A method for synthesizing dapoxetine chiral intermediates by enzyme catalysis

Abstract

The invention discloses an enzyme-catalyzed synthesis method of a dapoxetine chiral intermediate, wherein wet cells of recombinant Escherichia coli expressing aldehyde-keto reductase / alcohol dehydrogenase, wet cells of recombinant Escherichia coli expressing formate dehydrogenase, buffer, substrate, cosolvent, ammonium formate and coenzyme are added to a reaction container, reacted at 25 to 35°C for 2 to 24 hours, extracted, separated and rotary evaporated to obtain a dapoxetine chiral intermediate. The invention has mild reaction conditions, is environmentally friendly, has high regioselectivity and stereoselectivity, avoids chiral resolution and heavy metal residues in the product, and just makes up for the shortcomings of chemical methods, has high conversion rate, high chiral purity, small enzyme dosage and low preparation cost.

Description

Technical Field

[0001] The invention relates to the technical field of biosynthesis, and in particular to an enzyme-catalyzed synthesis method of a dapoxetine chiral intermediate. Background Art

[0002] Dapoxetine (trade name Priligy), chemical name S-(+)-N,N-dimethyl-a-[2-(naphthalenyloxy)ethyl]benyenemethanamine hydrochloride, is a fast-acting and metabolic selective serotonin reuptake inhibitor (SSRI), a drug used to treat premature ejaculation in men aged 16 to 84. Dapoxetine was first developed by Eli Lilly and Company, and was later sold to Johnson & Johnson in 2003. It was approved by the U.S. Food and Drug Administration for the treatment of premature ejaculation in 2004. It is currently the only first-line recommended drug for male premature ejaculation.

[0003] (R)-3-chlorophenylpropanol is an extremely important intermediate in the chemical synthesis process of dapoxetine. (R)-3-chlorophenylpropanol is currently mainly synthesized chemically. CN101012147A mentions a method for preparing (R)-3-chlorophenylpropanol, which reacts 3-chlorophenylpropionone with NaBH4 or KBH4 to produce (R)-3-chlorophenylpropanol, wherein the left-handed isomer of the chiral amino acid derivative includes left-handed tryptophan derivatives, left-handed isoleucine derivatives, etc. The cost of the amino acid derivatives is relatively high, and the method has certain limitations in actual production and application.

[0004] CN104496830A provides a new synthetic route for dapoxetine hydrochloride (the route is as follows), in which another method for synthesizing (R)-3-chlorophenylpropanol is mentioned. This method is to react 3-chlorophenylpropanone with (-) diisopinocampheyl chloroborane in an organic phase solvent at 10 to 80° C. to obtain the compound (R)-3-chlorophenylpropanol. This method uses a large amount of organic solvent, has certain pollution to the environment, and has high requirements for reaction conditions, which makes it difficult to operate.

[0005] (R)-3-Hydroxyphenylpropanol is also an important intermediate in the chemical synthesis of dapoxetine, and currently chemical synthesis is the main method. The existing chemical synthesis method has complicated steps, low product yield, high pollution, is not suitable for large-scale production, and has high cost.

[0006] The current biological preparation method has low conversion rate, insufficient chiral purity, and has defects in tolerance because the substrate is toxic to the enzyme. A large amount of enzyme is required to catalyze the substrate, which is not conducive to the cheap preparation of the intermediate. Summary of the invention

[0007] The purpose of the present invention is to provide an enzyme-catalyzed synthesis method of a chiral intermediate of dapoxetine, which has mild reaction conditions, is environmentally friendly, has high regioselectivity and stereoselectivity, avoids chiral resolution and heavy metal residues in the product, and just makes up for the shortcomings of the chemical method, has high conversion rate, high chiral purity, low enzyme dosage and low preparation cost.

[0008] The technical solution adopted by the present invention to solve its technical problem is:

[0009] An enzyme-catalyzed synthesis method for a dapoxetine chiral intermediate comprises adding wet cells of recombinant Escherichia coli expressing aldehyde-keto reductase / alcohol dehydrogenase, wet cells of recombinant Escherichia coli expressing formate dehydrogenase, a buffer, a substrate, a cosolvent, ammonium formate and a coenzyme into a reaction container, reacting at 25 to 35° C. for 2 to 24 hours, extracting, separating and rotary evaporating to obtain a dapoxetine chiral intermediate;

[0010] The substrate is 3-chloropropiophenone or 3-hydroxypropiophenone;

[0011] The dosage ratio of each raw material in 1L reaction system is: 10-200g of wet cells of recombinant Escherichia coli expressing aldehyde-keto reductase / alcohol dehydrogenase, 10-200g of wet cells of recombinant Escherichia coli expressing formate dehydrogenase, 20g-100g of substrate, 50-500mL of cosolvent, 0.2-1moL of ammonium formate, 0.1-2.5mmoL of coenzyme, and the remainder of buffer.

[0012] The buffer is selected from one of TEA buffer, PB buffer and Tris-HCl buffer.

[0013] The concentration of the TEA buffer is 0.05-0.20 M, and the pH value is 6-8; the concentration of the PB buffer is 0.05-0.10 M, and the pH value is 6-7; the concentration of the Tris-HCl buffer is 0.05-0.10 M, and the pH value is 7-8. Preferably, the concentration of the TEA buffer is 0.1 M and the pH value is 6.5.

[0014] The coenzyme is NADP + or NAD + .

[0015] The co-solvent is at least one of ethyl acetate, butyl acetate, dibutyl phthalate, DMSO, methyl tert-butyl ether, dimethyl phthalate, and methanol.

[0016] The aldehyde-keto reductase / alcohol dehydrogenase is selected from one of the mutants of AKR11, AKR19, AKR22, ADH1, ADH5, ADH11 and ADH1.

[0017] The amino acid sequences of AKR11, AKR19, AKR22, ADH1, ADH5 and ADH11 are shown in SEQ ID NO.1 to SEQ ID NO.6. The nucleotide sequences corresponding to the amino acids are shown in SEQ ID NO.7 to SEQ ID NO.12.

[0018] The mutant of ADH1 is selected from one of ADH1-S16A / G114A, ADH1-S16A / T118A, and ADH1-S16A / V157A;

[0019] ADH1-S16A / G114A means that the S at position 16 of the ADH1 amino acid sequence is changed to A, and the G at position 114 is changed to A;

[0020] ADH1-S16A / T118A means that the S at the 16th position of the ADH1 amino acid sequence is changed to A, and the T at the 118th position is changed to A; ADH1-S16A / V157A means that the S at the 16th position of the ADH1 amino acid sequence is changed to A, and the V at the 157th position is changed to A.

[0021] The dosage ratio of each raw material in the 1L reaction system is: 10-200g of wet recombinant Escherichia coli expressing aldehyde-keto reductase / alcohol dehydrogenase, 50-100g of wet recombinant Escherichia coli expressing formate dehydrogenase, 20g-100g of substrate, 50-500mL of cosolvent, 0.2-1moL of ammonium formate, 0.1-2.5mmoL of coenzyme, and the rest of buffer. The recombinant Escherichia coli wet cells expressing formate dehydrogenase, ammonium formate, and coenzyme constitute a coenzyme circulation system.

[0022] The dosage ratio of each raw material in 1L reaction system is: 10-200g of wet cells of recombinant Escherichia coli expressing aldehyde-keto reductase / alcohol dehydrogenase, 25-75g of wet cells of recombinant Escherichia coli expressing formate dehydrogenase, 20g-100g of substrate, 50-500mL of cosolvent, 0.2-1moL of ammonium formate, 0.1-2.5mmoL of coenzyme, and the remainder of buffer.

[0023] The beneficial effects of the present invention are as follows: the biocatalytic method not only has mild reaction conditions, is environmentally friendly, has high regioselectivity and stereoselectivity, but also avoids chiral resolution and heavy metal residues in the product, which just makes up for the shortcomings of the chemical method. The present invention provides 6 kinds of aldehyde-keto reductases or alcohol dehydrogenases AKR11, AKR19, AKR22, ADH1, ADH5, ADH11, and mutants of preferred enzymes, which can catalyze 3-chlorophenylacetone to generate (R)-3-chlorophenylpropanol, 3-hydroxyphenylacetone to generate (R)-3-hydroxyphenylpropanol, wherein the mutant ADH1-S16A / G114A of ADH1 for generating (R)-3-chlorophenylpropanol from 3-chlorophenylacetone has a reaction conversion rate of 99% and an optical purity of more than 99%. The method only requires one step of reaction to obtain the target product, and the reagents used are less, the reaction conditions are mild, the catalytic activity is high, the production cost is reduced, and it has broad application prospects and considerable market value. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the technical roadmap of the present invention;

[0025] Figure 2 This is the HPLC detection chart of 3-chloropropiophenone. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is further described in detail below through specific embodiments.

[0027] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the following embodiments, unless otherwise specified, are all conventional methods in the art.

[0028] In the method of the present invention, the aldehyde-keto reductase / alcohol dehydrogenase can be a free enzyme (e.g., in the form of enzyme powder), or in the form of cells expressing the aldehyde-keto reductase / alcohol dehydrogenase (e.g., wet bacteria). Or in other forms, such as the supernatant of cell disruption or whole cells expressing the aldehyde-keto reductase / alcohol dehydrogenase, or immobilized free enzyme powder.

[0029] In the preparation method of the aldehyde-keto reductase / alcohol dehydrogenase of the present invention, the enzyme protein expression host microorganism used can be selected from Escherichia coli BL21 (DE3), and the recombinant plasmid can be selected from pET28 plasmid. The molecular biological operations involved in the operation steps are all well-known conventional experimental operation methods 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), cultivation of bacterial cells in culture medium and enzyme production (i.e. fermentation).

[0030] In the present invention, the formula of LB liquid culture medium is: 10 g / L tryptone, 5 g / L yeast extract, and 5 g / L NaCl; the formula of LB solid culture medium is: 10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, and 15 g / L agar.

[0031] Example 1

[0032] Obtaining recombinant bacteria expressing aldehyde-keto reductase / alcohol dehydrogenase:

[0033] (1) The expression vectors of AKR11, AKR19, AKR22, ADH1, ADH5, and ADH11 were synthesized by Shanghai Bioengineering. The commercial plasmid pET28 was used to insert the target gene fragment into pET28 to complete the construction of the expression vector. Gene sequences: AKR11 (SEQ ID NO.7), AKR19 (SEQ ID NO.8), AKR22 (SEQ ID NO.9), ADH1 (SEQ ID NO.10), ADH5 (SEQ ID NO.11), and ADH11 (SEQ ID NO.12).

[0034] (2) Use Escherichia coli BL21 (DE3) to express the enzyme protein. Transform the constructed plasmids into the same strain of Escherichia coli cells in sequence and spread them on LB plate culture medium (add 50 mg / L kanamycin and 20 mg / L chloramphenicol, respectively). The specific implementation is as follows:

[0035] First, the bacteria transferred with plasmid pET28 were spread on LB plate medium containing kanamycin resistance (kanamycin 50 mg / L), cultured at 37°C for 12 hours, and then a single colony was transferred to LB liquid medium (kanamycin was additionally added to a final concentration of 50 mg / L), cultured at 37°C for 12 hours to prepare the competent state, and then the plasmid pET28 containing the target gene was transformed into the competent state, spread on LB plate medium containing chloramphenicol (20 mg / L) resistance, and cultured at 37°C for 12 hours to obtain recombinant bacteria.

[0036] Obtaining recombinant bacteria expressing formate dehydrogenase:

[0037] (1) The formate dehydrogenase expression vector was sent for synthesis (Shanghai Bioengineering Co., Ltd.). The commercial plasmid pET28 was used to insert the target gene fragment into pET28 to complete the construction of the expression vector. The gene sequence of formate dehydrogenase is GenBank No. AXT18256.1.

[0038] (2) Use Escherichia coli BL21 (DE3) to express the enzyme protein. Transform the constructed plasmids into the same strain of Escherichia coli cells in sequence and spread them on LB plate culture medium (add 50 mg / L kanamycin and 20 mg / L chloramphenicol, respectively). The specific implementation is as follows:

[0039] First, the bacteria transferred with plasmid pET28 were spread on LB plate medium containing kanamycin resistance (kanamycin 50 mg / L), cultured at 37°C for 12 hours, and then a single colony was transferred to LB liquid medium (kanamycin was additionally added to a final concentration of 50 mg / L), cultured at 37°C for 12 hours to prepare the competent state, and then the plasmid pET28 containing the target gene was transformed into the competent state, spread on LB plate medium containing chloramphenicol (20 mg / L) resistance, and cultured at 37°C for 12 hours to obtain recombinant bacteria.

[0040] Embodiment 2:

[0041] S1, AKR11, AKR19, AKR22, ADH1, ADH5, ADH11 wet bacterial preparation: The recombinant engineering bacteria AKR11, AKR19, AKR22, ADH1, ADH5, ADH11 prepared in Example 1 were inoculated (inoculation amount 1%) in 10mL LB liquid culture medium and cultured at 37°C and 180rpm for 8-10h. The bacterial solution was inoculated into 100mL LB culture medium with an inoculation amount of 1%, and cultured at 37°C and 180rpm for 2-2.5h (equivalent to an OD600 value of 0.6-0.8), and then IPTG was added at a final concentration of 0.1mM, and induced at 24°C and 180rpm for 12h. The cultured bacterial solution was poured into a 500ml large centrifuge cup, centrifuged at 8000rpm, 4°C for 10min at low temperature and high speed, the supernatant was discarded, the bacterial bodies were collected and weighed for subsequent use.

[0042] Preparation of wet formate dehydrogenase cells: The formate dehydrogenase recombinant engineering bacteria prepared in Example 1 were inoculated (inoculation volume 1%) in 10 mL LB liquid medium and cultured at 37 ° C, 180 rpm for 8-10 h. The bacterial solution was inoculated in 100 mL LB culture medium with an inoculation volume of 1%, and cultured at 37 ° C, 180 rpm for 2-2.5 h (equivalent to an OD600 value of 0.6-0.8), and then IPTG was added at a final concentration of 0.1 mM, and induced for 12 h at 24 ° C, 180 rpm. The cultured bacterial solution was poured into a 500 ml large centrifuge cup, centrifuged at 8000 rpm, 4 ° C for 10 min at low temperature and high speed, and the supernatant was discarded, and the cells were collected and weighed for subsequent use.

[0043] S2. Take 0.15 g of wet bacteria of AKR11, AKR19, AKR22, ADH1, ADH5 and ADH11 respectively and add them into 2 mL EP tubes, and set up a blank control group.

[0044] S3, add 0.15g of wet formate dehydrogenase bacteria to each EP tube, add 700μL of PB buffer (0.1M, pH7), 100μL of 7M ammonium formate solution, and 25mM NADP + Solution 100 μL, 3-chloropropiophenone methyl tert-ether solution 100 μL (3-chloropropiophenone system final concentration is 50 g / L), put the EP tube into a constant temperature mixer, 30°C, 1200 rpm shaking reaction for 4 hours.

[0045] S4. After the reaction is completed, 200 μL of the reaction solution is taken, 200 μL of ethyl acetate is added, the mixture is shaken and mixed for 2 min, centrifuged at 12000 rpm for 1 min, the supernatant is taken, and sent to HPLC for detection. The product is obtained after rotary evaporation.

[0046] After HPLC detection, the conversion rate and ee value of each aldehyde-keto reductase / alcohol dehydrogenase are shown in Table 1:

[0047] Aldehyde-keto reductase / alcohol dehydrogenase Conversion rate ee value AKR11 22.86％ R-type, 12.26% AKR19 >99％ R-type, 85.56% AKR22 56.34％ R-type, 66.76% ADH1 >99％ R-type, 89.26% ADH5 78.69％ R-type, 88.48% ADH11 46.88％ R-type, 42.36%

[0048] The results showed that AKR19 and ADH-1 had the highest conversion rates, and ADH-1 had better chirality with an R-type ee value of 89.26%.

[0049] Example 3: Construction and screening of aldehyde-keto reductase / alcohol dehydrogenase mutant library

[0050] 1. Starting strain:

[0051] The ADH1 screened out in Example 2 was used as the original strain, and the plasmid pET28b(+)-ADH-1 was activated and extracted, wherein the amino acid sequence of ADH-1 is shown in SEQ ID NO.4, and the coding gene sequence is shown in SEQ ID NO.10.

[0052] 2. Single mutation:

[0053] (1) Construction of mutation library

[0054] The preparation of the ADH-1 mutant library was achieved by site-directed mutagenesis. The vector pET28b(+)-ADH-1 in the original strain was used as a template, and alanine scanning primers (see Table 2) were designed for polymerase chain reaction (PCR). The recombinant plasmid digested with DpnⅠ was transferred to Escherichia coli BL21(DE3) competent cells, and the clones were inoculated into 10mL LB plate medium and cultured at 37℃ for 12-16h.

[0055] Table 2 Primer table:

[0056]

[0057] (2) HPLC screening

[0058] Randomly select the positive clones and original strains on the plate, inoculate them into 10mL LB liquid medium (add the corresponding antibiotic kanamycin, 50mg / L in the test tube), and culture them in a temperature-controlled shaker at 37°C and 200rpm for 8-10h. Inoculate the culture solution at a 1% inoculum volume into 100mL LB liquid medium (add the corresponding antibiotic kanamycin, 50mg / L in the shake flask), and also culture them in a temperature-controlled shaker at 37°C and 180rpm for 2-2.5h (equivalent to when the strain density OD 600 When the value is 0.6-0.8), add the inducer IPTG with a final concentration of 0.1mM, and continue to culture in a temperature-controlled shaker at 24°C and 180rpm for 12 hours. Pour the cultured bacterial solution into a 800ml large centrifuge cup, centrifuge it in a low-temperature high-speed centrifuge at 8000rpm and 4°C for 10min, discard the supernatant, collect the bacteria and weigh them for subsequent use.

[0059] HPLC was used to detect the concentration and chirality of 3-chloropropiophenone and 3-chlorophenylpropanol and calculate the conversion rate. The dominant strains were screened using the conversion rate and chirality as indicators.

[0060] The obtained dominant strains were sent to Hangzhou Qingke Biotechnology Co., Ltd. for sequencing and stored in a -80°C refrigerator. The dominant mutants finally screened were ADH1-S16A, ADH1-G114A, ADH1-T118A, ADH1-V157A, and ADH1-L211A.

[0061] 3. Iterative mutagenesis Using vectors pET28b(+)-ADH1-S16A, ADH1-G114A, ADH1-T118A, ADH1-V157A, and ADH1-L211A as templates, alanine scanning primers (primers are the same as in Table 2) were designed to perform polymerase chain reaction (PCR). Based on the single-point mutants, the dominant mutations were further screened according to the HPLC screening steps.

[0062] PCR reaction system (25 μL): 1 μL forward primer (100 μM), 1 μL reverse primer (100 μM), 12.5 μL 2× Phanta buffer (Novagen, China), 0.5 μL dNTP mixture (10 mM each), 1 μL plasmid template, 0.5 μL DNA polymerase Phanta (Novagen, China) and 8.5 μL ultrapure water.

[0063] The PCR program set according to the Phanta Super-Fidelity DNA polymerase manual (Novozyme, China) was as follows: 95°C pre-denaturation for 5 min, followed by 30 cycles (95°C denaturation for 15 s, 55°C annealing for 15 s, 72°C extension for 4 min), 72°C final extension for 10 min, and 16°C incubation.

[0064] Iterated mutants (double-site mutants) were obtained: ADH1-S16A / G114A, ADH1-S16A / T118A, ADH1-S16A / V157A, ADH1-S16A / L211A, ADH1-G114A / T118A, ADH1-G114A / V157A, ADH1-G114A / L211A, ADH1-T118A / V157A, ADH1-T118A / L211A, ADH1-V157A / L211A.

[0065] 4. Catalytic activity detection

[0066] The single mutant, iterative mutant and control strain bacterial liquid (WT, ADH1 as control) were used as catalysts, and 3-chlorophenylacetone was used as substrate to compare the catalytic activity of each mutant. The reaction system was selected as 1mL, the catalyst dosage was 100g / L of bacterial wet weight, the final substrate concentration was 50g / L, pH 7.0, 100mM PB buffer was used as the reaction medium, ammonium formate 0.7M, formate dehydrogenase 100g / L, NADPH 2.5mM, 0.1mL tertiary methyl ether was used as a cosolvent, 30°C, 1200rpm vortex oscillation reaction for 2h, 200μL of the reaction solution was added to 50μL 6M hydrochloric acid (stop reaction), 200μL ethyl acetate was added to extract twice, and the ethyl acetate phase was combined. For the ethyl acetate sample, the concentration and chirality of 3-chlorophenylacetone and 3-chlorophenylpropanol were detected by HPLC described in Example 1 and the conversion rate was calculated.

[0067] The screening results are shown in Table 3:

[0068] Mutants Conversion rate ee value WT >99％ R-type, 89.26% S16A 90.45％ R-type, 100% G114A 92.33％ R-type, 92.50% T118A 94.23％ R-type, 95.54% V157A >99％ R-type, 82.118% L211A 96.88％ R-type, 91.14% S16A / G114A >99％ R-type, 96.24% S16A / T118A >99％ R-type, 95.34% S16A / V157A >99％ R-type, 95.86%

[0069] Finally, the dominant strains of ADH1-mutants ADH1-S16A / G114A, S16A / T118A, and S16A / V157A were obtained through further screening.

[0070] Amino acid sequence: S16A / G114A is shown in SEQ ID NO.13, S16A / T118A is shown in SEQ ID NO.14, and S16A / V157A is shown in SEQ ID NO.15.

[0071] Example 4: Cosolvent Screening

[0072] Here are the steps:

[0073] S1. Add 1 g each of ADH1-S16A / G114A wet bacteria and formate dehydrogenase wet bacteria, 6 mL of PB buffer (0.1 M, pH 7), 1 mL of 7 M ammonium formate solution, and 10 mM NADP to the reaction bottle. + 1 mL of solution, 2 mL of 500 g / L 3-chloropropiophenone co-solvent solution. Ethyl acetate, butyl acetate, dibutyl phthalate, DMSO (dimethyl sulfoxide), methyl tert-butyl ether, dimethyl phthalate, methanol, etc. were used as co-solvents, respectively, and the reaction was carried out at 30°C and 800 rpm.

[0074] S2. After 2 hours of reaction, 1 mL of the reaction solution was taken and extracted with an equal volume of ethyl acetate. The upper organic phase was centrifuged and sent to HPLC for detection. The results are shown in Table 4:

[0075] Solvent Conversion rate ee value Ethyl acetate 25.78％ R-type, 88.06% Butyl acetate 68.34％ R-type, 94.24% Dibutyl phthalate 80.24％ R-type, 97.22% DMSO (Dimethyl Sulfoxide) >99％ R-type, 68.18% Methyl tert-butyl ether >99％ R-type, 96.02% Dimethyl phthalate >99％ R-type, 98.46% Methanol 80％ R-type, 98.12%

[0076] The results showed that the highest conversion rate was >99% when the cosolvent was dimethyl phthalate, and its chirality was R type with an ee value of 98.46%.

[0077] Example 5: Buffer and pH Optimization

[0078] Buffer Optimization:

[0079] S1. Add 1 g of ADH1-S16A / G114A wet bacteria and 1 g of formate dehydrogenase wet bacteria, 6 mL of buffer (0.1 M), 1 mL of 7 M ammonium formate solution, and 10 mM NADP to the reaction bottle. + 1 mL of solution, 2 mL of 500 g / L 3-chloropropiophenone methyl tert-butyl ether solution, wherein the buffer solution is set to pH 7.0 PB, Tris-HCl and TEA buffer, and the reaction is carried out at 30°C and 800 rpm.

[0080] S2. After 1 hour of reaction, 1 mL of the reaction solution was taken and extracted with an equal volume of ethyl acetate. The upper organic phase was centrifuged and sent to HPLC for detection. The results are shown in Table 5:

[0081] Buffer Conversion rate ee value pH 7.0 PB buffer 50.65％ R-type, 96.18% pH 7.0 Tris-HCl buffer 49.78％ R-type, 96.20% pH 7.0 TEA buffer 56.88％ R-type, 96.22%

[0082] The results showed that the conversion rate was highest when the buffer was TEA buffer at pH 7.0.

[0083] pH Optimization:

[0084] S1. Add 1 g of ADH1-S16A / G114A wet bacteria and 1 g of formate dehydrogenase wet bacteria, 6 mL of buffer (0.1 M), 1 mL of 7 M ammonium formate solution, and 10 mM NADP to the reaction bottle.+ 1 mL of solution, 2 mL of 500 g / L 3-chloropropiophenone methyl tert-butyl ether solution, wherein the buffer was set to TEA buffer at pH 6.0, 6.5, 7.0, 7.5, and 8.0, and the reaction was carried out at 30° C. and 800 rpm.

[0085] S2. After 2 hours of reaction, 1 mL of the reaction solution was taken and extracted with an equal volume of ethyl acetate. The upper organic phase was centrifuged and sent to HPLC for detection. The results are shown in Table 6:

[0086]

[0087]

[0088] The results showed that the conversion rate was the highest at >99% when the buffer was TEA buffer at pH 6.5, which was the most preferred.

[0089] Example 6: Screening of ADH1 mutants using 3-hydroxypropiophenone

[0090] S1. Add 1 g each of ADH1-mutants (ADH1-S16A, ADH1-G114A, ADH1-T118A, ADH1-V157A, ADH1-L211A) and formate dehydrogenase wet bacteria, 6 mL of PB buffer (0.1 M, pH 7), 1 mL of 7 M ammonium formate solution, and 10 mM NADP to the reaction bottle. + 1 mL of solution, 2 mL of 500 g / L methyl tert-butyl ether solution of substrate, wherein the substrate is set to 3-hydroxypropiophenone, and the reaction is carried out at 30°C and 800 rpm.

[0091] S2. After 1 hour of reaction, 1 mL of the reaction solution was taken and extracted with an equal volume of ethyl acetate. The upper organic phase was centrifuged and sent to HPLC for detection. The results are shown in Table 7:

[0092] ADH1 mutants Conversion rate ee value WT 50.56％ R-type, 97.34% S16A >99％ R-type, 98.66% G114A 70.34％ R-type, 98.96% T118A 92.56％ R-type, 97.48% V157A 90.56％ R-type, 98.9% L211A >99％ R-type, 80.36%

[0093] The results showed that the dominant ADH1-mutant strain ADH1-S16A had the best catalytic efficiency for 3-hydroxypropiophenone.

[0094] Embodiment 7:

[0095] Add 1g of ADH1-S16A / G114A wet bacteria and 1g of formate dehydrogenase wet bacteria, 6mL of PB buffer (0.1M, pH7), 1mL of 7M ammonium formate solution, and 10mM NADP to the reaction bottle. + 1 mL of solution, 2 mL of 500 g / L methyl tert-butyl ether solution of substrate, wherein the substrate is set to 3-chloropropiophenone, and the reaction is carried out at 30°C and 800 rpm.

[0096] After 6 h of reaction, 1 mL of the reaction solution was taken and extracted with an equal volume of ethyl acetate. The upper organic phase was centrifuged and sent to HPLC for detection.

[0097] According to the above conditions, single factor replacement is performed separately:

[0098]

[0099]

[0100] Sequence Listing:

[0101] SEQ ID NO.1

[0102] MKLLEGKVALITGATRGIGKGIAEMYAQQGAKIAFTYAGSVDKAKELEATLSSVTQIKGYQS

[0103] DASDFDAAQTLVDEVLKEFGKIDILINNAGITKDNLLLRMSKEDWDKVINVNLDSVFNLTK

[0104] AVIKPMMKAKEGSIINMTSVVGISGNAGQANYAASKAGVIGFTKSVALELGSRNIRCNAIAP

[0105] GFIQTEMTDALDDKAKDKWNEAIPMKKLGQTKDIANACVFLGSEMASYITGQTLNVDGGLLTHHHHHH*

[0106] SEQ ID NO.2

[0107] MILDETITLNSGVKIPKFALGTWMIDDDQAAEAVRNAIKMGYRHIDTAQAYDNERGVGEG

[0108] VRTAGIDRDKIFVTSKIAAEHKDYDVTKKSIDETLEKMGLDYIDMMLIHSPQPWKEVNQSD

[0109] NRYLEGNLAAWRAMEDAVNEGKIRTIGVSNFKKADLENIIKNSDTVPAVDQVLAHIGHTPF

[0110] NLLSFTHEHDIAVEAYSPVAHGAALDNPVIEKMAKKYNVSVPQLCIRYDWQIGMIVLPKTTNPEHMKENTEIDFEISEADMDLLRRVKPLDYGDFDIYPVYGGKMHHHHHH*

[0111] SEQ ID NO.3

[0112] MGSSHHHHHHSSGLVPRGSHMDYRKLGPSGTVVTAYCLGTMTFGAEADEAASHKLLDDY

[0113] FAWGGNFIDTADVYSAGKSEEIIGRWLKARPTEARQAIVATKGRFPMGNGPNDIGLSRRHLS

[0114] QALDDSLRRLGLEQIDLYQMHAWDALTPIEETLRFLDDAVSSGKIGYYGFSNYVGWHIAKA

[0115] SEIAKARGYTRPVTLQPQYNLLMRDIELEIVAACQDAGMGLLPWSPLGGGWLTGKYKRDE

[0116] MPTGATRLGENPNRGGESYAPRNAQERTWAIIGTVEEIAKARGVSMAQVALAWTAARPAIT

[0117] SVILGAETPEQLADNLGAMKVELSGEEMARLNEVSAPQPLDYPYGKGGINQRHRKIEGGRHHHHHH*

[0118] SEQ ID NO.4

[0119] MYRLLNKTAVITGGNSGIGLATAKRFVAEGAYVFIVGRRRKELEQAAAEIGRNVTAVKADV

[0120] TKLEDLDRLYAIVREQRGSIDVLFANSGAIEQKTLEEITPEHYDRTFDVNVRGLIFTVQKALP

[0121] LLRDGGSVILTSSVAGVLGLQAHDTYSAAKAAVRSLARTWTTELKGRSIRVNAVSPGAIDTP

[0122] IIENQVSTQEEADELRAKFAAATPLGRVGRPEELAAAVLFLASDDSSYVAGIELFVDGGLTQVHHHHHH*

[0123] SEQ ID NO.5

[0124] MSIPSSQYGFVFNKQSGLNLRNDLPVHKPKAGQLLLKVDAVGLCHSDLHVIYEGLDCGDN

[0125] YVMGHEIAGT

[0126] VAAVGDDVINYKVGDRVACVGPNGCGGCKYCRGAIDNVCKNAFGDWFGLGYDGGYQQY

[0127] LLVTRPRNLSRI

[0128] PDNVSADVAAASTDAVLTPYHAIKMAQVSPTSNILLIGAGGLGGNAIQVAKAFGAKVTVLD

[0129] KKKEARDQA

[0130] KKLGADAVYETLPESISPGSFSACFDFVSVQATFDVCQKYVEPKGVIMPVGLGAPNLSFNLG

[0131] DLALREIR

[0132] ILGSFWGTTNDLDDVLKLVSEGKVKPVVRSAKLKELPEYIEKLRNNAYEGRVVFNPHHHHHH*

[0133] SEQ ID NO.6

[0134] MANLRESSRDKSRWSLEGMTALVTGGSKGIGEAVVEELAMLGARVHTCARDETQLQESLR

[0135] EWQAKGFQVTTSVCDVSSRDQREKLMETVSSLFQGKLNILVNNAGTCITKPTIDYTSEDFSF

[0136] LMSTNLESSFHLSQLAHPLLKSSGLGSIVLISSVASVVHVNVGSIYGATKGAMNQLARNLAC

[0137] EWASDSIKVNSVCPGFISTPLASNYFRNEEFKKEVENIIPTGRVGEANEVSSLVAYLCLPAAASYVTGQTICVDGGFSVNGFTFKSLPLRHHHHHH*

[0138] SEQ ID NO.7

[0139] ATGAAACTGCTGGAAGGTAAAGTTGCGCTGATCACCGGCGCTACTCGTGGGCATCGGTAA

[0140] AGGTATCGCTGAAATGTACGCACAGCAGGGTGCAAAGATCGCGTTCACCTACGCTGGTT

[0141] CTGTAGACAAAGCAAAAGAACTGGAAGCGACTCTGTCCAGCGTTACTCAGATCAAGGG

[0142] CTACCAGTCTGACGCATCTGATTTCGACGCTGCTCAGACTTTGGTTGACGAAGTGCTGA

[0143] AAGAATTTGGCAAGATCGACATCTTGATCAACAACGCGGGTATCACCAAAGACAACCTG

[0144] CTGCTGCGTATGTCCAAAGAAGATTGGGACAAAGTTATCAACGTTAACCTGGATTCCGT

[0145] GTTTAACCTGACCAAAGCTGTTATCAAACCGATGATGAAAGCCAAAGAAGGTAGCATCA

[0146] TCAACATGACCTCTGTGGTTGGTATCTCTGGTAACGCGGGTCAGGCAAACTACGCGGCG

[0147] TCCAAAGCGGGTGTGATCGGTTTCACCCAAATCTGTTGCTCTGGAACTGGGTTCTCGTAA

[0148] CATCCGTTGCAATGCAATCGCACCGGGCTTCATCCAGACTGAAAATGACCGATGCACTGG

[0149] ATGACAAAGCGAAAGACAAGTGGAACGAAGCGATTCCAATGAAGAAACTGGGTCAGA

[0150] CCAAAGATATTGCTAACGCTTGCGTATTTCTGGGTTCTGAAATGGCGAGCTACATCACTG

[0151] GTCAGACCTTGAACGTTGATGGTGGTCTGCTGACCCACCATCATCACCATCACTAASEQ ID NO.8

[0152] ATGATTCTGGACGAAACCATCACTCTGAACTCTGGTGTTAAGATTCCGAAATTCGCACTG

[0153] GGTACTTGGATGATCGACGACGATCAGGCTGCTGAAGCTGTTCGTAACGCGATCAAGAT

[0154] GGGTTATCGTCACATCGATACCGCACAAGCGTACGACAACGAACGTGGTGTTGGTGAAG

[0155] GTGTTCGTACTGCTGGTATCGACCGTGACAAAATCTTCGTTACCTCCAAAATCGCTGCTG

[0156] AACACAAAGACTACGACGTTACCAAGAAATCCATCGACGAAACTCTGGAGAAGATGGG

[0157] TCTGGACTACATCGACATGATGCTGATCCACAGTCCGCAACCGTGGAAAGAAGTGAACC

[0158] AGTCTGACAACCGTTACCTGGAAGGTAATCTGGCTGCTTGGCGTGCAATGGAAGATGCG

[0159] GTTAACGAAGGTAAGATCCGTACCATCGGTGTTTCCAACTTCAAGAAAGCGGACCTGGA

[0160] GAACATCATCAAGAACTCTGACACCGTTCCAGCAGTAGACCAGGTACTGGCTCACATCG

[0161] GTCACACTCCGTTCAACCTGCTGAGCTTCACTCACGAACACGACATCGCTGTTGAAGCG

[0162] TACTCTCCGGTTGCACATGGTGCAGCTCTGGACAATCCGGTTATCGAGAAGATGGCGAA

[0163] GAAATACAACGTTTCTGTACCGCAGCTGTGCATCCGTTACGACTGGCAGATCGGTATGAT

[0164] CGTCTTGCCAAAGACCACCAACCCGGAACACATGAAAGAGAACACCGAAATCGACTTC

[0165] GAGATCAGCGAAGCTGACATGGACCTGCTTCGTCGTGTTAAACCGCTGGATTACGGTGA

[0166] CTTCGACATCTATCCGGTTTACGGTGGTAAGATGCACCACCATCACCACCACTAASEQ ID NO.9

[0167] ATGGGCTCTAGCCACCACCATCACCACCATTCTTCCGGTCTGGTTCCACGTGGTAGCCAC

[0168] ATGGACTACCGTAAACTGGGTCCGTCTGGTACTGTTGTTACTGCGTACTGTCTGGGCACC

[0169] ATGACCTTTGGCGCAGAAGCGGACGAAGCGGCTTCTCACAAACTGCTGGACGACTACT

[0170] TCGCTTGGGGTGGTAACTTCATCGACACCGCTGACGTTTACAGCGCGGGTAAATCTGAA

[0171] GAGATCATCGGTCGTTGGCTGAAAGCGCGTCCAACTGAAGCTCGTCAGGCTATCGTTGC

[0172] GACCAAAGGTCGTTTCCCGATGGGTAACGGTCCGAACGACATCGGTCTGAGCCGTCGTC

[0173] ACCTGAGCCAGGCACTGGATGACAGCCTGCGTCGTCTGGGTCTGGAACAGATCGACCT

[0174] GTACCAGATGCACGCATGGGACGCACTGACTCCGATCGAAGAAACTCTGCGTTTCTTGG

[0175] ACGACGCGGTTTCTAGCGGTAAGATCGGTTACTACGGTTTCTCCAACTACGTTGGTTGGC

[0176] ACATCGCTAAAGCGTCTGAAATCGCAAAGGCTCGTGGTTACACCCGTCCAGTTACTCTG

[0177] CAACCGCAGTACAACCTGCTGATGCGTGACATCGAACTGGAAATCGTTGCAGCTTGCCA

[0178] AGACGCGGGTATGGGTTTGCTTCCGTGGAGTCCACTGGGTGGTGGTTGGCTGACTGGTA

[0179] AATACAAACGTGACGAAATGCCGACTGGTGCTACTCGTCTGGGTGAGAACCCGAACCG

[0180] TGGTGGTGAAAGCTACGCACCACGTAACGCGCAGGAACGTACCTGGGCAATCATCGGTA

[0181] CTGTTGAAGAAATCGCGAAAGCTCGTGGTGTTAGCATGGCACAGGTTGCTCTGGCATGG

[0182] ACCGCAGCACGTCCGGCTATCACCTCTGTTATCTTGGGTGCTGAAACTCCAGAACAGCT

[0183] GGCTGACAACCTGGGTGCGATGAAAGTGGAACTGTCTGGTGAAGAAATGGCGCGTCTG

[0184] AACGAAGTTTCTGCGCCGCAACCACTGGATTATCCGTACGGTAAAGGTGGTATCAACCA

[0185] GCGTCACCGTAAGATCGAAGGTGGTCGTCATCACCATCATCACCACTAASEQ ID NO.10

[0186] GAACGGGTGAGTAACACGTGAGCAACCTGCCCTAGGCTTTGGGATAACCCCGGGAAAC

[0187] CGGGGCTAATAC

[0188] CGAATATTACCACCGGGCGCATGTTTGGTGGTGGAAAGTTTTTCGGCCTGGGATGGGCT

[0189] CGCGGCCTATCAGCTTGTTGGTGGGGTGATGGCCTACCAAGGCGACGACGGGTAGCCGG

[0190] CCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAG

[0191] GCAGCAGTGGGGAATATTGCACAATGGGCGGAAGCCTGATGCAGCGACGCCGCGTGAG

[0192] GGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGACGAAGCGAAAGTGACGGT

[0193] ACCTGCAGAAGAAGCGCCGGCCAACTACGTGCCAGCAGCCGCGGTAAGACGTAGGGCG

[0194] CGAGCGTTGTCCGGATTTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCGCGTCGACT

[0195] GTGAAAACCCGCGGCTCAACCGCGGGCCTGCAGTCGATACGGGCAGGCTAGAGTTCGG

[0196] TAGGGGAGACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACC

[0197] GGTGGCGAAGGCGGGTCTCTGGGCCGATACTGACGCTGAGGAGCGAAAGCGTGGGGA

[0198] GCGAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGTTGGGCGSEQ ID NO.11

[0199] GAATTCATGACCACTGCGAGCCATCCGGTTAAACTGACCAAACAGTTCAAGACCAAATC

[0200] CGGTTCTGACGTTTCTATCGCGACTGGTACTGGCACCAAATGGAAGAAAGACGCGAAA

[0201] GACGACGACATCAACCAGGAACTGGTTGACCAAATCTTGTTGGCACTGAAACTGGGTTT

[0202] CCGTCACATCGACACCGCAGAAGTTTACAACACCCAGGCGGAAGTTGGTGAAGCGGTT

[0203] AAACAGTCTGGTATTCCGCGTGAAGAACTGTGGATCACCACCAAATACAATCCGGGTTG

[0204] GCGTACCATCTCTGCTTCTTCTGCGAGTCCATCTGCGTCTATCGACAAAGCGCTGAAACA

[0205] GCTGGACACCGACTACATCGACCTGTACCTGATCCACCAGCCGTTCTTCACCGAAGAAT

[0206] CTACTCACGGTTACACTCTGGAAGATGTTTGGAACGTTCTGATCGACTACAAGAAACAG

[0207] GGTAAGATCCGTGAAATCGGTGTTTCCAACTTCGCGCAAGAACACATCGAACGTCTGAA

[0208] GAAGAATTTCTGAACCGGAGTTCTATCCGGTTGTTAACCAGATCGAATCTCATCCGTTCTT

[0209] GCAAGACCAGTCTAAAGGTATCACCGAATACTCTCAGGCGAACGGTATCTTGGTTGAAG

[0210] CGTTCAGTCCGCTGACTCCGGTTAGCCGTGTTGAGAAGAACGCGCTGACTGGCTACCTG

[0211] GACGAACTGTCCAAAAAGTACAACAAGACTGGTGGTCAAATCTTGCTGCGTTGGACTCT

[0212] GCAACGTGGTATTCTGCCGATCACCACTTCTGCGAAAGAAGATCGTATCAAAGAAGCTC

[0213] TGGACGTGTTCGACTTCGAACTGACTCAAGAAGAGTTCGACAAGATCGCGGAAATCGG

[0214] TAAAGAGAATCCGCACCGTGTGTTCTTCCACGACGAGTTCAAAGACCTGGAACACCATC

[0215] ACCACCACCACTAAGCGGCCGC

[0216] SEQ ID NO.12

[0217] ATGGCGAACCTGCGTGAATCTAGCCGTGATAAATCTCGTTGGAGCCTGGAAGGCATGAC

[0218] TGCTCTGGTAACTGGTGGCTCCAAAGGTATCGGCGAAGCAGTTGTAGAAGAACTGGCTA

[0219] TGCTGGGTGCTCGTGTTCACACCTGCGCTCGTGACGAAACTCAGCTGCAAGAATCTCTG

[0220] CGTGAATGGCAGGCTAAAGGCTTCCAGGTTACTACCTCCGTATGTGACGTTTCTAGCCGT

[0221] GATCAGCGTGAGAAACTGATGGAAACCGTTTCCAGCCTGTTCCAGGGTAAACTGAACAT

[0222] TCTGGTAAACAACGCTGGTACTTGCATCACCAAACCGACTATCGACTACACCTCTGAAG

[0223] ATTTCAGCTTTCTGATGAGCACCAACCTGGAATCCAGCTTCCATCTGAGCCAGCTCGCGC

[0224] ATCCGCTGCTGAAATCCAGCGGCCTGGGTTCCATCGTGCTGATCAGCTCTGTTGCTTCCG

[0225] TTGTACACGTTAACGTAGGTTCTATCTACGGTGCAACCAAAGGTGCGATGAACCAGCTG

[0226] GCGCGTAACCTGGCTTGCGAATGGGCGTCTGATTCCATCAAAGTTAACTCCGTGTGTCC

[0227] GGGCTTCATCTCCACTCCACTGGCATCTAACTACTTCCGTAACGAAGAGTTCAAGAAAG

[0228] AAGTTGAGAACATCATTCCGACTGGTCGTGTTGGCGAAGCAAACGAAGTTTCTAGCCTG

[0229] GTTGCATATCTGTGCTTGCCAGCAGCTTCTTACGTTACTGGTCAGACCATCTGCGTGGAT

[0230] GGTGGTTTCTCCGTTAACGGTTTCACCTTCAAATCTCTGCCGCTGCGCCATCATCACCAC

[0231] CACCACTAA

[0232] SEQ ID NO.13

[0233] MYRLLNKTAVITGGNAGIGLATAKRFVAEGAYVFIVGRRRKELEQAAAEIGRNVTAVKADV

[0234] TKLEDLDRLYAIVREQRGSIDVLFANSGAIEQKTLEEITPEHYDRTFDVNVRALIFTVQKALP

[0235] LLRDGGSVILTSSVAGVLGLQAHDTYSAAKAAVRSLARTWTTELKGRSIRVNAVSPGAIDTP

[0236] IIENQVSTQEEADELRAKFAAATPLGRVGRPEELAAAVLFLASDDSSYVAGIELFVDGGLTQVHHHHHH*

[0237] SEQ ID NO.14

[0238] MYRLLNKTAVITGGNAGIGLATAKRFVAEGAYVFIVGRRRKELEQAAAEIGRNVTAVKADV

[0239] TKLEDLDRLYAIVREQRGSIDVLFANSGAIEQKTLEEITPEHYDRTFDVNVRGLIFAVQKALP

[0240] LLRDGGSVILTSSVAGVLGLQAHDTYSAAKAAVRSLARTWTTELKGRSIRVNAVSPGAIDTP

[0241] IIENQVSTQEEADELRAKFAAATPLGRVGRPEELAAAVLFLASDDSSYVAGIELFVDGGLTQVHHHHHH*

[0242] SEQ ID NO.15

[0243] MYRLLNKTAVITGGNAGIGLATAKRFVAEGAYVFIVGRRRKELEQAAAEIGRNVTAVKADV

[0244] TKLEDLDRLYAIVREQRGSIDVLFANSGAIEQKTLEEITPEHYDRTFDVNVRGLIFTVQKALP

[0245] LLRDGGSVILTSSVAGVLGLQAHDTYSAAKAAARSLARTWTTELKGRSIRVNAVSPGAIDTP

[0246] IIENQVSTQEEADELRAKFAAATPLGRVGRPEELAAAVLFLASDDSSYVAGIELFVDGGLTQVHHHHHH*.

[0247] The above-described embodiment is only a preferred solution of the present invention and does not limit the present invention in any form. There are other variations and modifications without exceeding the technical solution described in the claims.

Claims

1. An enzyme-catalyzed synthesis method of a dapoxetine chiral intermediate, characterized in that: Add wet cells of recombinant Escherichia coli expressing aldehyde-keto reductase / alcohol dehydrogenase, wet cells of recombinant Escherichia coli expressing formate dehydrogenase, buffer, substrate, cosolvent, ammonium formate and coenzyme into a reaction container, react at 25-35° C. for 2-24 hours, extract, separate and rotary evaporate to obtain a dapoxetine chiral intermediate; The substrate is 3-chloropropiophenone or 3-hydroxypropiophenone; The dosage ratio of each raw material in 1L reaction system is: 10-200g of wet recombinant Escherichia coli expressing aldehyde-keto reductase / alcohol dehydrogenase, 10-200g of wet recombinant Escherichia coli expressing formate dehydrogenase, 20g-100g of substrate, 50-500mL of cosolvent, 0.2-1moL of ammonium formate, 0.1-2.5mmoL of coenzyme, and the rest of buffer; The aldehyde-keto reductase / alcohol dehydrogenase is a mutant of ADH1; the amino acid sequence of ADH1 is shown in SEQ ID NO.4; When the substrate is 3-chloropropiophenone, the mutant of ADH1 is selected from one of ADH1-S16A / G114A, ADH1-S16A / T118A, and ADH1-S16A / V157A; ADH1-S16A / G114A means that the S at position 16 of the ADH1 amino acid sequence is changed to A, and the G at position 114 is changed to A; ADH1-S16A / T118A means that the S at position 16 of the ADH1 amino acid sequence is changed to A, and the T at position 118 is changed to A; ADH1-S16A / V157A means that the S at position 16 of the ADH1 amino acid sequence is changed to A, and the V at position 157 is changed to A; When the substrate is 3-hydroxypropiophenone, the mutant of ADH1 is ADH1-S16A, wherein S at position 16 of the amino acid sequence of ADH1 is changed to A.

2. The enzyme-catalyzed synthesis method according to claim 1, characterized in that: The buffer is selected from one of TEA buffer, PB buffer and Tris-HCl buffer.

3. The enzyme-catalyzed synthesis method according to claim 2, characterized in that: The concentration of the TEA buffer is 0.05-0.20 M, and the pH value is 6-8; the concentration of the PB buffer is 0.05-0.10 M, and the pH value is 6-7; the concentration of the Tris-HCl buffer is 0.05-0.10 M, and the pH value is 7-8.

4. The enzyme-catalyzed synthesis method according to claim 1, characterized in that: The coenzyme is NADP + or NAD + .

5. The enzyme-catalyzed synthesis method according to claim 1, characterized in that: The co-solvent is at least one of ethyl acetate, butyl acetate, dibutyl phthalate, DMSO, methyl tert-butyl ether, dimethyl phthalate, and methanol.

6. The enzyme-catalyzed synthesis method according to claim 1, characterized in that: The dosage ratio of each raw material in 1L reaction system is: 10-200g of wet cells of recombinant Escherichia coli expressing aldehyde-keto reductase / alcohol dehydrogenase, 50-100g of wet cells of recombinant Escherichia coli expressing formate dehydrogenase, 20g-100g of substrate, 50-500mL of cosolvent, 0.2-1moL of ammonium formate, 0.1-2.5 mmoL of coenzyme, and the remainder of buffer.

7. The enzyme-catalyzed synthesis method according to claim 1, characterized in that: The dosage ratio of each raw material in 1L reaction system is: 10-200g of wet cells of recombinant Escherichia coli expressing aldehyde-keto reductase / alcohol dehydrogenase, 25-75g of wet cells of recombinant Escherichia coli expressing formate dehydrogenase, 20g-100g of substrate, 50-500mL of cosolvent, 0.2-1moL of ammonium formate, 0.1-2.5 mmoL of coenzyme, and the remainder of buffer.

Images