Thermotolerant Kluyveromyces mutant and method for preparing (S)-(4-chlorophenyl)-(pyridin-2-yl)-methanol

Abstract

The present invention provides a thermotolerant Kluyveromyces mutant and a method for preparing (S)-(4-chlorophenyl)-(pyridin-2-yl)-methanol, using Kluyveromyces thermotolerans ST thermotolerant Kluyveromyces ST, deposit number CCTCC NO: M 2022498, whole cell catalysis preparation of chiral drug bepotastine drug intermediates ( S )-(4-chlorophenyl)-(pyridine-2-yl)-methanol method, belongs to the field of biocatalysis technology. The invention adopts normal temperature and pressure plasma mutagenesis and adaptive evolution technology to screen out a thermotolerant Kluyveromyces mutant strain with advantages of toxic and high temperature resistance. The asymmetric reduction reaction is carried out in a two-phase aqueous system composed of PEG and sodium dihydrogen phosphate at a reaction temperature of 40°C, a substrate 4-chlorophenyl-(pyridine-2-yl)-methanone concentration of 50g / L, a bacterial concentration of 50g / L, and a pH of 8 for 36h. The product ( S The optical purity and yield of )-(4-chlorophenyl)-(pyridine-2-yl)-methanol can reach 99.2%ee and 92.9%, respectively. This method has the advantages of low temperature, simple operation, high product optical purity and yield, and has industrial application prospects.

Description

Technical Field

[0001] The invention relates to the field of biocatalysis technology, and specifically to the breeding of toxic and heat-resistant Kluyveromyces mutants and the application method thereof in the synthesis of chiral drug intermediate (S)-(4-chlorophenyl)-(pyridin-2-yl)-methanol. Background Art

[0002] Bepotastine drugs are a class of second-generation H1 antihistamines with high efficiency and low toxicity and side effects. They can be used to treat allergic rhinitis, neurological tinnitus, urticaria and other diseases. They have the advantages of rapid action, high selectivity, low side effects and significant efficacy. Due to the rapid increase in the incidence of allergic diseases in recent decades, the demand for bepotastine drugs, which play an important role in the treatment of allergic diseases, has continued to increase. As second-generation antihistamines, astemizole and terfenadine can induce cardiotoxic reactions, followed by cetirizine and loratadine. Bepotastine has attracted much attention due to its high efficiency and low toxicity. (S)-(4-chlorophenyl)-(pyridin-2-yl)-methanol ((S)-CPMA) is an important chiral intermediate for the synthesis of bepotastin drugs. The reports on its synthesis method are as follows: In 1996, Corey EJ et al. used catecholborane as a chiral positioning selector, BF3 and BBr3 as reducing agents to asymmetric reduce (4-phenyl)-(pyridin-2-yl)-methaone. The yield and ee value of the product (4-phenyl)-(pyridin-2-yl)-methanol were 19% and 30%, respectively; in 2003, Chen et al. C et al. used the chiral catalyst trans-RuCl2[(R)-xylbinap][(R)-daipen] to asymmetrically reduce CPMK to synthesize (S)-CPMA with an ee value of 60.6%; in 2006, Truppo MD et al. used ketoreductase (commercial enzyme) to enzymatically asymmetric reduce CPMK, and the optical purity of the product (S)-CPMA was only 60%. In 2012, Zhou Jieyu et al. used whole cells of Kluyveromyces yeast to asymmetric reduce CPMK in a two-phase aqueous system to synthesize (S)-CPMA. Under the condition of substrate concentration of 6g / L, the yield reached 92.1% and the ee value was 86.7%.

[0003] From the above data, it can be seen that the chemical reduction method for synthesizing (S)-(4-chlorophenyl)-(pyridin-2-yl)-methanol is not effective, the ee value and yield are relatively low, and the operation process is relatively complicated. Some chiral catalysts will introduce heavy metal elements during the reaction, which is not conducive to its application in the pharmaceutical industry. There are not many reports on biological methods. The yield of the reaction of preparing (S)-(4-chlorophenyl)-(pyridin-2-yl)-methanol by reduction of whole cells of Kluyveromyces in a two-phase aqueous solution reached 92.1%, but the ee value was only 86.7%, which did not meet the requirements as a pharmaceutical intermediate. Therefore, it is urgent to develop biocatalysts with strong substrate tolerance, high optical purity and high catalytic efficiency and their process research. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing (S)-(4-chlorophenyl)-(pyridin-2-yl)-methanol by breeding a mutant strain of Kluyveromyces resistant to toxins and heat and whole cell catalysis. A thermotolerant Kluyveromyces mutant strain with strong toxin resistance is bred by plasma mutagenesis at room temperature and pressure, and a whole cell catalysis method is used to study the asymmetric reduction of 4-chlorophenyl-(pyridin-2-yl)-methanone, which provides a feasible way for preparing (S)-(4-chlorophenyl)-(pyridin-2-yl)-methanol.

[0005] The technical scheme of the present invention is as follows: firstly, a microbial mutant strain resistant to substrate toxicity and having stereoselective carbonyl reductase activity is screened, and then the whole cell thereof is used as a catalyst to asymmetric reduce the prochiral 4-chlorophenyl-(pyridin-2-yl)-methanone in a two-phase aqueous reaction system composed of PEG and sodium dihydrogen phosphate to prepare (S)-(4-chlorophenyl)-(pyridin-2-yl)-methanol with high optical purity, and the steps are as follows:

[0006] Breeding of thermotolerant Kluyveromyces mutants: The wild thermotolerant Kluyveromyces strain obtained by screening was used as the original strain, and the concentration of the Kluyveromyces seed suspension was OD 600 =0.7-1, in a normal temperature and pressure plasma device, the temperature of ARTP is maintained at 20-25°C, the power is adjusted to 120W, and the flow rate of helium (He) is 3-10L / min; the metal slide is burned for at least 30-40s before the bacterial suspension is coated on the metal slide; the slide is placed in a mutagenesis chamber, and the distance between the emitter and the bacterial liquid is 1.5-3nm, preferably 2nm; the ARTP mutagenesis time is 100s-140s; after the mutagenesis is completed, the cells are cultured in YPD medium until OD 600The micro-droplet culture conditions are as follows: the number of droplets is 50, adaptive evolution is selected, the number of generations is 3, the generation time is 22h, the temperature is 28°C, the concentration of the influencing factor is 10g / L, 20g / L, 30g / L, 40g / L, 50g / L, 60g / L, 80g / L, 90 g / L and 100g / L, and the change of bacterial concentration is observed every day; the dilution of 10 is used to spread the plate method -6 ~10 -8 The supernatant was transferred to a YPD plate containing 50-100 g / L of CPMK, and each dilution was repeated 3 times and cultured in a 28°C incubator. After the bacterial colony grows, it is repeatedly streaked and separated on a YPD plate containing CPMK. After being observed under a microscope to determine that it is a pure single colony, it is inoculated on a preservation medium for preservation to obtain a microbial mutant strain with high stereoselective carbonyl reductase activity. Kluyveromyces thermotolerans ST was deposited in the China Center for Type Culture Collection on April 27, 2022, with a deposit number of CCTCC NO: M 2022498, and the deposit address is Wuhan University, Hubei, Wuhan.

[0007] Another technical solution of the present invention is to use the thermotolerant Kluyveromyces mutant strain in the preparation of (S)-(4-chlorophenyl)-(pyridin-2-yl)-methanol.

[0008] A method for preparing (S)-(4-chlorophenyl)-(pyridin-2-yl)-methanol comprises the following steps:

[0009] (1) Cultivation of wet cells of thermotolerant Kluyveromyces yeast mutants;

[0010] (2) Preparation of a two-phase aqueous reaction system consisting of PEG and sodium dihydrogen phosphate;

[0011] (3) In a two-phase aqueous reaction system composed of PEG and sodium dihydrogen phosphate, wet cells of a thermotolerant Kluyveromyces mutant, 4-chlorophenyl-(pyridin-2-yl)-methanone and glucose were added to carry out an enzyme-catalyzed reaction to obtain (S)-(4-chlorophenyl)-(pyridin-2-yl)-methanol.

[0012] The yield of (S)-CPMA can reach over 90%.

[0013] In step (1), after the thermotolerant Kluyveromyces mutant is fermented in a culture medium, the fermentation liquid is centrifuged to obtain wet cells of the thermotolerant Kluyveromyces mutant;

[0014] The culture medium comprises 10-100 g / L of glucose, 5-20 g / L of yeast extract powder, 1-10 g / L of potassium dihydrogen phosphate, and 0.2-2 g / L of magnesium sulfate heptahydrate;

[0015] The culture conditions are fermentation culture at room temperature for 1-3 days.

[0016] In step (2), PEG includes PEG4000 or PEG2000;

[0017] In the two-phase aqueous reaction system, the mass fraction of PEG is 10-30%, and the mass fraction of sodium dihydrogen phosphate is 10-30%.

[0018] As a preferred solution, in the two-phase aqueous reaction system, the mass fraction of PEG is 15%, and the mass fraction of sodium dihydrogen phosphate is 15%.

[0019] In step (3), the concentration of the wet cells of the thermotolerant Kluyveromyces mutant is 10-100 g / L; the concentration of 4-chlorophenyl-(pyridin-2-yl)-methanone is 10-100 g / L; and the concentration of glucose is 10-60 g / L.

[0020] As a preferred embodiment, in the step (3), the concentration of the wet cells of the thermotolerant Kluyveromyces mutant is 40 g / L; the concentration of 4-chlorophenyl-(pyridin-2-yl)-methanone is 40 g / L; and the concentration of glucose is 10 g / L.

[0021] The enzyme catalytic reaction conditions are 25-45°C, pH between 6-9, and 50-250 rpm for 3-72h. The best effect is achieved when the reaction temperature is 40°C, pH is 8, the rotation speed is 150 rpm, and the reaction time is 36h.

[0022] The preparation process of (S)-(4-chlorophenyl)-(pyridin-2-yl)-methanol also includes a product post-treatment step, specifically, filtering the product obtained in step (3) through a membrane, collecting the filtrate, extracting the collected filtrate with ethyl acetate for 3-4 times, drying the obtained product, centrifuging and filtering the filtrate, evaporating and concentrating the filtrate, and crystallizing at -5 to 5°C to obtain (S)-(4-chlorophenyl)-(pyridin-2-yl)-methanol. The optical purity is greater than 99.2%.

[0023] The reaction system selected by the method of the present invention is a two-phase aqueous reaction system prepared by a certain mass fraction of PEG4000 and a certain mass fraction of NaH2PO4. In the PEG 4000 (15%, W / W) / NaH2PO4 (15%, W / W) two-phase aqueous reaction system at 25°C, the ee value and yield of the final product can reach 99.19% and 92.93% respectively.

[0024] Detection of conversion reaction products: After extraction, drying and 0.22 μm pore size organic membrane filtration, the product yield and enantiomeric excess were determined by liquid chromatography. The specific conditions were as follows: Daicel Chiralcel OB-H (5 μm, 250 mm × 4.6 mm) liquid chromatography column, mobile phase of n-hexane: isopropanol (80:20, V / V), flow rate of 1 mL / min, column temperature of 35 °C, UV detection wavelength of 254 nm, injection volume of 20 μL, retention time of 8 min.

[0025] The optical purity of the product was evaluated by the enantiomeric excess value.

[0026]

[0027] A S : Liquid chromatography analysis of S-configuration product peak area; A R : Liquid chromatography analysis of R-configuration product peak area.

[0028] Beneficial effects of the present invention:

[0029] The invention screens and obtains a thermotolerant Kluyveromyces mutant of a microbial strain with high substrate concentration tolerance and good stereoselectivity, and asymmetric reduction of 1 g / L of 4-chlorophenyl-(pyridin-2-yl)-methanone substrate is carried out in a PEG 4000 (15%, W / W) / NaH2PO4 (15%, W / W) two-phase aqueous reaction system, and the ee value of the generated product (S)-(4-chlorophenyl)-(pyridin-2-yl)-methanol reaches 99.2%, and the yield reaches 92.9%. Compared with chemical methods and other enzyme methods, the invention has the following advantages: ① the generated (S)-(4-chlorophenyl)-(pyridin-2-yl)-methanol has a high enantiomeric excess value, a yield of 92.93%, and an ee of 99.19%; ② the ability of substrate CPMK tolerance is strong, which can reach 100 g / L; ③ the reaction conditions are mild and environmentally friendly; there are few side reactions, and the reduction can be completed in one step.

[0030] Deposit of biological material samples: The above-mentioned Kluyveromyces thermotoleransST strain is currently deposited in the China Center for Type Culture Collection, abbreviated as CCTCC, address: Wuhan University, China, deposit number CCTCCM 2022498, deposit date April 27, 2022. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 HPLC spectra of (S)-CPMA and (R)-CPMA.

[0032] Figure 2 This is the HPLC spectrum of CPMK. DETAILED DESCRIPTION

[0033] Example 1

[0034] Screening of thermotolerant Kluyveromyces yeast mutants with asymmetric reduction activity of 4-chlorophenyl-(pyridin-2-yl)-methanone

[0035] Using the thermotolerant Kluyveromyces obtained by screening in a certain enterprise environment as the original strain, a microbial mutant strain with high stereoselectivity was obtained through plasma mutagenesis at normal temperature and pressure.

[0036] The main culture media used in the experiment include:

[0037] Enrichment medium (g / L): glucose 20, CPMK 20, peptone 20, yeast extract 20, agar

[0038] Screening plate medium (g / L): glucose 20, CPMK 20-100, peptone 20, yeast extract 20

[0039] Fermentation medium (g / L): glucose 10-100, yeast extract 5-20, potassium dihydrogen phosphate 1-10, magnesium sulfate heptahydrate 0.2-2, inoculation amount 1-5%, fermentation culture 1-3 days

[0040] The specific steps for screening microbial strains are:

[0041] The concentration of Kluyveromyces seed suspension was OD 600 =0.7-1, in a normal temperature and pressure plasma device, the temperature of ARTP is maintained at 20-25°C, the power is adjusted to 120W, and the flow rate of helium (He) is 3-10L / min; the metal slide is burned for at least 30-40s before the bacterial suspension is coated on the metal slide; the slide is placed in a mutagenesis chamber, and the distance between the emitter and the bacterial liquid is 1.5-3nm, preferably 2nm; the ARTP mutagenesis time is 100s-140s; after the mutagenesis is completed, the cells are cultured in YPD medium until OD 600 is 0.1; the micro-droplet culture conditions are as follows: the number of droplets is 50, adaptive evolution is selected, the number of generations is 3, the generation time is 22h, the temperature is 28°C, the concentrations of the influencing factors are 10g / L, 20g / L, 30g / L, 40g / L, 50g / L, 60g / L, 80g / L, 90g / L and 100g / L, respectively, and the changes in bacterial concentration are observed every day.

[0042] The dilution was 10 -6 ~10 -8The supernatant was transferred to a YPD plate containing 50-100 g / L of CPMK, and each dilution was repeated 3 times and cultured in an incubator at 28°C. After the bacterial colony grew out, it was repeatedly streaked and separated on a YPD plate containing CPMK. After being observed under a microscope to determine that it was a pure single colony, it was screened again to obtain the strain SX41 with the highest conversion rate and the best toxin resistance (see Table 1), which was inoculated on a preservation medium for preservation to obtain a microbial mutant strain with high stereoselective carbonyl reductase activity. The thermotolerant Kluyveromyces thermotolerans ST was deposited in the China Center for Type Culture Collection on April 27, 2022, with a preservation number of CCTCC NO: M 2022498, and the preservation address is Wuhan University, Wuhan, Hubei.

[0043] Table 1 Mutant strains with asymmetric reduction activity of (4-chlorophenyl)-(pyridin-2-yl)-methanone with a conversion rate of more than 50%

[0044] Strain No. Yield(%) ee(%) SX3 62.5 98.5 SX5 58.3 99.3 SX10 65.3 99.2 SX35 75.1 99.2 SX41 85.3 99.5 SX47 70.2 98.2 SX50 69.6 99.1

[0045] Example 2 Effect of bacterial concentration on asymmetric reduction CPMK reaction in a two-phase aqueous reaction system

[0046] Cultivation of wet cells of thermotolerant Kluyveromyces mutants: The culture medium composition is as follows (g / L): 50 glucose, 16 yeast extract powder, 8 potassium dihydrogen phosphate, 0.8 magnesium sulfate heptahydrate. The culture was fermented for 1 day, and the fermentation liquid was centrifuged to obtain wet cells of thermotolerant Kluyveromyces mutants (hereinafter referred to as wet cells).

[0047] 10g / L, 20g / L, 40g / L, 50g / L, 60g / L, 70g / L, 80g / L, and 90g / L wet cells of thermotolerant Kluyveromyces mutants were suspended in 1L aqueous biphase consisting of 15% PEG 4000 and 15% NaH2PO4 containing 1% glucose and 40g / L CPMK. The reaction was carried out at 40°C, pH 7.2, 150rpm, and aeration volume of 0.6L / min for 24 h, and HPLC detection was performed to calculate the ee value and yield of the reaction. The results are shown in Table 2.

[0048] Table 2 Effect of bacterial concentration on asymmetric reduction CPMK reaction in two-phase aqueous reaction system

[0049] Bacteria concentration (g / L) Yield(%) ee(%) 10 82.71 98.94 20 79.05 99.12 40 92.93 99.19 50 70.23 99.10 60 70.21 99.20 70 70.11 99.23 80 69.61 99.37 90 69.35 99.24

[0050] As can be seen from Table 2, when the bacterial concentration increases from 10g / L to 40g / L, the reaction yield increases rapidly, from 82.71% to 92.93%, and the ee value increases from 98.94% to 99.19%; however, when the bacterial concentration increases from 40g / L to 50g / L, the yield and ee value of the product both decrease. When the concentration exceeds 60g / L, the yield does not change much, and the ee value changes slightly, at about 99.20%. The above results show that the optimal bacterial concentration in the reduction reaction system is 40g / L.

[0051] Example 3 Effect of reaction temperature on asymmetric reduction CPMK reaction in a two-phase aqueous reaction system

[0052] 40 g / L wet bacteria were suspended in 1 L aqueous biphase consisting of 15% PEG 4000 and 15% NaH2PO4 containing 1% glucose and 40 g / L CPMK. The reaction was carried out at 30°C, 35°C, 40°C, 45°C, pH 7.2, 150 rpm, and aeration volume of 0.6 L / min for 24 h. HPLC detection was performed and the ee value and yield of the reaction were calculated. The results are shown in Table 3.

[0053] Table 3 Effect of reaction temperature on asymmetric reduction CPMK reaction in a two-phase aqueous reaction system

[0054] Temperature(℃) Yield(%) ee(%) 30 76.72 98.53 35 76.45 98.56 40 81.75 98.39 45 27.64 92.75

[0055] It can be seen from Table 3 that within the temperature range of 30°C to 35°C, the ee value (98.53%-98.56%) and the yield (76.45%-76.72%) are relatively stable. When the temperature reaches 40°C, the yield reaches a maximum value of 81.75%. When the temperature continues to rise to 45°C, the ee value and the yield decrease significantly, decreasing to 27.64% and 92.75%, respectively. This indicates that the catalytic effect is best when the reaction temperature is 40°C.

[0056] Example 4 Effect of rotation speed on asymmetric reduction CPMK reaction in a two-phase aqueous reaction system

[0057] 10 g / L wet bacteria were suspended in 1 L of a biphasic aqueous solution consisting of 15% PEG 4000 and 15% NaH2PO4 containing 1% glucose and 40 g / L CPMK. The reaction was carried out at 50 rpm, 100 rpm, 150 rpm, 200 rpm, pH 7.2, 40°C, and an aeration volume of 0.6 L / min for 24 h. HPLC detection was performed and the ee value and yield of the reaction were calculated. The results are shown in Table 4.

[0058] Table 4 Effect of rotation speed on asymmetric reduction CPMK reaction in two-phase aqueous reaction system

[0059] Speed ​​(rpm) Yield(%) ee(%) 50 74.18 98.44 100 79.60 98.61 150 72.55 98.52 200 69.03 98.60

[0060] It can be seen from Table 4 that when the rotation speed increases from 100 rpm to 200 rpm, the yield decreases from 79.60% to 69.03%, and the ee value remains basically unchanged, which indicates that the rotation speed is preferably 100 rpm.

[0061] Example 5 Effect of substrate concentration on asymmetric reduction CPMK reaction in a two-phase aqueous reaction system

[0062] 40 g / L wet bacteria were suspended in 1 L aqueous biphase consisting of 15% PEG 4000 and 15% NaH2PO4 containing 1% glucose and 30 g / L, 40 g / L, 50 g / L, 60 g / L, 70 g / L, 80 g / L, 90 g / L CPMK, and then reacted at 40°C, pH 7.2, 100 rpm, and aeration 0.4 L / min for 24 hours. HPLC detection was performed and the reaction ee value and yield were calculated. The results are shown in Table 5.

[0063] Table 5 Effect of substrate concentration on asymmetric reduction CPMK reaction in a two-phase aqueous reaction system

[0064] Substrate concentration (g / L) Yield(%) ee(%) 30 67.74 98.60 40 78.56 98.56 50 92.1 99.2 60 79.84 98.14 70 71.85 98.11 80 65.37 97.23 90 64.51 95.68

[0065] It can be seen from Table 5 that when the substrate concentration increases from 30 to 50 g / L, the yield gradually increases and can reach 92.1%. However, as the substrate concentration continues to increase, the yield drops sharply and the ee value also drops to a certain extent. The yield is the highest when the substrate concentration is 50 g / L, which indicates that the substrate concentration should be 50 g / L.

[0066] Example 6 Effect of the mass fraction of PEG4000 on the asymmetric reduction CPMK reaction in a two-phase aqueous reaction system

[0067] 40 g / L wet bacteria were suspended in 1 L of a biphasic solution consisting of PEG 4000 and 15% NaH2PO4 with mass fractions of 12%, 15%, 18%, and 21%, respectively, containing 1% glucose and 50 g / L CPMK. The mixture was then reacted at 40°C, pH 7.2, 100 rpm, and aeration volume of 0.4 L / min for 24 h. HPLC detection was performed and the reaction ee value and yield were calculated. The results are shown in Table 5.

[0068] Table 6 Effect of the mass fraction of PEG4000 on the asymmetric reduction CPMK reaction in a two-phase aqueous reaction system

[0069]

[0070] It can be seen from Table 6 that when the mass fraction of PEG4000 increases from 12% to 21%, the yield gradually increases and can reach 80.21%. However, as the mass fraction of PEG4000 continues to increase, the yield decreases instead, and the ee value also decreases to a certain extent. The yield is the largest when the mass fraction of PEG4000 is 15%, which indicates that a mass fraction of PEG4000 of 15% is more appropriate.

[0071] Example 7 Effect of the mass fraction of NaH2PO4 on the asymmetric reduction CPMK reaction in a two-phase aqueous reaction system

[0072] 40 g / L wet bacteria were suspended in 1 L aqueous biphasic solution containing 1% glucose and 50 g / L CPMK, which consisted of 9%, 12%, 15%, 18% NaH2PO4 and 15% PEG 4000 by mass, respectively. The mixture was then reacted at 40°C, pH 7.2, 100 rpm, and aeration volume of 0.4 L / min for 24 h. HPLC detection was performed and the reaction ee value and yield were calculated. The results are shown in Table 5.

[0073] Table 7 Effect of the mass fraction of NaH2PO4 on the asymmetric reduction CPMK reaction in a two-phase aqueous reaction system

[0074]

[0075] It can be seen from Table 7 that when the mass fraction of NaH2PO4 increases from 9% to 18%, the yield gradually increases and can reach 80.46%. However, when the mass fraction of NaH2PO4 continues to increase, the yield decreases instead, and the ee value also decreases. The yield is the largest when the mass fraction of NaH2PO4 is 15%, which shows that the catalytic effect of NaH2PO4 with a mass fraction of 15% is the best.

Claims

1. A strain of thermotolerant Kluyveromyces yeast ( Kluyveromyces thermotolerans )ST strain, deposited in the China Center for Type Culture Collection on April 27, 2022, with the deposit number CCTCC NO: M 2022498, and the deposit address is Wuhan University, Wuhan, Hubei.

2. The thermotolerant Kluyveromyces ST strain according to claim 1 is used in the preparation of ( S )-(4-chlorophenyl)-(pyridin-2-yl)-methanol.

3. A S )-(4-chlorophenyl)-(pyridin-2-yl)-methanol preparation method, characterized in that, The steps include: (1) Cultivating wet cells of the thermotolerant Kluyveromyces ST strain according to claim 1; (2) Preparation of a two-phase aqueous reaction system consisting of PEG and sodium dihydrogen phosphate; PEG is PEG4000 or PEG2000; In the two-phase aqueous reaction system, the mass fraction of PEG is 10-30%, and the mass fraction of sodium dihydrogen phosphate is 10-30%; (3) In a two-phase aqueous reaction system composed of PEG and sodium dihydrogen phosphate, wet cells of the thermotolerant Kluyveromyces ST strain, 4-chlorophenyl-(pyridin-2-yl)-methanone and glucose were added to carry out an enzyme-catalyzed reaction to obtain ( S )-(4-chlorophenyl)-(pyridin-2-yl)-methanol; the wet cell concentration of the thermotolerant Kluyveromyces ST strain is 10-100 g / L; the concentration of 4-chlorophenyl-(pyridin-2-yl)-methanone is 10-100 g / L; the concentration of glucose is 10-60 g / L, The enzyme catalytic reaction conditions are 25-45° C., pH between 6-9, and a rotation speed of 50-250 rpm for 3-72 hours.

4. According to claim 3 ( S )-(4-chlorophenyl)-(pyridin-2-yl)-methanol preparation method, characterized in that, In step (1), after the thermotolerant Kluyveromyces ST strain is fermented in the culture medium, the fermentation liquid is centrifuged to obtain wet cells of the thermotolerant Kluyveromyces ST strain; The culture medium comprises 10-100 g / L of glucose, 5-20 g / L of yeast extract powder, 1-10 g / L of potassium dihydrogen phosphate, and 0.2-2 g / L of magnesium sulfate heptahydrate; The culture conditions are fermentation culture at room temperature for 1-3 days.

5. According to claim 3 ( S )-(4-chlorophenyl)-(pyridin-2-yl)-methanol preparation method, characterized in that, In the two-phase aqueous reaction system, the mass fraction of PEG is 15%, and the mass fraction of sodium dihydrogen phosphate is 15%.

6. According to claim 3 ( S )-(4-chlorophenyl)-(pyridin-2-yl)-methanol preparation method, characterized in that, In step (3), the wet cell concentration of the thermotolerant Kluyveromyces ST strain is 40 g / L; the concentration of 4-chlorophenyl-(pyridin-2-yl)-methanone is 40 g / L; and the concentration of glucose is 10 g / L.

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