plasmid containing target gene fragment into bacterial cells (i.e., transformation), bacterial culture in culture medium and enzyme production (i.e., fermentation).

[0026] In this invention, the LB liquid culture medium formula is: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl; the LB solid culture medium formula is: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, and 20 g / L agar.

[0027] Example 1: Obtaining recombinant genetically engineered bacteria containing alcohol dehydrogenase and formate dehydrogenase The seven alcohol dehydrogenases involved are derived from *Bacillus subtilis* BGSC1A1, *Thermoonaerobium brockii*, *Gluconobacter oxydans*, *Comamonas testosteroni*, *Lactobacillus kefir*, *Deinococcus marmoris*, and *Cochlearia officinalis*. These alcohol dehydrogenases are named ADH-1, ADH-2, ADH-3, ADH-4, ADH-5, ADH-6, and ADH-7, respectively, and their nucleotide sequences are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7. As shown in NO:7, the synthesis of the whole gene was completed by Beijing Qingke Biotechnology Co., Ltd., and the gene was codon optimized. Corresponding restriction sites were added to both ends of the gene, and the gene was constructed into the corresponding vector pET28 to obtain the recombinant plasmid vector of alcohol dehydrogenase. The obtained recombinant plasmid vector was then transformed into Escherichia coli BL21(DE3) using conventional methods to obtain recombinant genetically engineered bacteria BL21(DE3) / pET28a-ADH-1, BL21(DE3) / pET28a-ADH-2, BL21(DE3) / pET28a-ADH-3, BL21(DE3) / pET28a-ADH-4, BL21(DE3) / pET28a-ADH-5, BL21(DE3) / pET28a-ADH-6, and BL21(DE3) / pET28a-ADH-7. The specific method for obtaining the recombinant genetically engineered bacteria is referred to the method in the applicant's prior application 202411002189.8.

[0028] The preparation of the formate dehydrogenase (FDH) recombinant genetically engineered bacteria is described in Example 1 of the applicant's prior application 202411002189.8.

[0029] Example 2: Obtaining wet cells of recombinant genetically engineered bacteria containing alcohol dehydrogenase and formate dehydrogenase The recombinant genetically engineered bacteria of alcohol dehydrogenase and formate dehydrogenase obtained in Example 1 were inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C. Then, they were inoculated at a 2% inoculation rate (v / v) into LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C and 150 rpm until the bacterial cell concentration reached OD500. 600=Approximately 0.6, add IPTG to a final concentration of 0.1 mM, induce culture at 28℃ for 12 h, collect the bacterial cells by centrifugation at 4℃ and 12000 rpm for 10 min, wash the wet bacterial cells with 0.85% physiological saline, and store at -20℃ for later use.

[0030] Example 3: Screening of alcohol dehydrogenases from various sources Using the recombinant genetically engineered alcohol dehydrogenase strain prepared in Example 2 as a catalyst, racemic 3-ethylbicyclo[3.2.0]hept-3-en-6-one (confirmed by HPLC) was produced. Figure 1 The reaction was catalyzed by the enzyme NADPH and regenerated using recombinant genetically engineered bacteria containing formate dehydrogenase FDH. The specific reaction conditions were as follows: The wet cells of recombinant genetically engineered bacteria containing alcohol dehydrogenases ADH-1, ADH-2, ADH-3, ADH-4, ADH-5, ADH-6, and ADH-7 (final concentration 20 g / L) and recombinant genetically engineered bacteria containing formate dehydrogenase FDH (final concentration 50 g / L) were resuspended in 9 mL of phosphate buffer (0.1 M, pH 7.0). Then, NADP sodium salt and ammonium formate (final concentration 700 mM / L) were dissolved in 1 mL of phosphate buffer. Methyl tert-butyl ether was used to dissolve racemic 3-ethylbicyclo[3.2.0]hep-3-en-6-one (commercially available, from Bidex Pharmaceuticals), and then added to the reaction system. The final concentration of the substrate was 50 g / L. The reaction was carried out at 30 °C and 1200 rpm for 12 h. Samples were taken for analysis, and the results are shown in Table 1. ADH-1, ADH-2, ADH-3, ADH-5, ADH-6, and ADH-7 showed low activity towards the substrate. Among them, ADH-4 showed the highest activity and selectivity. However, ADH-4 catalyzed the formation of a non-target alcohol, retaining (1R,5S)-3-ethylbicyclo[3.2.0]hep-3-en-6-one. Therefore, ADH-4 was selected as the parent for subsequent experiments.

[0031] Table 1 Results of seven alcohol dehydrogenases catalyzing racemic 3-ethylbicyclo[3.2.0]hept-3-en-6-one .

[0032] Example 4 Construction of mutant libraries To enhance the catalytic activity of alcohol dehydrogenase ADH-4, this invention explores mutation sites of alcohol dehydrogenase and ultimately selects some reasonable sites for site-directed mutagenesis. Among them, the preferred mutations are: S at position 94 mutated to Y, V at position 141 mutated to F, A at position 185 mutated to W, I at position 16 mutated to M, Y at position 153 mutated to S, and A at position 194 mutated to R.

[0033] To mutate the sites in the parental ADH-4 sequence, corresponding primers were designed, and the primer sequences are shown in Table 2.

[0034] Table 2: Primer Design Table .

[0035] Using the recombinant plasmid pET28a-ADH-4 containing the target gene fragment as a template, the template was amplified using the overlap extension PCR method. The PCR amplification system was (50 µL): template DNA 0.1 ng-1 ng, 2× Phanta Max Buffer (Novozymes Biotechnology) 25 µL, dNTPs (10 mM each) 1 µL, upstream and downstream of the mutant primers 1 µL each, PhantaMax Super-Fidelity DNA Polymerase (Novozymes Biotechnology) 1 U, and the remainder ddH2O was added to the total volume. PCR reaction parameters: (1) 95 °C pre-denaturation for 30 s; (2) 95 °C denaturation for 30 s; (3) 65 °C annealing for 30 s; (4) 72 °C extension for 6 min, steps (2)-(4) were repeated 30 times; (5) 72 °C complete extension for 7 min, and stored at 16 °C. After the PCR product showed a positive result by 0.9% agarose gel electrophoresis, 20 µL of the PCR reaction solution was taken, and 1 µL of the restriction enzyme Dpn I was added for digestion at 37 °C for 3 h to remove the template plasmid DNA. The DNA was then inactivated at 65 °C for 10 min. The cells were heat-shocked and transformed into E. coli BL21(DE3) competent cells. After recovery, the cells were plated on LB agar plates containing 50 μg / mL kanamycin and cultured overnight. Each plate yielded a mutant library of approximately 300 clones. Subsequently, 4-5 clones were picked and cultured on LB medium at 37 °C for 8 h. The bacterial culture was then sequenced to obtain the recombinant engineered alcohol dehydrogenase. E.coli BL21(DE3) / pET28a-ADH-4-S94Y、 E.coli BL21(DE3) / pET28a-ADH-4-V141F、 E.coli BL21(DE3) / pET28a-ADH-4-A185W、 E.coli BL21(DE3) / pET28a-ADH-4-S94Y / V141F / A185W、 E.coli BL21(DE3) / pET28a-ADH-4-S94Y / V141F / A185W / I16M、 E.coli BL21(DE3) / pET28a-ADH-4-S94Y / V141F / A185W / I16M / Y153S、 E. coli BL21(DE3) / pET28a-ADH-4-S94Y / V141F / A185W / I16M / Y153S / A194R.

[0036] The culture and induction process for the mutant is the same as in Example 2.

[0037] Example 5: Screening of alcohol dehydrogenase mutants Using the alcohol dehydrogenase mutant prepared in Example 4 as a catalyst, a racemic 3-ethylbicyclo[3.2.0]hepta-3-en-6-one reaction was carried out, and the coenzyme NADPH was cyclically regenerated using recombinant genetically engineered bacteria containing formate dehydrogenase FDH. The specific reaction conditions were as follows: Various alcohol dehydrogenase mutant recombinant genetically engineered bacteria wet cells (final concentration 10 g / L) and formate dehydrogenase FDH recombinant genetically engineered bacteria wet cells (final concentration 50 g / L) were resuspended in 9 mL of phosphate buffer (0.1 M, pH 7.0). Then, NADP sodium salt and ammonium formate (final concentration 700 mM / L) were dissolved in 1 mL of phosphate buffer. Methyl tert-butyl ether was used to dissolve racemic 3-ethylbicyclo[3.2.0]hepta-3-en-6-one, which was then added to the reaction system. The final concentration of the substrate was 50 g / L. The reaction was carried out at 30 °C and 1200 rpm for 0.5 h. Samples were taken for analysis, and the results are shown in Table 3. Compared with the parent ADH-4, the activities of each mutant were significantly improved, with the catalytic activities of the mutants increasing by 1.71, 1.42, 1.68, 3.85, 6.16, 9.24, and 15.71 times, respectively. The mutant ADH-4-S94Y / V141F / A185W / I16M / Y153S / A194R exhibited the highest activity. Therefore, ADH-4-S94Y / V141F / A185W / I16M / Y153S / A194R (abbreviated as ADH-4-M7) was selected as the parent for subsequent experiments.

[0038] Table 3. Increased activity of alcohol dehydrogenase mutants .

[0039] Example 6 Temperature Optimization Using alcohol dehydrogenase ADH-4-M7 as a catalyst, 50 g / L racemic 3-ethylbicyclo[3.2.0]hepta-3-en-6-one was catalyzed under the following conditions: 18 mL phosphate buffer (0.1 M, pH 7.0), 2 mL methyl ether, 1.0 g of racemic 3-ethylbicyclo[3.2.0]hepta-3-en-6-one, 0.4 g of recombinant *E. coli* wet cells containing ADH-4-M7, 0.4 g of recombinant *E. coli* wet cells containing formate dehydrogenase, 0.88 g of ammonium formate (final concentration 700 mM / L), and 0.008 g of sodium NADP coenzyme (final concentration 1 mM / L). The reaction was carried out at 20-50℃ for 1 h, and the conversion rate was measured. The results are shown in Table 4. The highest conversion rate of alcohol dehydrogenase ADH-4-M7 was observed at 30℃, and the reaction was subsequently carried out at 30℃.

[0040] Table 4 Results of catalytic reaction of racemic 3-ethylbicyclo[3.2.0]hepta-3-en-6-one at different temperatures .

[0041] Example 7 pH Optimization Using alcohol dehydrogenase ADH-4-M7 as a catalyst, 50 g / L racemic 3-ethylbicyclo[3.2.0]hepta-3-en-6-one was catalyzed under the following conditions: 18 mL of different pH buffers (0.1 M, pH 5.0-10.0), 2 mL of methyl ether, 1.0 g of racemic 3-ethylbicyclo[3.2.0]hepta-3-en-6-one, 0.4 g of recombinant Escherichia coli wet cells containing ADH-4-M7, 0.4 g of recombinant Escherichia coli wet cells containing formate dehydrogenase, 0.88 g of ammonium formate (final concentration 700 mM / L), and 0.008 g of sodium NADP coenzyme (final concentration 1 mM / L). The reaction was carried out at 30℃ for 1 h, and the conversion rate was measured. The results are shown in Table 5. The alcohol dehydrogenase ADH-4-M7 showed the highest conversion rate at pH 7.0, and the reaction was subsequently carried out at pH 7.0.

[0042] Table 5 Results of catalytic reaction of racemic 3-ethylbicyclo[3.2.0]hepta-3-en-6-one at different pH values .

[0043] Example 8: Optimization of cell and substrate concentrations Using alcohol dehydrogenase ADH-4-M7 as a catalyst, 50-300 g / L racemic 3-ethylbicyclo[3.2.0]hepten-3-en-6-one was catalyzed under the following conditions: 18 mL phosphate buffer (0.1 M, pH 7.0), 2 mL methyl ether, 1.0 g-6.0 g of racemic 3-ethylbicyclo[3.2.0]hepten-3-en-6-one, 0.2-1.2 g of recombinant Escherichia coli wet cells containing ADH-4-M7, 0.8 g of recombinant Escherichia coli wet cells containing formate dehydrogenase, 0.88 g of ammonium formate (final concentration 700 mM / L), and 0.008 g of sodium NADP coenzyme (final concentration 1 mM / L). After reacting at 30℃ for 12 h, samples were taken for liquid chromatography analysis. The results are shown in Table 6. At a substrate concentration of 50 g / L and with 10 g / L bacterial cells as a catalyst, after reacting at 30℃ for 12 h, the non-target ketone was nearly completely converted, and the remaining (1R,5S)-3-ethylbicyclo[3.2.0]hept-3-en-6-one... ee The value was 99.4%. As the substrate concentration increased, the required cell volume increased significantly. Substrate concentrations of 50 g / L, 75 g / L, 100 g / L, 125 g / L, 150 g / L, 175 g / L, 200 g / L, 225 g / L, and 250 g / L required 10-50 g / L of cells for complete reaction. Substrate concentrations of 275 g / L and 300 g / L failed to achieve complete conversion at higher cell concentrations. The ee value of (1R,5S)-3-ethylbicyclo[3.2.0]hept-3-en-6-one did not meet the target. Considering cost and operational procedures, a substrate concentration of 250 g / L and a cell concentration of 50 g / L were chosen as the conditions for subsequent large-scale reactions.

[0044] Table 6: Optimization of ADH-4-M7 cell concentration and racemic 3-ethylbicyclo[3.2.0]hepta-3-en-6-one concentration .

[0045] Example 9: Catalysis of racemic 3-ethylbicyclo[3.2.0]hept-3-en-6-one by recombinant Escherichia coli containing alcohol dehydrogenase ADH-4-M7 (1R,5S)-3-ethylbicyclo[3.2.0]hepta-3-en-6-one was prepared using alcohol dehydrogenase ADH-4-M7 in a 3L system. Racemic 3-ethylbicyclo[3.2.0]hepta-3-en-6-one (750 g, 150 g / L) was dissolved in 300 mL of methyl ether. Resuspension of recombinant *E. coli* cells containing alcohol dehydrogenase ADH-4-M7 (final concentration 50 g / L) was then carried out in 2.7 L of phosphate buffer (0.1 M, pH 7.0). Subsequently, resuspension of recombinant *E. coli* cells containing formate dehydrogenase FDH (final concentration 40 g / L) was carried out. Then, sodium NADP (final concentration 1 mM / L) and ammonium formate (final concentration 700 mM / L) were dissolved in the resuspension. The mixture was incubated at 30°C for 10-20 min. The substrate dissolved in methyl ether was then added to the reaction mixture. The reaction was carried out at rpm and 30℃ for 12 h. According to TLC monitoring, the substrate was completely reacted after 12 h. After two extractions with 1 / 2 volume of ethyl acetate, followed by drying with anhydrous sodium sulfate and rotary evaporation, 338 g of crude (1R,5S)-3-ethylbicyclo[3.2.0]hept-3-en-6-one was finally obtained. The detection chromatogram is shown below. Figure 2 The yield was 45.3% (theoretical yield 50%). ee The value is 99.1%, and the purity is 99.5%.

[0046] Example 10: Preparation of (1R,5S)-3-ethylbicyclo[3.2.0]hept-3-en-6-one in a 50 L system (1R,5S)-3-ethylbicyclo[3.2.0]hepta-3-en-6-one was prepared using alcohol dehydrogenase ADH-4-M7 in a 50 L system. The substrate 3-ethylbicyclo[3.2.0]hepta-3-en-6-one (12.5 kg, 250 g / L) was dissolved in 5 L of methyl ether. Recombinant *E. coli* wet cells containing alcohol dehydrogenase ADH-4-M7 (final concentration 50 g / L) were resuspended in 45 L of phosphate buffer (0.1 M, pH 7.0). Subsequently, resuspended recombinant *E. coli* wet cells containing formate dehydrogenase FDH (final concentration 40 g / L). Then, sodium NADP (final concentration 1 mM / L) and ammonium formate (final concentration 700 mM / L) were dissolved in the resuspended bacterial solution. The mixture was incubated at 30 °C for 10–20 min. The substrate dissolved in methyl ether was then added to the reaction mixture. The reaction was carried out at 30℃ and rpm for 12 h. According to TLC monitoring, the substrate was completely reacted after 12 h. After two extractions with 1 / 2 volume of ethyl acetate, followed by drying with anhydrous sodium sulfate and rotary evaporation, 5586 g of crude (1R,5S)-3-ethylbicyclo[3.2.0]hept-3-en-6-one was finally obtained. The detection chromatogram is shown below. Figure 2The yield was 44.7% (theoretical yield 50%). ee The value is 99.0%, and the purity is 99.4%.

[0047] Example 11 The difference between this embodiment and Example 10 is that the final concentration of the wetted Escherichia coli recombinant formate dehydrogenase FDH is 20 g / L, the final concentration of sodium NADP coenzyme is 0.5 mM / L, and the final concentration of ammonium formate is 400 mM / L.

[0048] Example 12 The difference between this embodiment and Example 10 is that the final concentration of the formate dehydrogenase FDH recombinant Escherichia coli wet cells is 50 g / L.

[0049] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications are possible without departing from the technical solutions described in the claims.

Claims

1. A method for the enzymatic synthesis of an intermediate of meloxicam, characterized in that, The enzyme catalysis reaction is carried out under stirring condition with racemic 3-ethylbicyclo[3.2.0]hept-3-en-6-one as a substrate, the genetically engineered bacteria producing alcohol dehydrogenase as an enzyme catalyst, the genetically engineered bacteria producing formic acid dehydrogenase, ammonium formate and coenzyme, and the intermediate (1R, 5S)-3-ethylbicyclo[3.2.0]hept-3-en-6-one of mefloquine is obtained after purification and separation of the reaction product. The amount of each raw material in 1L reaction system is as follows: 50g-300g of substrate, 10-60g of wet bacteria of the genetically engineered bacteria producing alcohol dehydrogenase, 20-50g of wet bacteria of the genetically engineered bacteria producing formic acid dehydrogenase, 400-700mM of ammonium formate, 0.5-1.0mM of coenzyme, and the rest of buffer solution.

2. A process for the enzymatic synthesis of a meloxicam intermediate according to claim 1, characterized in that, The temperature of the enzyme catalysis reaction is 5-50℃, and the time is 2-24 hours.

3. A process for the enzymatic synthesis of a meloxicam intermediate according to claim 1, characterized in that, The coenzyme is NADP sodium salt.

4. The process for the enzymatic synthesis of a meloxicam intermediate according to claim 1, characterized in that, The buffer solution is phosphate buffer solution, and the concentration of the phosphate buffer solution is 0.05-0.10M, and the pH value is 6-8.

5. The process for the enzymatic synthesis of a meloxicam intermediate according to claim 1, characterized in that, The genetically engineered bacteria producing alcohol dehydrogenase is recombinant Escherichia coli producing alcohol dehydrogenase, and the genetically engineered bacteria producing formic acid dehydrogenase is recombinant Escherichia coli producing formic acid dehydrogenase.

6. The process for the enzymatic synthesis of a meloxicam intermediate according to claim 1, characterized in that, The alcohol dehydrogenase is ADH4, and the amino acid sequence of the ADH4 is shown in SEQ ID NO:

8.

7. The process according to claim 1, characterized in that, The alcohol dehydrogenase is a mutant of ADH4, and the mutant of ADH4 is selected from one or more of the following: The mutant ADH-4-S94Y is obtained by mutating S at the 94th position of the amino acid sequence of ADH4 to Y; The mutant ADH-4-V141F is obtained by mutating V at the 141th position of the amino acid sequence of ADH4 to F; The mutant ADH-4-A185W is obtained by mutating A at the 185th position of the amino acid sequence of ADH4 to W; The mutant ADH-4-S94Y / V141F / A185W is obtained by mutating S at the 94th position of the amino acid sequence of ADH4 to Y, mutating V at the 141th position of the amino acid sequence of ADH4 to F, and mutating A at the 185th position of the amino acid sequence of ADH4 to W; The mutant ADH-4-S94Y / V141F / A185W / I16M is obtained by mutating S at the 94th position of the amino acid sequence of ADH4 to Y, mutating V at the 141th position of the amino acid sequence of ADH4 to F, mutating A at the 185th position of the amino acid sequence of ADH4 to W, and mutating I at the 16th position of the amino acid sequence of ADH4 to M; The mutant ADH-4-S94Y / V141F / A185W / I16M / Y153S is obtained by mutating S at the 94th position of the amino acid sequence of ADH4 to Y, mutating V at the 141th position of the amino acid sequence of ADH4 to F, mutating A at the 185th position of the amino acid sequence of ADH4 to W, mutating I at the 16th position of the amino acid sequence of ADH4 to M, and mutating Y at the 153th position of the amino acid sequence of ADH4 to S; The mutant ADH-4-S94Y / V141F / A185W / I16M / Y153S / A194R is obtained by mutating S at the 94th position of the amino acid sequence of ADH4 to Y, mutating V at the 141th position of the amino acid sequence of ADH4 to F, mutating A at the 185th position of the amino acid sequence of ADH4 to W, mutating I at the 16th position of the amino acid sequence of ADH4 to M, mutating Y at the 153th position of the amino acid sequence of ADH4 to S, and mutating A at the 194th position of the amino acid sequence of ADH4 to R.

8. An alcohol dehydrogenase for use in the enzymatic synthesis of an intermediate of meloxicam, characterized in that, The alcohol dehydrogenase is a mutant of ADH4, and the mutant of ADH4 is selected from one or more of the following: Mutant ADH-4-S94Y, which is mutant of ADH4 amino acid sequence at position 94 S to Y, the amino acid sequence is shown as SEQ ID NO. 16; Mutant ADH-4-V141F, which is mutant of ADH4 amino acid sequence at position 141 V to F, the amino acid sequence is shown as SEQ ID NO. 18; Mutant ADH-4-A185W, which is mutant of ADH4 amino acid sequence at position 185 A to W, the amino acid sequence is shown as SEQ ID NO. 20; Mutant ADH-4-S94Y / V141F / A185W, which is mutant of ADH4 amino acid sequence at position 94 S to Y, position 141 V to F, position 185 A to W, the amino acid sequence is shown as SEQ ID NO. 22; Mutant ADH-4-S94Y / V141F / A185W / I16M, which is mutant of ADH4 amino acid sequence at position 94 S to Y, position 141 V to F, position 185 A to W, position 16 I to M, the amino acid sequence is shown as SEQ ID NO. 24; Mutant ADH-4-S94Y / V141F / A185W / I16M / Y153S, which is mutant of ADH4 amino acid sequence at position 94 S to Y, position 141 V to F, position 185 A to W, position 16 I to M, position 153 Y to S, the amino acid sequence is shown as SEQ ID NO. 26; Mutant ADH-4-S94Y / V141F / A185W / I16M / Y153S / A194R, which is mutant of ADH4 amino acid sequence at position 94 S to Y, position 141 V to F, position 185 A to W, position 16 I to M, position 153 Y to S, position 194 A to R, the amino acid sequence is shown as SEQ ID NO.

28.

9. A recombinant vector, characterized in that, The coding gene containing the alcohol dehydrogenase of claim 8.

10. A recombinant host cell, characterized in that, The recombinant vector of claim 9.

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