A ketoreductase and its application

Abstract

The present invention discloses a ketoreductase. The present invention also discloses its application in the in vitro biosynthesis of (R)-6-hydroxy-8-chlorooctanoic acid ethyl ester. As a biocatalyst, it converts 8-chloro-6-oxooctanoic acid ethyl ester into (R)-6-hydroxy-8-chlorooctanoic acid ethyl ester with excellent chiral selectivity, bringing the corresponding chirality into the synthesis route, greatly reducing production costs, eliminating the environmental impact generated during the by-product treatment process, and is simple to operate, mild in conditions, and short in time. The conversion rate and product purity are both above 87%, making it suitable for industrial application.

Description

Technical Field

[0001] The present invention relates to the field of biochemical engineering, and in particular to a ketoreductase and application thereof in in vitro biosynthesis of (R)-6-hydroxy-8-chlorooctanoic acid ethyl ester. Background Art

[0002] (R)-6-Hydroxy-8-chlorooctanoic acid ethyl ester is a chiral intermediate of (R)-α-lipoic acid. Lipoic acid (LA, commonly referred to as α-lipoic acid) is a water-insoluble white or pale yellow crystal widely distributed in plant and animal tissues. α-Lipoic acid is a safe, effective, and potent antioxidant, known as the "universal antioxidant." It has been shown to relieve fatigue, slow aging, and prevent memory loss. Lipoic acid has two enantiomers, with (R)-α-lipoic acid being much more effective than (S)-α-lipoic acid. (R)-α-lipoic acid belongs to the vitamin B class, acting as a coenzyme in multiple enzyme complexes and playing a key role in the tricarboxylic acid cycle and photosynthesis. Clinically, (R)-α-lipoic acid is increasingly used to treat diseases such as neurological diabetes and ischemia-reperfusion. In addition, (R)-α-lipoic acid is also effective in treating a variety of diseases and can effectively inhibit the spread of HIV-1 in cells.

[0003] As lipoic acid is increasingly used in medicine, food and health products, the global annual demand for lipoic acid continues to grow, currently at a rate of approximately 10%. However, its production is far behind demand. The huge supply gap has given lipoic acid an important market position in domestic and foreign markets.

[0004] Currently, lipoic acid is primarily produced through chemical synthesis, with two methods depending on the raw materials: the adipic acid method and the cyclohexanone method. However, both methods involve complex processes and employ chemical raw materials, along with the use of extensive amounts of toxic catalysts during the synthesis process. This raises serious questions about the safety of the product and poses significant environmental risks. Furthermore, chemically synthesized lipoic acid is a mixture of equal amounts of (R)-α-lipoic acid and (S)-α-lipoic acid, requiring further separation to obtain the biologically active (R)-α-lipoic acid. Furthermore, the price difference between the mixed lipoic acid and (R)-α-lipoic acid is significant. Summary of the Invention

[0005] Purpose of the invention: In view of the shortcomings of the prior art, the first technical problem to be solved by the present invention is to provide a ketoreductase.

[0006] The technical problem that the present invention also aims to solve is to provide the application of the above-mentioned ketoreductase in the in vitro biosynthesis of (R)-6-hydroxy-8-chlorooctanoic acid ethyl ester and a specific preparation method.

[0007] Technical solution: In order to solve the nucleotide problem of the above-mentioned technology, the present invention provides a ketoreductase, wherein the amino acid sequence of the ketoreductase is selected from SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5.

[0008] The ketoreductase with the amino acid sequence of SEQ ID NO.1 was named SCR-1, the ketoreductase with the amino acid sequence of SEQ ID NO.2 was named SCR-2, the ketoreductase with the amino acid sequence of SEQ ID NO.3 was named SCR-3, the ketoreductase with the amino acid sequence of SEQ ID NO.4 was named SCR-4, and the ketoreductase with the amino acid sequence of SEQ ID NO.5 was named SCR-5.

[0009] The present invention also provides a gene or nucleic acid encoding the above-mentioned ketoreductase.

[0010] Furthermore, the ketoreductase gene or nucleic acid mentioned above, the nucleotide sequence of the ketoreductase gene or nucleic acid is selected from SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10.

[0011] The present invention also provides the use of the ketoreductase in in vitro biosynthesis of (R)-6-hydroxy-8-chlorooctanoic acid ethyl ester.

[0012] The present invention also provides an in vitro biosynthesis method of (R)-6-hydroxy-8-chlorooctanoic acid ethyl ester, which comprises the following steps:

[0013] (1) Obtaining the above-mentioned ketoreductase gene;

[0014] (2) constructing a recombinant vector containing the ketoreductase gene, and transferring the recombinant vector into Escherichia coli, and culturing the recombinant Escherichia coli to obtain a recombinant Escherichia coli bacterial solution;

[0015] (3) Lysing the recombinant Escherichia coli bacterial solution to obtain a crude enzyme solution;

[0016] (4) 8-chloro-6-oxooctanoic acid ethyl ester, phosphate buffer solution, crude enzyme solution, and coenzyme factors are mixed, and the reaction is completed to obtain (R)-6-hydroxy-8-chloroooctanoic acid ethyl ester.

[0017] Furthermore, the E. coli bacterial solution lysis condition in step (2) is centrifugation at 20,000-35,000 g / min for 30-60 min.

[0018] Furthermore, step (3) also includes purification of the crude enzyme solution.

[0019] Furthermore, the purification treatment of the crude enzyme solution includes: passing the crude enzyme solution through a Ni column and eluting it with imidazole solutions of different gradients, then passing the Ni column eluate with the highest enzyme content through a Q column, and then eluting it with salt solutions (mainly NaCl) of different gradients to obtain a preliminarily purified enzyme-containing solution, and dialyzing the preliminarily purified enzyme-containing solution to obtain a purified enzyme solution.

[0020] Furthermore, step (3) also includes preparing the purified enzyme solution into immobilized enzyme.

[0021] Furthermore, in the process of preparing the immobilized enzyme, the immobilization carrier may be selected from but not limited to amino resin and epoxy resin; the pH value of the reaction system is 4-9 and the reaction time is 1-6 hours.

[0022] Furthermore, the coenzyme factor in step (4) is NADH or NADPH, preferably NADPH, the mass ratio of the coenzyme factor to 8-chloro-6-oxooctanoic acid ethyl ester is 0.5-5:1, and the mass ratio of the crude enzyme solution to 8-chloro-6-oxooctanoic acid ethyl ester is 0.002-0.01:1.

[0023] Furthermore, the reaction conditions in step (4) are 25-45°C, preferably 30°C; the reaction time is 2-12h, and the pH value of the reaction system is 5-9, preferably 6.5-7.5.

[0024] Furthermore, step (4) further includes a purification step: adding an organic solvent such as ethyl acetate, xylene or n-heptane to the (R)-6-hydroxy-8-chlorooctanoate solution for extraction, drying the organic phase, and obtaining a white crystalline product by concentration and recrystallization.

[0025] The reaction formula of the above biocatalytic reaction is:

[0026]

[0027] Beneficial effects: Compared with the prior art, the present invention has the following outstanding significant advantages: the present invention discloses a ketoreductase, which serves as a biocatalyst to convert 8-chloro-6-oxooctanoic acid ethyl ester into (R)-6-hydroxy-8-chloroooctanoic acid ethyl ester, has excellent chiral selectivity, brings the corresponding chirality into the synthesis route, greatly reduces production costs, eliminates the environmental impact generated during the by-product treatment process, and is simple to operate, mild in conditions, and short in time. The conversion rate and product purity are both above 87%, making it suitable for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the high performance liquid chromatogram of 8-chloro-6-oxooctanoic acid ethyl ester.

[0029] Figure 2 This is the high performance liquid chromatogram of (R)-6-hydroxy-8-chlorooctanoic acid ethyl ester.

[0030] Figure 3 The present invention provides a high performance liquid chromatogram of the process of preparing (R)-6-hydroxy-8-chlorooctanoic acid ethyl ester using the biocatalytic synthesis method of the present invention. DETAILED DESCRIPTION

[0031] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0032] The biological materials used in the following examples, including the pET28a vector and E. coli DE3 competent cells, were purchased commercially. To prepare PBS buffer, weigh 40 g NaCl, 1 g KCl, 7.2 g Na₂HPO₄, and 1.2 g KH₂PO₄ and dissolve them in 800 mL of distilled water. Adjust the solution to 7.0 with HCl, and finally add distilled water to a volume of 1 L to obtain 50 mM PBS buffer.

[0033] Example 1

[0034] S1 vector construction and cloning: The gene with the nucleotide sequence shown in SEQ ID NO. 6 was seamlessly ligated with the vector pET28a to obtain the recombinant vector pET28a-SCR-1. The recombinant vector was treated as follows: 10 μl of the recombinant vector was added to 100 μl of E. coli BL21 (DE3) competent cells in an ice bath, followed by an ice bath for 30 min, a heat shock at 42°C for 60 s, and another ice bath for 5 min. 300 μl of 37°C antibiotic-free LB medium was added to the tube and the cells were shaken at 37°C and 200 rpm for 1 h. The cells were then plated on a kanamycin-resistant solid LB plate and incubated at 37°C. After colonies grew, single colonies were picked with an autoclaved toothpick and marked on the kanamycin-resistant LB plate for seed preservation. The corresponding bacteria and the marked area on the plate were marked accordingly.

[0035] S2 Verification: Then, 20 μl of PCR Mix with T7 universal primers was stirred evenly and PCR amplification was performed. The PCR reaction conditions were as follows: 95°C for 15 min, denaturation at 94°C for 15 s, annealing at 55°C for 15 s, and extension at 72°C for 1 min. 30 cycles were performed, and finally, 72°C was incubated for 5 min. After PCR amplification, electrophoresis was performed to observe the results. Positive clones were obtained, which were the recombinant E. coli strain containing the target enzyme gene: pET28a-SCR-1 recombinant E. coli;

[0036] Expression and extraction of S3 enzyme: pET28a-SCR-1 recombinant Escherichia coli was picked into LB medium containing kanamycin resistance, cultured at 37°C to an OD of about 1.0, and then IPTG was added at a final concentration of 0.2 mM. The culture was induced at 28°C for 16 hours. The bacteria were then centrifuged at 7000g / min for 6 minutes to collect the cells. After discarding the supernatant culture medium, the cells were resuspended in 100mM PBS solution at a ratio of cell weight: PBS solution = 1g:5ml. The resuspended cells were crushed with a high-pressure cell disruptor to obtain an enzyme-containing lysate. After centrifugation at 35000g / min for 30 minutes, the supernatant was extracted to obtain a crude enzyme solution of the target enzyme.

[0037] S4 purification: The enzyme-containing supernatant is passed through a Ni column, and then eluted with different gradients of imidazole solution. The Ni column eluate with the highest enzyme content is then passed through a Q column, and then eluted with different gradients of salt solution (mainly NaCl) to obtain a preliminarily purified enzyme-containing solution. The preliminarily purified enzyme-containing solution is dialyzed for 12 hours to finally obtain a purified enzyme solution of the target enzyme;

[0038] S5 reaction: In a 25 mL glass reaction bottle, add 2 g of 8-chloro-6-oxooctanoic acid ethyl ester substrate, 7.1 g of NADPH, and 5 mL of 50 mM PBS buffer. Then, add 5 mL of ketoreductase solution (13.2 mg / mL) to the reaction bottle to initiate the reaction. The reaction solution temperature was 30°C, the pH of the reaction solution was controlled at 7.0, and the reaction was stirred for 9 h.

[0039] After the reaction is completed, the reaction solution is adjusted to alkaline (pH>11) and extracted twice with equal volumes of ethyl acetate. The organic phases are combined and filtered through a 0.22 μm filter. 10 μl of the solution is collected and analyzed by high performance liquid chromatography. The conversion rate is calculated to be >92% and the ee value is >99%. The organic phase is then dried over anhydrous magnesium sulfate, concentrated by rotary evaporation, and recrystallized by cooling to obtain 1.72 g of the product with a purity of 99.7%.

[0040] Example 2

[0041] The crude enzyme solution prepared in step S3 of Example 1 was used as a reaction raw material to carry out the following reaction.

[0042] In a 25 mL glass reaction bottle, add 2 g of 8-chloro-6-oxooctanoic acid ethyl ester substrate, 7.1 g of NADPH, and 5 ml of 50 mM PBS buffer. Then, add 5 ml of crude ketoreductase enzyme solution to the reaction bottle to start the reaction. The reaction solution temperature is 30°C, the pH of the reaction solution is controlled to 7.0, and the reaction is stirred evenly for 9 hours.

[0043] After completion of the reaction, the solution was adjusted to alkaline (pH > 11) and extracted twice with equal volumes of ethyl acetate. The organic phases were combined and filtered through a 0.22 μm filter. 10 μl of the solution was analyzed by high-performance liquid chromatography (HPLC) to determine a conversion of >89% and an EE of >99%. The organic phase was then dried over anhydrous magnesium sulfate, concentrated by rotary evaporation, and recrystallized upon cooling to yield 1.66 g of the product with a purity of 99.5%.

[0044] Example 3

[0045] The crude enzyme solution prepared in step S3 of Example 1 was used as a reaction raw material to carry out the following reaction.

[0046] In a 25 mL glass reaction bottle, add 2 g of 8-chloro-6-oxooctanoic acid ethyl ester substrate, 6.4 g of NADH, and 5 ml of 50 mM PBS buffer. Then, add 5 ml of crude ketoreductase enzyme solution to the reaction bottle to start the reaction. The reaction solution temperature is 30°C, the pH of the reaction solution is controlled to 7.0, and the reaction is stirred evenly for 9 hours.

[0047] After completion of the reaction, the solution was adjusted to alkaline (pH > 11) and extracted twice with equal volumes of ethyl acetate. The organic phases were combined and filtered through a 0.22 μm filter. 10 μl of the solution was analyzed by high-performance liquid chromatography (HPLC) to determine a conversion of >81% and an EE of >99%. The organic phase was then dried over anhydrous magnesium sulfate, concentrated by rotary evaporation, and recrystallized upon cooling to yield 1.54 g of the product with a purity of 99.2%.

[0048] Example 4

[0049] The crude enzyme solution prepared in step S3 of Example 1 was used as a reaction raw material to carry out the following reaction.

[0050] In a 25 mL glass reaction bottle, add 2 g of 8-chloro-6-oxooctanoic acid ethyl ester substrate, 7.1 g of NADPH, and 2 ml of 50 mM PBS buffer. Then, add 8 ml of crude ketoreductase enzyme solution to the reaction bottle to start the reaction. The reaction solution temperature is 30°C, the pH of the reaction solution is controlled to 7.0, and the reaction is stirred evenly for 9 hours.

[0051] After the reaction is completed, the reaction solution is adjusted to alkaline (pH>11) and extracted twice with equal volumes of ethyl acetate. The organic phases are combined and filtered through a 0.22 μm filter. 10 μl of the solution is collected and analyzed by high performance liquid chromatography. The conversion rate is >93% and the ee value is >99%. The organic phase is then dried over anhydrous magnesium sulfate, concentrated by rotary evaporation, and recrystallized by cooling to obtain 1.69 g of the product with a purity of 99.7%.

[0052] Example 5

[0053] The crude enzyme solution prepared in step S3 of Example 1 was used as a reaction raw material to carry out the following reaction.

[0054] In a 25 mL glass reaction bottle, add 2 g of substrate, 7.1 g of NADPH, and 2 ml of 50 mM PBS buffer. Then, add 8 ml of crude ketoreductase enzyme solution to the reaction bottle to start the reaction. The reaction liquid temperature is 30°C, the pH of the reaction liquid is controlled to 7.0, and the reaction is stirred evenly for 6 hours.

[0055] After the reaction is completed, the reaction solution is adjusted to alkaline (pH>11) and extracted twice with equal volumes of ethyl acetate. The organic phases are combined and filtered through a 0.22 μm filter. 10 μl of the solution is collected and analyzed by high performance liquid chromatography. The conversion rate is calculated to be >92% and the ee value is >99%. The organic phase is then dried over anhydrous magnesium sulfate, concentrated by rotary evaporation, and recrystallized by cooling to obtain 1.63 g of the product with a purity of 99.7%.

[0056] Example 6

[0057] In step S3, the PBS buffer used to resuspend the cells was replaced with triethanolamine buffer. The remaining preparation methods were the same as in Example 1. The final reaction results, as determined by HPLC, showed a substrate conversion rate of >89% and an EE value of >99%. The organic phase was then dried over anhydrous magnesium sulfate, concentrated by rotary evaporation, and recrystallized upon cooling to yield 1.61 g of the product with a purity of 99.3%.

[0058] Example 7

[0059] In step S1, the ketoreductases selected were SCR-2, SCR-3, SCR-4, and SCR-5, respectively. S2 and S3 were the same as in Example 1. Step S3 was followed by the preparation method of Example 2, using the crude enzyme solution as the reaction raw material.

[0060] The final reaction results are as follows:

[0061] Ketoreductase Conversion Rate % ee value% Product g purity% SCR-2 ＞95％ >99 1.79 99.4％ SCR-3 ＞90％ >99 1.33 99.6％ SCR-4 ＞87％ >99 1.22 99.3％ SCR-5 ＞90％ >99 1.35 99.1％

[0062] Example 8

[0063] Steps S1-S4 are the same as in Example 1.

[0064] Immobilization of S5 enzyme: The amino resin was washed three times with 50 mM PBS buffer and filtered. A 2% glutaraldehyde solution was prepared with 50 mM PBS solution, and the amino resin and 2% glutaraldehyde were mixed in a ratio of amino resin:2% glutaraldehyde = 1:4 (mass / volume ratio). The mixture was then stirred / shaken at 23°C for 1 hour and filtered to obtain the amino resin. The mixture was washed three times with 50 mM PBS buffer and filtered. The enzyme solution prepared in S4 containing approximately 200 mg of ketoreductase was mixed with 5 g of amino resin. The mixture was shaken at 80 rpm at 23°C for 18 hours. The amino resin was then collected by filtration (the filtrate can be used to determine the enzyme immobilization rate), washed three times with 50 mM PBS buffer, and filtered to obtain the immobilized ketoreductase.

[0065] S6. Filling of immobilized enzyme: filling the immobilized enzyme into the column reactor to obtain a column reactor containing immobilized ketoreductase.

[0066] Preparation of S7 reaction solution: 20 g of 8-chloro-6-oxooctanoic acid ethyl ester substrate and 75 g of NADPH were dissolved in 100 ml of 50 mM PBS buffer to prepare a reaction solution, and the pH of the reaction solution was controlled to 7.0.

[0067] S8 reaction: The reaction liquid is allowed to flow through a column reactor containing immobilized ketoreductase. The reaction temperature is controlled at 30°C and the flow time (i.e., reaction time) is controlled to be 4 hours. Then, the liquid flowing out of the reactor is collected to obtain a solution containing ethyl (R)-6-hydroxy-8-chlorooctanoate.

[0068] The collected solution was diluted 10-fold and filtered through a 0.22 μm filter. 10 μL of the supernatant was analyzed by high-performance liquid chromatography (HPLC); the substrate conversion was calculated to be >82%. The organic phase was then dried over anhydrous magnesium sulfate, concentrated by rotary evaporation, and recrystallized upon cooling to yield 1.55 g of the product with a purity of 99.5%.

[0069] Example 9

[0070] The column reactor in step S6 was replaced with a tank reactor to obtain a tank reactor containing immobilized ketoreductase. The reaction was stirred at a speed of 100 rpm for 4 hours. The remaining steps were the same as in Example 8.

[0071] The collected solution was diluted 10-fold and filtered through a 0.22 μm filter. 10 μL of the supernatant was analyzed by HPLC; the substrate conversion was calculated to be >87%. The organic phase was then dried over anhydrous magnesium sulfate, concentrated by rotary evaporation, and recrystallized upon cooling to yield 1.61 g of the product with a purity of 99.4%.

[0072] Example 10

[0073] The remaining steps are the same as in Example 8, and step S5 is as follows:

[0074] Immobilization of S5 enzyme: The epoxy resin was washed three times with 50 mM PBS buffer and then filtered. Then, the enzyme solution prepared in S4 containing approximately 200 mg of ketoreductase was mixed with 7 g of epoxy resin. After mixing, the mixture was shaken at 200 rpm at 23°C for 18 hours and then allowed to stand for 20 hours. The epoxy resin was then filtered to collect (the filtrate can be used to determine the enzyme immobilization rate), washed three times with 50 mM PBS buffer, and filtered to obtain immobilized ketoreductase.

[0075] The collected solution was diluted 10-fold and filtered through a 0.22 μm filter. 10 μL of the supernatant was analyzed by HPLC; the substrate conversion was calculated to be >90%. The organic phase was then dried over anhydrous magnesium sulfate, concentrated by rotary evaporation, and recrystallized upon cooling to yield 1.71 g of the product with a purity of 99.5%.

[0076] Example 11

[0077] The column reactor in step S6 was replaced with a tank reactor to obtain a tank reactor containing immobilized ketoreductase. The reaction was stirred at a speed of 100 rpm for 4 hours. The remaining steps were the same as in Example 10.

[0078] The collected solution was diluted 10-fold and filtered through a 0.22 μm filter. 10 μL of the supernatant was analyzed by HPLC; the substrate conversion was calculated to be >94%. The organic phase was then dried over anhydrous magnesium sulfate, concentrated by rotary evaporation, and recrystallized upon cooling to yield 1.76 g of the product with a purity of 99.3%.

[0079] Example 12

[0080] Steps 1-5 are the same as in Example 10, and the remaining steps are as follows:

[0081] The immobilized enzyme was loaded into a tank reactor to obtain a tank reactor containing immobilized ketoreductase; 3 L of reaction solution was prepared by adding 600 g of substrate, 2.3 kg of NADPH, and 3 L of 50 mM PBS buffer, and then controlling the pH of the reaction solution to 7.0; the reaction solution was poured into the tank reactor and stirred at 100 rpm for 4 hours. The temperature of the tank reactor was maintained at 30° C. during the entire reaction process; the liquid flowing out of the reactor was collected as a solution containing (R)-6-hydroxy-8-chlorooctanoic acid ethyl ester. Each 100 ml of reaction solution was used as a batch for the above reaction and collected. After all the collected solution was completed, it was tested.

[0082] The collected solution was diluted 10-fold and filtered through a 0.22 μm filter. 10 μL of the supernatant was collected and analyzed by high-performance liquid chromatography. The substrate conversion rates for batches 1-3 were all >85%, for batches 4-15, all >80%, and for batches 15-30, all >65%. The reaction solutions from all batches were collected, and the organic phase was dried over anhydrous magnesium sulfate, concentrated by rotary evaporation, and recrystallized upon cooling to yield 435.9 g of the product with a purity of 98.2%.

[0083] Example 13

[0084] In step S1, the ketoreductases selected were SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, and SEQ ID NO. 5, respectively. The remaining steps were the same as in Example 11. The final reaction results were as follows:

[0085]

[0086]

[0087] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A ketoreductase, characterized in that The amino acid sequence of the ketoreductase is SEQ ID NO.

3.

2. A gene or nucleic acid encoding the ketoreductase according to claim 1.

3. The ketoreductase gene or nucleic acid according to claim 2, wherein The nucleotide sequence of the ketoreductase gene or nucleic acid is SEQ ID NO.

8.

4. Use of the ketoreductase according to claim 1, or the gene or nucleic acid according to claim 2 or 3, in in vitro biosynthesis of ethyl (R)-6-hydroxy-8-chlorooctanoate.

5. An in vitro biosynthesis method of (R)-6-hydroxy-8-chlorooctanoic acid ethyl ester, characterized in that: The following steps are involved: (1) Obtaining the ketoreductase gene according to claim 2 or 3; (2) constructing a recombinant vector containing the gene of the ketoreductase, and transferring the recombinant vector into Escherichia coli, and culturing the recombinant Escherichia coli to obtain a bacterial liquid; (3) Lysing the recombinant E. coli bacterial solution to obtain crude enzyme solution; (4) 8-chloro-6-oxooctanoic acid ethyl ester, phosphate buffer solution, crude enzyme solution, and coenzyme factors are mixed, and the reaction is completed to obtain (R)-6-hydroxy-8-chloroooctanoic acid ethyl ester.

6. The synthesis method according to claim 5, characterized in that The E. coli bacterial solution lysis condition in step (2) is centrifugation at 20,000-35,000 g / min for 30-60 min.

7. The synthesis method according to claim 5, characterized in that Step (3) also includes purification of the crude enzyme solution.

8. The synthesis method according to claim 7, characterized in that Step (3) also includes preparing the purified enzyme solution into immobilized enzyme.

9. The synthesis method according to claim 5, characterized in that In step (4), the coenzyme factor is NADH or NADPH, the mass ratio of the coenzyme factor to 8-chloro-6-oxooctanoic acid ethyl ester is 0.5-5:1, and the mass ratio of the crude enzyme solution to 8-chloro-6-oxooctanoic acid ethyl ester is 0.002-0.01:

1.

10. The synthesis method according to claim 5, characterized in that The reaction conditions in step (4) are 25-45°C, the reaction time is 2-12h, and the pH value of the reaction system is 5-9.

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