Method for preparing (s)-1, 1-bis (4-fluorophenyl)-2-propanol using enzymatic reactions and use of ketoreductases

Abstract

The invention discloses a method for preparing (S)-1, 1-bis (4-fluorophenyl)-2-propanol by utilizing an enzymatic reaction and application of ketoreductase, and the method comprises the following steps: catalyzing a substrate 1, 1-bis (4-fluorophenyl)-acetone in a reaction system by using the ketoreductase as shown in SEQ ID NO: 16 to generate the (S)-1, 1-bis (4-fluorophenyl)-2-propanol. According to the method disclosed by the invention, the cost of used raw materials is reduced, the production process is more environment-friendly, and the conversion rate of a substrate is improved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular, relates to a method for preparing (S)-1,1-bis(4-fluorophenyl)-2-propanol by using an enzymatic reaction and the use of ketoreductase. Background Art

[0002] Florylpicoxamid is a second-generation pyridine amide fungicide developed by Dow AgroSciences. This product can be used in multiple growth stages of crops and can improve crop yield and quality. The structural formula of Florylpicoxamid is shown in Formula I below.

[0003]

[0004] WO2018009618A1 reports a method for preparing Florylpicoxamid, and the specific process is shown in the following formula II:

[0005]

[0006] Specifically, (R)-ethyl lactate reacts with p-fluorophenylmagnesium bromide to obtain compound H, which is reduced to compound K, which is then esterified with G to obtain L, which is then hydrolyzed and amidated with pyridinecarboxylic acid compound B to obtain Florylpicoxamid. Among them, compound K (S)-1,1-bis(4-fluorophenyl)-2-propanol is an important intermediate. This method uses a method for chemically reducing chiral compound H to obtain compound K. This method uses a chemical reduction method, and the raw materials chiral ethyl lactate, reducing agent and TFA are relatively expensive, and the environmental pollution is serious.

[0007] CN111057725A provides a method for preparing a Florylpicoxamid intermediate (S)-1,1-bis(4-fluorophenyl)-2-propanol using 1,1-bis(4-fluorophenyl)-2-propanone as a raw material, and replaces the chemical method in the prior art with an enzyme-catalyzed method. The chemical method used is used to prepare 1,1-bis(4-fluorophenyl)-2-propanone, and then ketoreductase is used to catalyze 1,1-bis(4-fluorophenyl)-2-propanone to prepare (S)-1,1-bis(4-fluorophenyl)-2-propanol. The ketoreductase provided by this invention has a low conversion rate, and the reaction conditions are not perfect, which is not suitable for industrial production. Summary of the invention

[0008] In order to solve the problems of high raw material cost and low enzyme-catalyzed substrate conversion rate in the prior art for preparing (S)-1,1-bis(4-fluorophenyl-2-propanol), the present invention provides a method for preparing (S)-1,1-bis(4-fluorophenyl)-2-propanol by enzymatic reaction and the use of ketoreductase. The preparation method of the present invention reduces the cost of raw materials, makes the production process more environmentally friendly, and improves the substrate conversion rate of the existing enzyme-catalyzed method.

[0009] In order to solve the above technical problems, the first aspect of the present invention provides a method for preparing (S)-1,1-bis(4-fluorophenyl)-2-propanol by enzymatic reaction, the method comprising the following steps: using a ketoreductase or a variant thereof as shown in SEQ ID NO: 16 to catalyze 1,1-bis(4-fluorophenyl)-acetone in a reaction system to produce (S)-1,1-bis(4-fluorophenyl)-2-propanol. Wherein, the variant has one or more amino acid residues substituted, deleted or inserted in the amino acid sequence as shown in SEQ ID NO: 16, and the function of the ketoreductase is maintained or improved; the amino acid sequence of the variant preferably has at least 99% sequence homology with the amino acid sequence as shown in SEQ ID NO: 16.

[0010] In the reaction system, the ketoreductase is used to catalyze the compound of formula 1 to obtain the compound of formula 2: (S)-1,1-bis(4-fluorophenyl)-2-propanol.

[0011]

[0012] In some embodiments of the invention, the ketoreductase is a free ketoreductase and / or a whole cell ketoreductase; and / or,

[0013] The initial concentration of the 1,1-bis(4-fluorophenyl)-acetone in the reaction system is 0.04-1.5M, for example, 0.2M.

[0014] The ketoreductase is obtained by expressing a recombinant prokaryotic cell or a recombinant eukaryotic cell containing a gene encoding the ketoreductase; the recombinant prokaryotic cell is preferably an E. coli BL21 cell.

[0015] In some preferred embodiments of the present invention, the free ketoreductase comprises a crude ketoreductase enzyme solution or a purified ketoreductase enzyme solution; and / or, the whole-cell ketoreductase is used in the form of wet bacteria;

[0016] In some more preferred embodiments of the present invention, when the amount of the free ketone reductase added in the reaction system is calculated based on the mass of the wet bacteria producing the free ketone reductase, the ratio of the amount of the free ketone reductase added to the molar amount of the 1,1-bis(4-fluorophenyl)-acetone added is (100-500) g:1 mol (i.e., (0.1-0.5) g:1 mmol), for example, 175 g:1 mol (i.e., 0.175 g:1 mmol);

[0017] The molar ratio of the added mass of the whole-cell ketoreductase in the reaction system to the added 1,1-bis(4-fluorophenyl)-acetone is (10-100) g:1 mol (i.e. (0.01-0.1) g:1 mmol), for example, 50 g:1 mol (i.e. 0.05 g:1 mmol).

[0018] In some embodiments of the present invention, the reaction system further comprises a reduced coenzyme.

[0019] In some preferred embodiments of the present invention, the reduced coenzyme is NADH and / or NADPH.

[0020] In the present invention, the reduced coenzyme serves as a hydrogen donor.

[0021] In some embodiments of the present invention, the reduced coenzyme is obtained by the following steps: reducing an oxidized coenzyme such as NAD in the presence of a dehydrogenase and a hydrogen donor + and / or NADP + .NAD + NAD·Na 2 Add in the form of NADP + Take NADP·Na 2 Add in the form of .

[0022] In some preferred embodiments of the present invention, in the reaction system using the free ketoreductase, the added molar amount of the oxidized coenzyme accounts for 0.8%-3%, for example 1%, of the added molar amount of the 1,1-bis(4-fluorophenyl)-acetone; in the reaction system using the whole-cell ketoreductase, the added molar amount of the oxidized coenzyme accounts for 0.02% to 0.1%, for example 0.04%, of the added molar amount of the 1,1-bis(4-fluorophenyl)-acetone.

[0023] In some embodiments of the present invention, the dehydrogenase is free glucose dehydrogenase or whole-cell glucose dehydrogenase, and the hydrogen donor is preferably glucose; and / or, the dehydrogenase is free isopropanol dehydrogenase or whole-cell isopropanol dehydrogenase, and the hydrogen donor is preferably isopropanol; and / or, the dehydrogenase is free glucose-6-phosphate dehydrogenase or whole-cell glucose-6-phosphate dehydrogenase, and the hydrogen donor is preferably D-glucose-6-phosphate; and / or, the dehydrogenase is free formate dehydrogenase or whole-cell formate dehydrogenase, and the hydrogen donor is preferably formate.

[0024] In some preferred embodiments, when the amount of free glucose dehydrogenase added in the reaction system is calculated according to the mass of the wet bacteria producing the free glucose dehydrogenase, the ratio of the added molar amount of the free glucose dehydrogenase to the added molar amount of 1,1-bis(4-fluorophenyl)-acetone is (100-500) g:1 mol (i.e., (0.1-0.5) g:1 mmol), for example, 150 g:1 mol (i.e., 0.15 g:1 mmol); or, the amount of whole-cell glucose dehydrogenase added in the reaction system is calculated according to When calculating the added mass of wet bacteria that produce the whole-cell glucose dehydrogenase, the ratio of the added molar amount of the wet bacteria to the added molar amount of the 1,1-bis(4-fluorophenyl)-acetone is (10-100) g:1 mol (i.e. (0.01-0.1) g:1 mmol), for example, 10 g:1 mol (i.e. 0.01 g:1 mmol); and / or, the ratio of the added molar amount of the glucose to the 1,1-bis(4-fluorophenyl)-acetone in the reaction system is (1-4):1, for example (1-3):1.

[0025] In some more preferred embodiments of the present invention, the NCBI accession number of the glucose dehydrogenase is NP_388275.1.

[0026] In some embodiments of the present invention, the reaction system further comprises a buffer; the buffer is preferably a phosphate buffer.

[0027] In some preferred embodiments of the present invention, the pH of the phosphate buffer is 6.5-7.5, such as 7.0.

[0028] In some more preferred embodiments of the present invention, in the reaction system using free ketoreductase, the concentration of phosphate is 40-60 mM, such as 50 mM; in the reaction system using whole-cell ketoreductase, the concentration of phosphate is 10-30 mM, such as 20 mM.

[0029] In some embodiments of the present invention, the reaction system further comprises a co-solvent, which is preferably DMSO or isopropanol.

[0030] In some preferred embodiments of the present invention, the added volume of the isopropanol accounts for 5%-20% of the total volume of the reaction system, such as 5%, 10% or 20%.

[0031] In some embodiments of the present invention, the temperature of the enzymatic reaction is 20-40°C, such as 30°C.

[0032] In some embodiments of the present invention, the enzymatic reaction is carried out under shaking, and the rotation speed of the shaking is preferably 50-500 rpm, such as 200 rpm or 400 rpm.

[0033] In some embodiments of the present invention, the enzymatic reaction time is 10-72 hours, such as 16 hours.

[0034] In some embodiments of the present invention, the method further comprises: performing an extraction and / or concentration process after the enzymatic reaction, and the extraction is preferably performed using ethyl acetate.

[0035] In order to solve the above technical problems, the second aspect of the present invention provides a ketoreductase or a genetically recombinant cell expressing the ketoreductase for use in preparing (S)-1,1-bis(4-fluorophenyl)-2-propanol using 1,1-bis(4-fluorophenyl)-acetone as a substrate, wherein the amino acid sequence of the ketoreductase is as shown in SEQ ID NO: 16, or one or more amino acid residues are substituted, deleted or inserted in the amino acid sequence as shown in SEQ ID NO: 16, and the function of the ketoreductase is maintained or improved. The amino acid sequence of the variant preferably has at least 99% sequence homology with the amino acid sequence as shown in SEQ ID NO: 16.

[0036] In some preferred embodiments of the present invention, the ketoreductase is a free ketoreductase and / or a whole-cell ketoreductase.

[0037] In some more preferred embodiments of the present invention, the free ketoreductase includes a crude ketoreductase enzyme solution or a purified ketoreductase enzyme solution; and the whole-cell ketoreductase is used in the form of wet bacteria.

[0038] In order to solve the above technical problems, the third aspect of the present invention provides a use of a ketoreductase or a genetically recombinant cell expressing the ketoreductase in the preparation of a pyridineamide fungicide, Florylpicoxamid, wherein the amino acid sequence of the ketoreductase is as shown in SEQ ID NO: 16, or one or more amino acid residues are substituted, deleted or inserted in the amino acid sequence as shown in SEQ ID NO: 16, and the function of the ketoreductase is maintained or improved. The amino acid sequence of the variant preferably has at least 99% sequence homology with the amino acid sequence as shown in SEQ ID NO: 16.

[0039] In some preferred embodiments of the present invention, the ketoreductase is a free ketoreductase and / or a whole-cell ketoreductase.

[0040] In some more preferred embodiments of the present invention, the free ketoreductase includes a crude ketoreductase enzyme solution or a purified ketoreductase enzyme solution; and the whole-cell ketoreductase is used in the form of wet bacteria.

[0041] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0042] The reagents and raw materials used in the present invention are commercially available.

[0043] The positive and progressive effects of the present invention are:

[0044] (1) There is no need to add expensive chiral raw materials, reducing agents and trifluoroacetic acid (TFA), which reduces the cost of raw materials and makes the production process more environmentally friendly.

[0045] (2) Optimize the enzymes and catalytic conditions used to increase the conversion rate, thereby increasing production, reducing production costs, and reducing environmental pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is the HPLC spectrum of the reaction solution after the K160 whole-cell enzyme reaction was completed.

[0047] Figure 2 This is the chiral HPLC spectrum of the reference substance of the racemate of compound 2 (1,1-bis(4-fluorophenyl)-2-propanol).

[0048] Figure 3 This is the HPLC spectrum of the product ee value determination after the K160 whole cell enzyme reaction is completed. DETAILED DESCRIPTION

[0049] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0050] The chiral analysis of (S)-1,1-bis(4-fluorophenyl)-2-propanol in the present invention is performed by high performance liquid chromatography (HPLC), and the specific analysis method is as follows:

[0051] Chromatographic column: Daicel Chiralpak IG (4.6mm*250mm, 5μm); mobile phase: n-hexane:ethanol=90:10 (volume ratio); detection wavelength: 210nm; flow rate: 1.0mL / min; column temperature: 25℃; 25mg sample, diluted to 50mL with ethanol, injection volume is 5μL.

[0052] The concentration of the substrate 1,1-bis(4-fluorophenyl)-2-propanone in the reaction solution and the analysis of the substrate conversion rate were performed by high performance liquid chromatography. The substrate conversion rate analysis method is as follows:

[0053] Chromatographic column: Agilent Eclipse plus C18 (3.5μm, 150×4.6mm); mobile phase: 0.1% TFA aqueous solution as mobile phase A, 0.1% TFA acetonitrile solution as mobile phase B, gradient elution according to the following conditions: 0min (90% A+10% B), 10min (100% B), 11min (100% B), 11.5min (90% A+10% B), 16min (90% A+10% B); detection wavelength: 210nm; flow rate: 1.0mL / min; column temperature: 30℃.

[0054] The conversion rate of the substrate is calculated as follows: conversion rate of the substrate = amount of substrate converted to product / total amount of substrate × 100%. The amount of substrate converted to product is calculated based on the amount of product generated. The amount of product generated is calculated using the area normalization method based on the peak area ratio of the product in the HPLC spectrum.

[0055] pET28a was purchased from Novagen; NdeI enzyme and HindIII enzyme were purchased from Thermo Fisher; EcoliBL21 (DE3) competent cells were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.

[0056] The experimental methods in the present invention are all conventional methods unless otherwise specified. For details of gene cloning operations, please refer to "Molecular Cloning Experiment Guide" compiled by J. Sambrook et al.

[0057] The molecular formula and molecular weight of each compound are shown in Table 1.

[0058] Table 1 Molecular formula and molecular weight of each compound

[0059] Compound Molecular formula Molecular weight 1,1-Bis(4-fluorophenyl)-2-propanone <![CDATA[C 15 H 12 F 2 O]]> 246.2 <![CDATA[NADP·Na 2 ]]> <![CDATA[C 21 A 26 N 7 So 2 O 17 Q 3 ]]> 787.37 <![CDATA[NAD·Na 2 ]]> <![CDATA[C 21 A 27 N 7 O 14 Q 2 So 2 ]]> 709.4 glucose <![CDATA[C 6 H 12 O 6 ]]> 180.16

[0060] Example 1 Acquisition of KRED enzyme and preparation of recombinant plasmid

[0061] Nucleotide sequences and amino acid sequences of multiple ketoreductases (Ketoreductase, KRED) from different species were obtained from NCBI. The sources of these ketoreductases and information such as GeneBank accession numbers are shown in Table 2. The genes of the above multiple ketoreductases were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd. (No. 698, Xiangmin Road, Songjiang District, Shanghai). The genes of the above multiple ketoreductases were cloned into pET28a vectors using restriction sites Nde I and Hind III, respectively, to obtain multiple recombinant plasmids containing the above ketoreductase genes.

[0062] Table 2 List of relevant information of ketoreductase

[0063]

[0064]

[0065] Among them, the coenzyme of K014, K024, K025, K085, and K160 is NADP + The coenzyme of K082, K083, K110, K353, K354, and K375 is NAD + The enzyme K014 in the present application is the same as the K014 disclosed in the specification of CN111057725A.

[0066] Example 2 Preparation of Ketoreductase Crude Enzyme Solution

[0067] The multiple recombinant plasmids containing the ketoreductase gene constructed in Example 1 were respectively transferred into Ecoli BL21 (DE3) competent cells, spread on resistant LB plates containing 50 μg / mL kanamycin, and inverted and cultured at 37°C for 16 hours. After the culture was completed, a single colony was picked and inoculated into resistant LB medium containing 50 μg / mL kanamycin and cultured at 37°C for 5 hours. According to the inoculum amount of 1% (v / v), it was transferred to TB medium containing 50 μg / mL kanamycin and cultured at 37°C and 220rpm until the OD 600 At around 0.4-0.6, add IPTG at a final concentration of 0.1 mM and induce at 25°C for 16 h. Centrifuge at 4000 rpm for 20 min, discard the supernatant, and collect the wet cells.

[0068] The collected wet cells were added to 50mM PBS buffer with a pH of 7.0 at a ratio of 1g:10mL (homogenization ratio of 1:10) to suspend the cells, and the suspended cells were broken by a homogenizer (4°C, 600bar) to obtain a homogenized solution. The homogenized solution was centrifuged at 12000rpm for 5min, and the supernatant was taken to obtain a crude enzyme solution containing ketoreductase, which was stored at -20°C for later use. Ketoreductase is an intracellular enzyme. After the bacterial cells are homogenized, the ketoreductase in them is released into the supernatant.

[0069] The concentration of enzyme protein in the crude ketoreductase solution was determined using a Bradford kit (purchased from Shanghai Jierui Bioengineering Co., Ltd.).

[0070] The enzymatic activity of the crude enzyme solution of ketoreductase was determined as follows:

[0071] A 1mL enzyme activity assay reaction system was used: at 30°C, 980μL of 50mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer (containing 50mM 1,1-bis(4-fluorophenyl)-acetone and 10% isopropanol as a cosolvent) at pH 7.0 was first added to the container, followed by 10μL of 25mM NADPH, and finally 10μL of crude enzyme solution. The change in OD value at 340nm was measured by UV spectrophotometer. The enzyme activity of KRED in the crude enzyme solution was calculated, and the results are shown in Table 3.

[0072] KRED unit enzyme activity is defined as the amount of enzyme required to consume 1 μmol NADPH per minute under specific reaction conditions (30°C, pH 7.0).

[0073] Table 3 Enzyme activity determination results

[0074] Crude enzyme solution number Enzyme protein concentration (mg / mL) Enzyme activity (U / mL) Unit protease activity (U / mg) K014 crude enzyme solution 4.18 9.49 2.27 K024 crude enzyme solution 7.18 9.46 1.32 K025 crude enzyme solution 5.83 4.67 0.8 K082 crude enzyme solution 6.16 3.94 0.64 K083 crude enzyme solution 8.15 5.39 0.66 K085 crude enzyme solution 6.25 7.4 1.18 K110 crude enzyme solution 4.61 5.23 1.13 K160 crude enzyme solution 3.89 20.3 5.22 K353 crude enzyme solution 4.52 0.12 0.03 K354 crude enzyme solution 3.96 0.35 0.09 K375 crude enzyme solution 4.55 0.89 0.2

[0075] In Table 3, unit protease activity (U / mg) refers to the enzyme activity per unit mass (mg) of enzyme protein in the crude enzyme solution. As shown in Table 3, among the above-mentioned crude enzyme solutions, the unit protease activity of K160 crude enzyme solution is the highest.

[0076] Example 3 Preparation of Glucose Dehydrogenase Crude Enzyme Solution

[0077] According to the glucose dehydrogenase (GDH) gene sequence from Bacillus subtilis 168 (NCBI accession number NP_388275.1), the glucose dehydrogenase (GDH) gene was fully synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. (No. 211 Pubin Road, Research and Innovation Park, Jiangbei New District, Nanjing, Jiangsu Province), with restriction sites NdeI and HindIII, and connected to the vector pET28a. The synthesized GDH gene was transformed into the host E. coli BL21 (DE3) competent cells to obtain an engineered strain containing the GDH gene.

[0078] After streaking and activating the engineered strains containing the GDH gene on plates, pick a single colony and inoculate it into 5 mL LB liquid medium containing 50 μg / mL kanamycin and shake culture at 37°C for 4 h. Transfer the inoculum to 150 mL of freshly prepared TB liquid medium containing 50 μg / mL kanamycin at a 1 v / v% inoculum and shake culture at 37°C until OD 600 When the pH value reached about 0.8, IPTG was added to a final concentration of 0.1 mM, and the culture was induced at 25°C for 16 h. After the culture was completed, the culture solution was centrifuged at 4500 rpm for 20 min, the supernatant was discarded, and the precipitate (i.e., wet bacteria) was collected and stored in a -20°C ultra-low temperature refrigerator for later use.

[0079] 10 g of the collected wet cells were resuspended in 50 mL of 0.05 M pH 7.0 sodium phosphate buffer (homogenization ratio of 1:5), and crushed by high pressure homogenization to obtain a crude GDH enzyme solution. 3-3.5% of the total volume of 10% PEI flocculant was slowly added to the crude GDH enzyme solution, stirred for 10 min, and centrifuged at 4000 rpm for 20 min to obtain the supernatant enzyme solution for the next step of determining GDH enzyme activity. The enzyme protein concentration in the crude GDH enzyme solution was determined using a Bradford kit (purchased from Shanghai Jierui Bioengineering Co., Ltd.).

[0080] GDH enzyme activity detection method: 1mL reaction system, 25℃ condition, first add 880μL pH7.0 0.1M disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (containing 400mM glucose), then add 100μL 25mM NADP + solution, and finally add 20 μL of supernatant enzyme solution that has been diluted 200 times, and measure the OD value at 340 nm with a UV spectrophotometer.

[0081] GDH enzyme activity is defined as: the amount of enzyme required to generate 1 μmol NAD(P)H per minute at pH 7.0 and 25°C is 1 enzyme activity unit (1U).

[0082] The enzyme activity of the crude GDH enzyme solution was measured to be 1480 U / mL, the enzyme protein concentration was 30 mg / mL, and the unit protease activity (U / mg) was 49.33 U / mg.

[0083] Example 4 Using ketoreductase crude enzyme solution to catalyze substrate production of (S)-1,1-bis(4-fluorophenyl)-2-propanol

[0084]

[0085] In the above formula, compound 1 is the substrate 1,1-bis(4-fluorophenyl)-2-propanone, which is prepared according to patent CN111057725A. Compound 2 is the product (S)-1,1-bis(4-fluorophenyl)-2-propanol.

[0086] The steps of this embodiment are as follows:

[0087] Obtain 100 mg 1,1-bis(4-fluorophenyl)-2-propanone (0.4 mmol), 3.15 mg NADP·Na 2 (0.004mmol) or 2.75mg NAD·Na 2 (0.004mmol), add 0.1mL isopropanol (as a cosolvent), 0.3mL of crude GDH enzyme solution obtained by the method of Example 3 (444U; equivalent to 0.06g GDH wet bacteria), 220mg glucose (1.221mmol), 0.5mL 200mM PBS (pH7.0), adjust the pH to 7.0 with 1M NaOH aqueous solution, add 0.7mL of crude enzyme solution of one of the ketoreductases obtained by the method of Example 2 (equivalent to 0.07g ketoreductase wet bacteria), add deionized water to make up the volume to 2mL. A plurality of enzyme catalytic reaction systems with a total volume of 2mL are obtained.

[0088] Among them, in each enzyme-catalyzed reaction system, the initial concentration of the substrate 1,1-bis(4-fluorophenyl)-2-propanone after addition but before the reaction begins is 0.2 mol / L. The added molar amount of the oxidized coenzyme is 1% of the added molar amount of the substrate. The initial added concentration of the oxidized coenzyme in the reaction system is 2 mM. The ratio of the crude enzyme solution of ketoreductase to the added molar amount of 1,1-bis(4-fluorophenyl)-2-propanone is 0.175 g:1 mmol based on the mass of the wet bacteria of ketoreductase. The enzyme activity of GDH in the reaction system is 222 U / mL. The ratio of the added enzyme activity of GDH to the added molar amount of the substrate is 1100 U:1 mmol. When the amount of free glucose dehydrogenase (GDH crude enzyme solution) added in the reaction system is calculated based on the mass of the wet bacteria that produces free glucose dehydrogenase, its ratio to the added molar amount of 1,1-bis(4-fluorophenyl)-propanone is 0.15 g:1 mmol. The ratio of the molar amount of glucose added to the reaction system to the molar amount of substrate added to the reaction system is 3: 1. The concentration of PBS in the reaction system is 50 mM. The addition amount of various crude enzyme solutions is shown in Table 4.

[0089] Table 4 Addition amount of various crude enzyme solutions

[0090]

[0091] 2 mL of the enzyme catalytic reaction system was placed at 30°C and 200 rpm for 16 hours. After the reaction was completed, the reaction solution was extracted with ethyl acetate, and the extract was dried. The dried product was dissolved in methanol and injected into HPLC to detect the product generation amount and ee value. The results of the substrate conversion rate are shown in Table 5. The results show that NAD + The dependent K353, K354, K375, K082, K083, and K110 enzymes cannot catalyze 1,1-bis(4-fluorophenyl)-2-propanone (substrate) to produce (S)-1,1-bis(4-fluorophenyl)-2-propanol (product); NADP + The dependent K024, K025, and K085 cannot catalyze the substrate to obtain the product; NADP + The dependent K160 and K014 can catalyze the substrate to obtain the product.

[0092] Table 5 Conversion rate of substrate and ee value of product when catalyzed by different ketoreductases

[0093]

[0094]

[0095] Example 5 Recombinant Escherichia coli whole-cell ketoreductase catalyzes substrate to produce (S)-1,1-bis(4-fluorophenyl)-2-propanol The steps of this example are as follows:

[0096] Obtain 2.5 g of substrate 1,1-bis(4-fluorophenyl)acetone (10.154 mmol) and 3 mg of NADP·Na 2 (0.004 mmol), 10 mL of isopropanol (as a cosolvent), 0.5 mL of the crude GDH enzyme solution obtained by the method of Example 3 (in the form of NADP + as a cofactor, 740U; equivalent to 0.1g GDH wet bacteria), 5.5g glucose (30.528mmol), 5mL 200mM PBS buffer (pH7.0), adjust the pH to 7.0 with 1M NaOH aqueous solution, add 0.5g ketoreductase wet bacteria (including whole cell ketoreductase) obtained by the method of Example 2, and add deionized water to make up to 50mL. Thus, an enzyme catalytic reaction system with a total volume of 50mL is obtained.

[0097] Among them, in this reaction system, the initial concentration of the substrate 1,1-bis(4-fluorophenyl)-2-propanone after addition but before the reaction starts is 0.2 mol / L. The ratio of the added amount of whole-cell ketoreductase to the added molar amount of the substrate is 0.05g:1mmol based on the mass of the wet bacteria of ketoreductase. The added concentration (g / mL) of whole-cell ketoreductase in the reaction system is 0.01g / mL based on the mass of the wet bacteria of ketoreductase. The added molar amount of the oxidized coenzyme is 0.04% of the added molar amount of the substrate. The initial added concentration of the oxidized coenzyme in the reaction system is 0.08mM. The enzyme activity of GDH in the reaction system is 14.8U / mL. The ratio of the added enzyme activity of GDH to the added molar amount of the substrate is 72.88U:1mol. The ratio of the added molar amount of glucose in the reaction system to the added molar amount of the substrate in the reaction system is 3:1. The concentration of PBS in the reaction system is 20mM.

[0098] 50 mL of the enzyme catalytic reaction system was stirred at 30°C and 400 rpm for 16 hours. The pH of the reaction was controlled at 6-7 with saturated sodium carbonate. After the reaction was completed, the reaction solution was extracted with ethyl acetate, and the extract was dried. The dried product was dissolved in methanol and injected into HPLC. The HPLC results are as follows: Figure 1 The detection results of substrate conversion rate are shown in Table 6.

[0099] Table 6 Substrate conversion rate when whole cell ketoreductase is catalyzed

[0100] Whole cell enzyme number Coenzyme Conversion rate of substrate ee value of the product K160 whole cell enzyme <![CDATA[NADP + ]]> 85％ 99.8％ K014 whole cell enzyme <![CDATA[NADP + ]]> 57％ 99.7％

[0101] Figure 1This is the HPLC spectrum of the reaction liquid after the K160 whole cell catalytic reaction was completed. The retention times were 9.551 min and 8.871 min, respectively, which were consistent with the peak positions of the substrate and product. It also showed that 1,1-bis(4-fluorophenyl)-2-propanol was obtained in the reaction, and the conversion rate of the substrate was measured to be 85%.

[0102] Figure 3 Taking K160 whole cell enzyme as an example, after the reaction is completed, the chiral HPLC spectrum of the obtained product is measured, and the retention time is 8.307min as its peak position. The chiral HPLC spectrum of the racemic reference substance of 1,1-bis(4-fluorophenyl)-2-propanol is Figure 2 , retention time is 8.311min and 11.197min. The peak positions of 8.307min and 8.311min are consistent, so it can be known that the reaction has obtained chiral 1,1-bis(4-fluorophenyl)-2-propanol.

[0103] The optical rotation values ​​of the obtained product were -33° and -17.9° respectively, with methanol and chloroform as solvents. The substrate has no optical activity and will not interfere with the results. According to the literature, it is speculated that the product is an S-type single configuration. According to the references Journal of Organic Chemistry, Vol. 68, nb. 11, 2003, 4600-4603 and Tetrahedron, vol. 33, 1977, 507-510, it is further known that the obtained product is an S-configuration, that is, the product is (S)-1,1-bis(4-fluorophenyl)-2-propanol.

[0104] The sequences used in the present invention are as follows:

[0105] SEQ ID NO: 1:

[0106]

[0107] SEQ ID NO:2:

[0108] MTDLFKPLPEPPTELGRLRVLSKTAGIRVSPLILGGASIGDAWSGFMGSMNKEQAFELLDAFYEAGGNCIDTANSYQNEESEIWIGEWMASRKLRDQIVIATKFTGDYKKYEVGGGKSANYCGNHKRSLHVSVRDSLRKLQTDWIDILYIHWWDYMSSIEEVMDSLHILVQQGKVLYLGVSDTPAWVVSAANYYATSHGKTPFSVYQGKWNVLNRDFERDIIPMARHFGMALAPWDVMGGGRFQSKKAMEERKKNGEGLRTFVGGPEQTELEVKISEALTKIAEEHGTESVTAIAIAYVRSKAKNVFPLIGGRKIEHLKQNIEALSIKLTPEQIEYLESIVPFDVGFPKSLIGDDPAVTKKLSPLTSMSARIAFDN

[0109] SEQ ID NO:3:

[0110] ATGAAACGTGTTAATGCATTTAATGATCTGAAACGTATTGGTGATGATAAAGTTACCGCCATTGGTATGGGTACCTGGGGTATTGGTGGTCGTGAAACCCCGGATTATAGCCGCGATAAAGAATCAATTGAAGCAATTCGCTATGGTCTGGAACTGGGTATGAATCTGATTGATACCGCAGAATTTTATGGTGCCGGTCATGCAGAAGAAATTGTGGGTGAAGCGATTAAAGAATTTGAACGTGAAGATATTTTTATTGTTAGCAAAGTGTGGCCGACCCATTTTGGTTATGAAGAAGCCAAAAAAGCCGCCCGTGCCAGCGCGAAACGCCTGGGTACCTATATTGATCTGTATCTGCTGCATTGGCCGGTTGATGATTTTAAAAAGATTGAAGAAACCCTGCATGCACTGGAAGATCTGGTTGATGAAGGTGTGATTCGCTATATTGGTGTTAGCAATTTTAATCTGGAACTGCTGCAGCGTAGCCAGGAAGTGATGCGTAAATATGAAATTGTTGCAAATCAGGTTAAATATAGCGTTAAAGATCGCTGGCCGGAAACCACCGGTCTGCTGGATTATATGAAACGTGAAGGTATTGCACTGATGGCCTATACCCCGCTGGAAAAAGGTACCCTGGCACGTAATGAATGCCTGGCCAAAATTGGTGAAAAATATGGTAAAACCGCCGCCCAGGTTGCCCTGAATTATCTGATTTGGGAAGAAAATGTGGTTGCAATTCCGAAAGCCAGCAATAAAGAACATCTGAAAGAAAATTTTGGTGCGATGGGTTGGCGTCTGAGCGAAGAAGATCGTGAAATGGCACGTCGTTGTGTT

[0111] SEQ ID NO:4:

[0112] MKRVNAFNDLKRIGDDKVTAIGMGTGWGIGGRETPDYSRDKESIEAIRYGLELGMNLIDTAEFYGAGHAEEIVGEAIKEFEREDIFIVSKVWPTHFGYEEAAKKAARASAKRLGTYIDLYLLHWPVDDFKKIEETLHALEDLVDEGVIRYIGVSNFNLELLQRSQEVMRKYEIVANQVKYSVKDRWPETTGLLDYMKREGIALMAYTPLEKGTLARNECLAKIGEKYGKTAAQVALNYLIWEENVVAIPKASNKEHLKENFGAMGWRLSEEDREMARRCV

[0113] SEQ ID NO:5:

[0114] ATGGGTATTCCGAAACGTAAACTGGGTGAACGTGGCCCGGAAGTGAGCGCAATTGGTCTGGGTTGCATGCGTATGAGCTTTGGTCAGAAAAAACTGCCGGATCGTAAAGAAATGATTAAACTGATCCGCACCGCGGTTGAACTGGGCATTAATTTCTTTGATACCGCCGAAGTGTATGGCCCGTATACCAATGAAGAACTGGTTGGCGAAGCACTGGAACCGTTTAAAGGTGAAGTGGTGATTGCCACCAAATTTGGCTTTGAACTGTATGAAGATGGCCGTCCGGGTTGGAAAGGTCTGAATAGTAATCCGGAACATATTAAAAAAGCCGTTGAAGGTAGCCTGCGCCGTCTGCGTGTGGAAGCAATTGATATTCTGTATCAGCATCGTGTTGATCCGAATGTTCCGATTGAAGAAGTTGCAGGCGCAGTGAAAGAACTGATTGAAGAAGGTAAAGTGAAACATTTTGGTCTGTGCGAAGCAAGCGCCGAAACCATTCGTCGTGCACATAAAGTGTGCCCGGTGGATGTGGTGCAGTATGAATATAGCATGTGGTGGCGTAAACCGGAAGAAGAACTGCTGCCGACCTGCGAAGAACTGGGCATTGGCTTTGTGGCATATTCTCCGCTGGGCAAAGGCTTTCTGACCGGTGCCATTGGTGAAAATAGCAAATTTGATGAAGAAGATAGCCGTAGTCGCATTCCGCGTTTTCAGAAAGAAAATCTGCGTGAAAATCTGGCCCTGGTTGAACTGCGTAAAACCATTGCAGAACGCAAAGGTGCGACGCCGAGCCAGATTGCCCTGGCCTGGCTGCTGGCCCAGAAACCGTGGATTGTGCCGATTCCGGGTACCACCAAACTGAGCCATCTGCTGGAAAATATTGGTGGTGCATTTGTGGAACTGACCCCGGAAGAACTGCAGGAAATTAATGATGCCCTGAGCCGTATTGAAATTAAAGGTAGCCGTTATCCGGAAGATATGGAAAAAATGACCTATCTG

[0115] SEQ ID NO:6:

[0116] MGIPKRKLGERGPEVSAIGLGCMRMSFGQKKLPDRKEMIKLIRTAVELGINFFDTAEVYGPYTNEELVGEALEPFKGEVVIATKFGFELYEDGRPGWKGLNSNPEHIKKAVEGSLRRLRVEAIDILYQHRVDPNVPIEEVAGAVKELIEEGKVKHFGLCEASAETIRRAHKVCPVDVVQYEYSMWWRKPEEELLPTCEELGIGFVAYSPLGKGFLTGAIGENSKFDEEDSRSRIPRFQKENLRENLALVELRKTIAERKGATPSQIALAWLLAQKPWIVPIPGTTKLSHLLENIGGAFVELTPEELQEINDALSRIEIKGSRYPEDMEKMTYL

[0117] SEQ ID NO:7:

[0118] ATGGTTCCGAAATTTTATAAACTGAGCAATGGTTTTAAAATTCCTAGCATTGCACTGGGTACCTATGATATTCCGCGTAGCCAGACCGCCGAAATTGTTTATGAAGGTGTGAAATGTGGTTATCGTCATTTTGATACCGCAGTTCTGTATGGTAATGAAAAGAAGTTGGTGATGGTATTATTAAATGGCTGAATGAAGATCCGGGCAATC ATAAACGTGAAGAAATCTTTTATACCACCAAACTGTGGAATAGCCAGAATGGTTATAAACGTGCAAAAGCAGCCATTCGTCAGTGTCTGAATGAAGTTAGTGGTCTGCAGTATATTGATCTGCTGCTGATTCATAGCCCGCTGGAAGGCAGTAAACTGCGTCTGGAAACCTGGCGCGCAATGCAGGAAGCAGTTGATGAAGGCCTGGTGAAA AGCATTGGTGTTAGCAATTATGGTAAAAAACATATTGATGAACTGCTGAATTGGCCGGAACTGAAACATAAACCGGTGGTTAATCAGATTGAAATTAGCCCGTGGATTATGCGTCAGGAACTGGCCGATTATTGTAAAAGTAAAGGTCTGGTTGTTGAAGCATTTGCACCGCTGTGTCATGGTTATAAAATGACCAATCCGGATCTGCTGA AAGTTTGTAAAGAAGTTGATCGTAATCCGGGTCAGGTTCTGATTCGTTGGAGCCTGCAGCATGGTTATCTGCCGCTGCCGAAAACCAAAACCGTTAAACGTCTGGAAGGTAATCTGGCAGCCTATAATTTTGAACTGAGTGATGAACAGATGAAATTTCTGGATCATCCGGATGCCTATGAACCGACCGATTGGGAATGTACCGATGCACCG

[0119] SEQ ID NO:8:

[0120] MVPKFYKLSNGFKIPSIALGTYDIPRSQTAEIVYEGVKCGYRHFDTAVLYGNEKEVGDGIIKWLNEDPGNHKREEIFYTTKLWNSQNGYKRAKAAIRQCLNEVSGLQYIDLLLIHSPLEGSKLRLETWRAMQEAVDEGLVKSIGVSNYGKKHIDELLNWPELKHKPVVNQIEISPWIMRQELADYCKSKGLVVEAFAPLCHGYKMTNPDLLKVCKEVDRNPGQVLIRWSLQHGYLPLPKTKTVKRLEGNLAAYNFELSDEQMKFLDHPDAYEPTDWECTDAP

[0121] SEQ ID NO:9:

[0122] ATGCCGGCCACCCTGCATGATAGCACCAAAATTCTGAGCCTGAATACCGGTGCACAGATTCCGCAGATTGGTCTGGGTACCTGGCAGTCAAAAGAAAATGATGCCTATAAAGCAGTGCTGACAGCCCTGAAAGATGGTTATCGCCATATTGATACCGCAGCAATTTATCGCAATGAAGATCAGGTTGGTCAGGCAATTAAAGATAGCGGTGTTCCGCGTGAAGAAATTTTTGTGACCACCAAACTGTGGTGTACGCAGCATCATGAACCGGAAGTGGCCCTGGATCAGTCACTGAAACGTCTGGGTCTGGATTATGTGGATCTGTATCTGATGCATTGGCCGGCACGTCTTGATCCGGCCTATATTAAAAATGAAGATATTCTGAGCGTTCCGACGAAAAAAGATGGTAGCCGTGCCGTTGATATTACCAATTGGAATTTTATTAAAACCTGGGAACTGATGCAGGAACTGCCGAAAACCGGTAAAACGAAAGCCGTGGGTGTTTCAAATTTTTCAATTAATAATCTGAAAGATCTGCTGGCCAGCCAGGGTAATAAACTGACCCCGGCAGCCAATCAGGTTGAAATTCATCCGCTGCTGCCGCAGGATGAACTGATTAATTTTTGTAAAAGCAAAGGCATTGTTGTTGAAGCATATAGCCCGCTGGGTAGCACCGATGCCCCGCTGCTGAAAGAACCGGTGATTCTGGAAATTGCGAAGAAAAATAATGTTCAGCCGGGTCATGTGGTTATTAGCTGGCATGTGCAGCGCGGTTATGTTGTTCTGCCGAAAAGCGTGAATCCGGATCGCATTAAAACCAATCGTAAAATTTTTACCCTGAGCACCGAAGATTTTGAAGCAATTAATAATATTAGCAAAGAAAAAGGTGAAAAACGCGTGGTGCATCCGAATTGGAGCCCGTTTGAAGTTTTTAAA

[0123] SEQ ID NO:10:

[0124] MPATLHDSTKILSLNTGAQIPQIGLGTWQSKENDAYKAVLTALKDGYRHIDTAAIYRNEDQVGQAIKDSGVPREEIFVTTKLWCTQHHEPEVALDQSLKRLGLDYVDLYLLMHWPARLDPAYIKNEDILSVPTKKDGSRAVDITNWNFIKTWELMQELPKTGKTKAVGVSNFSINNLKDLLASQGNKLTPAANQVEIHPLLPQDELINFCKSKGIVVEAYSPLGSTDAPLLKEPVILEIAKKNNVQPGHVVISWHVQRGYVVLPKSVNPDRIKTNRKIFTLSTEDFEAINNISKEKGEKRVVHPNWSPFEVFK

[0125] SEQ ID NO:11:

[0126]

[0127] SEQ ID NO:12:

[0128] MTTDTTVFVSGATGFIALHIMNDLLKAGYTVIGSGRSQEKNDGLLKKFNNNPKLSMEIVEDIAAPNAFDEVFKKHGKEIKIVLHTASPFHFETTNFEKDLLTPAVNGTKSILEAIKKYAADTVEKVIVTSSTAALVTPTDMNKGDLVITEESWNKDTWDSCQANAVAAYCGSKKFAEKTAWEFLKENKSSVKFTLSTINPGFVFGPQMFADSLKHGINTSSGIVSELIHSKVGGEFYNYCGPFIDVRDVSKAHLVAIEKPECTGQRLVLSEGLFCCQEIVDILNEEFPQLKGKIATGEPATGPSFLEKNSCKFDNSKTKKLLGFQFYNLKDCIVDTAAQMLEVQNEA

[0129] SEQ ID NO:13:

[0130]

[0131] SEQ ID NO:14:

[0132] MKAIQIIQPGKPPELREVEKPTPRPGQVLLKVTAAGACHSDDFVLNLPEEGFPYPLPMTLGHEGAGVVAEVGTGVTGISEGTSVAVYGAWGCGVCHFCARGLENYCSRAGELGITPPGLGNPGAMAEYLLVDDARHLVPLGDLDPVAAVPLTDAGLTPYHAIKPSLPKLVGGTTAVVIGAGGLGHVGIQLLRHLTPSRVIALDVSDDKLAFAREVGAHEVVLSDADAVANVRKITGNDGATAVFDFVGLQPTLDIAMGVVGTMGDVVIVGIGDMVATAKVGFFTQPYEVSVRAPYWGARDELIEVLDLARDGVLEVAVERFSLDDGVEAYRRLAANDLRGRAVVVPD

[0133] SEQ ID NO:15:

[0134] CATATGAACGCGCTGGTTACCGGTGGTAGCCGTGGCATCGGTGAAGCGATCGCGACCAAACTGGCGGAAGATGGCTACAGCGTTACCATCGCGTCTCGTGGCATCGATCAGCTGAACAAAGTTAAAGCGAAACTGCCGGTTGTTCGTGAAGGCCAGACCCACCACGTTTGGCAGCTGGATCTGAGCGACGCGGAAGCGGCGTCTAGCTTCAAAGGCGCGCCGCTGCCGGCGAGCAGCTACGATGTTCTGGTTAACAACGCGGGCGTTACCGATCCGTCTCCGATCGCGAAACAGTCTGATAGCGAAATCCACAAACTGTTCAGCGTTAACCTGCTGAGCCCGGTTGCGCTGACCAAAACCTACGTTCAGGCGGTTACCGGTAAACCGCGTGAAACCCCGGCGCACATCATCTTCATCAGCAGCGGCGTTGCGATCCGTGGTTACCCGAACGTTGCGGTTTACAGCGCGACCAAATCTGGTCTGGATGGTTTCATGCGTTCCCTGGCGCGTGAACTGGGCCCGGAAGGCGTTCACGTTAACACCGTTTCTCCGGGCCTGACCAAAACTGAAATGGCGTCTGGCGTTAGCCTGGATGATTTCCCGCCGAGCCCGATCGGCGGCTGGATCCAGCCGGAAGCGATCGCTGATGCGGTTCGTTACCTGGTTAAATCTAAAAACATCACCGGTACCATCCTGAGCGTTGATAACGGCATCACCGTTAAGCTTSEQ ID NO:16:

[0135] MNALVTGGSRGIGEAIATKLAEDGYSVTIASRGIDQLNKVKAKLPVVREGQTHHVWQLDLSDAEAASSFKGAPLPASSYDVLVNNAGVTDPSPIAKQSDSEIHKLFSVNLLSPVALTKTYVQAVTGKPRETPAHIIFISSGVAIRGYPNVAVYSATKSGLDGFMRSLARELGPEGVHVNTVSPGLTKTEMASGVSLDDFPPSPPIGGWIQPEAIADAVRYLVKSKNITGTILSVDNGITV

[0136] SEQ ID NO:17:

[0137] CATATGAAAGTTATCTTCTTCTCTATGCACCCGTATGAAGAAGAATTTCTGGGCCCGATCCTGCCGAGCGATTGGGACGTTGAAATGACCCCGGACTTCCTGGATGAAACCACTGTTGAAAAAGCAAAAGGTGCGCAGGTTGTTTCTCTGTTCGTTTCCGATAAAGCTGATGGCCCGGTGCTGGAAGCTCTGCACAGCTACGGTGTTGGTCTGCTGGCGCTGCGCTCTGCGGGTTATGACCACATCGACATCGAAACCGCAAAACGTCTGGGTATCAAAGTTGTTAACGTTCCGGCGTATAGCCCGCACGCGATCGCAGACCACACCCTGGCTATCATGCTGGCACTGATCCGCCGTCTGCATCGTGCGCACGATAAAGTTCGTCTGGGCGATTTCGATCTGGATGGTCTGATGGGTTTCGATCTGAACGGCAAAGTGGCTGGCGTGATCGGTCTGGGTAAAATCGGCCGTCTGGTTGCGACCCGTCTGAAAGCGTTCGGTTGCAAAGTGCTGGGCTACGATCCGTATATCCAGCCGGAAATCGTTGAAAACGTGGATCTGGACACCCTGATCACCCAGGCTGACATCATCAGCATTCACTGCCCGCTGACTCGTGAAAACTTTCACATGTTCAACGAAGAAACCTTCAAACGTATGAAACCGGGCGCTATCCTGGTTAACACCGCTCGTGGTGGCCTGATTGATACCAAAGCGCTGCTGGAAGCGCTGAAAAGCGGCAAACTGGGTGGCGCGGCGCTGGATGTTTACGAATACGAACGTGGCCTGTTCTTCAAAAACCACCAGAAAGAAGGCATCAAAGATCCGTATCTGGCGCAGCTGCTGGGTCTGGCAAACGTTGTGCTGACCGGTCACCAGGCGTTCCTGACCCGTGAAGCGGTTAAAAACATCGAAGAAACCACCGTTGAAAACATCCTGGAATGGCAGAAAAACCCGCAGGCTAAACTGAAAAACGAAATCTAAGCTT

[0138] SEQ ID NO:18:

[0139] MKVIFFSMHPYEEEFLGPILPSDWDVEMTPDFLDETTVEKAKGAQVVSLFVSDKADGPVLEALHSYGVGLLALRSAGYDHIDIETAKRLGIKVVNVPAYSPHAIADHTLAIMLALIRRLHRAHDKVRLGDFDLDGLMGFDLNGKVAGVIGLGKIGRLVATRLKAFGCKVLGYDPYIQPEIVENVDLDTLITQADIISIHCPLTRENFHMFNEETFKRMKPGAILVNTARGGLIDTKALLEALKSGKLGGAALDVYEYERGLFFKNHQKEGIKDPYLAQLLGLANVVLTGHQAFLTREAVKNIEETTVENILEWQKNPQAKLKNEI

[0140] SEQ ID NO:19:

[0141]

[0142] SEQ ID NO:20:

[0143] MKVAVFSAKSYDRQFLDAANAAQGHPHLLTYYDVLLRPKTAALVEGHDAICAFVNDDLGAQTLERLAELGVRLVTLRCTGFNNVDLATAARLGITVTRVSVYSPYSVAEHTVGLMLMLNRKLHRAYNRVRDDNFSLEGLMGFDLHGCTVGIIGTGKIGQIVAQILHGFGCHLYGYDPYPSQAFREIGTYTTLETLLAASEIITLHCPLLPENEHLINATTIAQMKRGVMLINTSRGKLVDTKAVIEGIKSGQIGYLGIDVYEEEDSLFFQDLSDTVIQDDTFQLLQSFPNVVITAHQAFFTRNALTDIARTTIENLTCFEQGLPLANEVKQASP

[0144] SEQ ID NO:21:

[0145] CATATGATGGGCGAATTTGATGGTAAAGTGGTTATCGTTACCGGCGGCACCAAAGGTATCGGTCGTGCGGCGGTTAAAAAATTCTGCGAAGCGGGTGCGACCGCGATCATCGTGAACCGCAGCGAAAAAGAAGGCAAAGAATACGAAAAAGAACTGGTTGAATCCGGCTACAAAGCGGAAGCGATCGCTGCGGATGTGAGCAAAGTTGAAGATATTAAAAACATGGTGGAAATCGTTCTGCAAAAATACGGCAAAATCGATGTTCTGGTGAACTGCGCGGGTGTTAACCGTCGCAAAAAAGCGATCGAATACAACGAAGAAGATTGGGACTTCATGGTTGACATCAACCTGAAAGGCGTTTTCTTCACCTGCCTGGAAGTGGGCAAAACCATGATCGAACGTGGCGAAGGCGCGATCGTTAACATCTCTAGCATCCAGGGTGAAGTTGTTCTGCCGGAACGTAGCATCTACGCGATCACCAAAGGCGGTGTGAAACAGCTGACCAAAGCGCTGGCGGTTGAATGGAGCAAATTCGGTGTTCGTGTTAACGCGATCTCCCCGGCGTTCGTTGAAACCCCGATGGTTGAAAAAGTGCTGGAAGATGAAAACTGGCGTAACATCATCCTGAGCCACACCCCGATGGGCCGTGTGGGCAAACCGGAAGAAATCGCGGAAGTTATCCTGTTCCTGGCGAGCGAACGTGCGAGCTACATCACCGGCGCGAACATCATGGTTGATGGCGGCTGGACCGCGTTCTAAAAGCTT

[0146] SEQ ID NO:22:

[0147] MGEFDGKVVIVTGGTKGIGRAAVKKFCEAGATAIIVNRSEKEGKEYEKELVESGY

[0148] KAEAIAADVSKVEDIKNMVEIVLQKYGKIDVLVNCAGVNRRKKAIEYNEEDWDFMV

[0149] DINLKVFFTCLEVGKTMIERGEGAIVNISSIQGEVVLPERSIYAITKGGVKQLTKALAV

[0150] EWSKFGVRVNAISPAFVETPMVEKVLEDENWRNIILSHTPMGRVGKPEEIAEVILFLASE

[0151] RASYITGANIMVDGGWTAF。

Claims

1. A method for preparing (S)-1,1-bis(4-fluorophenyl)-2-propanol by enzymatic reaction, characterized in that: The method comprises the following steps: using the ketoreductase shown in SEQ ID NO: 16 to catalyze 1,1-bis(4-fluorophenyl)-acetone in a reaction system to produce (S)-1,1-bis(4-fluorophenyl)-2-propanol.

2. The method according to claim 1, characterized in that The ketoreductase is free ketoreductase and / or whole cell ketoreductase; and / or, The initial concentration of the 1,1-bis(4-fluorophenyl)-acetone in the reaction system is 0.04-1.5M; Preferably, the free ketoreductase comprises a crude ketoreductase enzyme solution or a purified ketoreductase enzyme solution; and / or, the whole-cell ketoreductase is used in the form of wet cells; More preferably, when the amount of the free ketone reductase added in the reaction system is calculated based on the mass of the wet bacteria producing the free ketone reductase, the ratio of the amount of the free ketone reductase added to the molar amount of the 1,1-bis(4-fluorophenyl)-acetone added is (100-500) g:1 mol; The molar ratio of the added mass of the whole-cell ketoreductase in the reaction system to the added 1,1-bis(4-fluorophenyl)-acetone is (10-100) g:1 mol.

3. The method according to claim 1 or 2, characterized in that The reaction system also contains reduced coenzyme; The reduced coenzyme is preferably NADH and / or NADPH.

4. The method according to claim 3, characterized in that The reduced coenzyme is obtained by the following steps: reducing an oxidized coenzyme such as NAD in the presence of a dehydrogenase and a hydrogen donor + and / or NADP + ; Preferably, in the reaction system using the free ketoreductase, the added molar amount of the oxidized coenzyme accounts for 0.8%-3% of the added molar amount of the 1,1-bis(4-fluorophenyl)-acetone; in the reaction system using the whole-cell ketoreductase, the added molar amount of the oxidized coenzyme accounts for 0.02% to 0.1% of the added molar amount of the 1,1-bis(4-fluorophenyl)-acetone.

5. The method according to claim 4, characterized in that The dehydrogenase is free glucose dehydrogenase or whole-cell glucose dehydrogenase, and the hydrogen donor is preferably glucose; and / or, the dehydrogenase is free isopropanol dehydrogenase or whole-cell isopropanol dehydrogenase, and the hydrogen donor is preferably isopropanol; and / or, the dehydrogenase is free glucose-6-phosphate dehydrogenase or whole-cell glucose-6-phosphate dehydrogenase, and the hydrogen donor is preferably D-glucose-6-phosphate; and / or, the dehydrogenase is free formate dehydrogenase or whole-cell formate dehydrogenase, and the hydrogen donor is preferably formate; Preferably, when the amount of the free glucose dehydrogenase added in the reaction system is calculated according to the mass of the wet bacteria that produce the free glucose dehydrogenase, the ratio of the amount of the free glucose dehydrogenase added in the reaction system to the molar amount of 1,1-bis(4-fluorophenyl)-acetone added is (100-500) g:1 mol; or, when the amount of the whole-cell glucose dehydrogenase added in the reaction system is calculated according to the mass of the wet bacteria that produce the whole-cell glucose dehydrogenase, the ratio of the amount of the free glucose dehydrogenase added in the reaction system to the molar amount of 1,1-bis(4-fluorophenyl)-acetone added is (10-100) g:1 mol; and / or, the ratio of the molar amounts of glucose added to the 1,1-bis(4-fluorophenyl)-acetone added in the reaction system is (1-4):1; More preferably, the NCBI accession number of the glucose dehydrogenase is NP_388275.

1.

6. The method according to any one of claims 1 to 5, characterized in that: The reaction system further comprises a buffer; the buffer is preferably a phosphate buffer; Preferably, the pH of the phosphate buffer is 6.5-7.5; More preferably, in the reaction system using free ketoreductase, the concentration of phosphate is 40-60 mM; in the reaction system using whole-cell ketoreductase, the concentration of phosphate is 10-30 mM.

7. The method according to any one of claims 1 to 6, characterized in that: The reaction system further comprises a co-solvent; the co-solvent is preferably DMSO or isopropanol; Preferably, the added volume of the isopropanol accounts for 5%-20% of the total volume of the reaction system.

8. The method according to any one of claims 1 to 7, characterized in that: The temperature of the enzymatic reaction is 20-40°C; and / or, The enzymatic reaction is carried out in an oscillating state, and the rotation speed of the oscillation is preferably 50-500 rpm; and / or, The enzymatic reaction time is 10-72 hours; and / or, The method further comprises: performing an extraction and / or concentration process after the enzymatic reaction, and the extraction is preferably performed using ethyl acetate.

9. Use of a ketoreductase or a genetically recombinant cell expressing the ketoreductase in preparing (S)-1,1-bis(4-fluorophenyl)-2-propanol using 1,1-bis(4-fluorophenyl)-acetone as a substrate, wherein the amino acid sequence of the ketoreductase is as shown in SEQ ID NO: 16; Preferably, the ketoreductase is a free ketoreductase and / or a whole-cell ketoreductase; More preferably, the free ketoreductase includes a crude ketoreductase enzyme solution or a purified ketoreductase enzyme solution; and the whole-cell ketoreductase is used in the form of wet bacteria.

10. Use of a ketoreductase or a genetically recombinant cell expressing the ketoreductase in the preparation of a pyridineamide fungicide, Florylpicoxamid, wherein the amino acid sequence of the ketoreductase is as shown in SEQ ID NO: 16; Preferably, the ketoreductase is a free ketoreductase and / or a whole-cell ketoreductase; More preferably, the free ketoreductase includes a crude ketoreductase enzyme solution or a purified ketoreductase enzyme solution; and the whole-cell ketoreductase is used in the form of wet bacteria.

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