Method for preparing (S)-3-chlorophenylpropanol

Abstract

The invention discloses a method for preparing (S)-3-chlorophenylpropanol, which comprises the following steps: catalyzing 3-chloropropiophenone by using alcohol dehydrogenase so as to prepare the (S)-3-chlorophenylpropanol, and the reaction system of the method comprises the alcohol dehydrogenase, the 3-chloropropiophenone and reduced coenzyme; the amino acid sequence of the alcohol dehydrogenase is selected from one or more of amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7. According to the method provided by the invention, the 3-chloropropiophenone with high concentration (100mg / mL) can be converted into the (S)-3-chlorophenylpropanol, the conversion rate of 100% can be achieved after the reaction is carried out for 6 hours, the ee value can be up to 99.88%, and the method is high in conversion rate, high in stereoselectivity, simple in reaction condition, relatively low in cost and beneficial to industrial production.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological catalysis, and particularly relates to a method for preparing (S)-3-chloro-1-phenyl-1-propanol, a biological material, an application of an alcohol dehydrogenase in preparing (S)-3-chloro-1-phenyl-1-propanol and a composition. BACKGROUND

[0002] Dapoxetine, usually in the form of hydrochloride, with the chemical name of S-(+)-N,N-dimethyl-a-[2-(naphthalenyloxy)ethyl]benyenemethanamine hydrochloride, and the trade name of Priligy, is a selective serotonin reuptake inhibitor, which is originally widely used for treating depression and related emotional disorders, and is later developed for treating premature ejaculation in men, and has the advantages of fast drug effect and small side effects. (S)-3-chloro-1-phenyl-1-propanol ((S)-CPPO) is an important intermediate in the chemical synthesis of dapoxetine, and its chemical structural formula is shown as formula 1.

[0003]

[0004] Chemical synthesis method is complicated, has low product yield, and may cause great environmental pollution, therefore, the biological catalysis method for synthesizing (S)-3-chloro-1-phenyl-1-propanol, which has mild reaction conditions, is friendly to the environment, and has high stereoselectivity, is more and more widely concerned. (S)-3-chloro-1-phenyl-1-propanol can be prepared by enzymatic reduction of 3-chloro-1-phenyl-1-propanone (3-CPP), and currently there are some studies on the method:

[0005] In 2010, Yun Hee Choi et al. expressed a variety of microbial reductases in E. coli for the preparation of (S)-3-chloro-1-phenyl-1-propanol. Among them, the reductase (alcohol dehydrogenase) YOL151W from Saccharomyces cerevisiae showed the highest reaction activity, and when coupled with a coenzyme regeneration system using glucose dehydrogenase, 30 mM (i.e. 5.06 g / L) of 3-chloro-1-phenyl-1-propanone could be completely converted, and the ee value was 100% (Yun, H C, et al. Asymmetric synthesis of (S)-3-chloro-1-phenyl-1-propanol using Saccharomyces cerevisiae reductase with high enantioselectivity. Appl. Microbiol. Biot. 2010; 87(1): 185-193.). However, the substrate concentration used in this method is low, which cannot meet the needs of industrial production.

[0006] In 2018, Yunping Tang et al. expressed the carbonyl reductase (alcohol dehydrogenase) derived from Novosphingobium aromaticivorans in E. coli, and used the recombinant E. coli wet cells to catalyze the asymmetric reduction of 3-chloro-1-phenyl-1-propanone, while using glucose dehydrogenase to catalyze the regeneration of coenzyme NADH. In PBS buffer at pH 7.0, 100 g / L of 3-chloro-1-phenyl-1-propanone could be converted to (S)-3-chloro-1-phenyl-1-propanol after 12 h of reaction, with a conversion rate of 100% and an ee value of 99.6% (Tang Y, et al. Efficient synthesis of a (S)-fluoxetine intermediate using carbonyl reductase coupled with glucose dehydrogenase. Bioresour Technol. 2018; 250: 457-463.). However, the reaction time of this method is long, and the ee value of the product can be further improved.

[0007] In addition, CN112708641A discloses a method for preparing (S)-3-chlorophenylpropanol by asymmetric reduction reaction of 3-chlorophenylpropanone as raw material under the catalysis of carbonyl reductase (alcohol dehydrogenase). About 100 g / L of 3-chlorophenylpropanone can be completely converted, and the ee value is greater than 99.9% under the condition of 45°C oil bath, 200 rpm mechanical stirring and reaction for 24 h. However, the reaction time is long, the reaction temperature is high, and the concentration of the added cosolvent dimethyl sulfoxide in the reaction system is high, which is 30% (v / v), and the reaction cost is increased. SUMMARY

[0008] The technical problem to be solved by the present application is that the existing method for preparing (S)-3-chlorophenylpropanol by enzyme catalysis of 3-chlorophenylpropanone has low substrate concentration, long reaction time, low ee value and high cost. In order to solve the above technical problem, the present application provides a method for preparing (S)-3-chlorophenylpropanol by using alcohol dehydrogenase.

[0009] The present application is to solve the above technical problems by the following technical solutions:

[0010] The first aspect of the present application provides a method for preparing (S)-3-chlorophenylpropanol, which comprises: in a reaction system comprising alcohol dehydrogenase, 3-chlorophenylpropanone and reduced coenzyme, using the alcohol dehydrogenase to catalyze 3-chlorophenylpropanone, thereby preparing (S)-3-chlorophenylpropanol; the amino acid sequence of the alcohol dehydrogenase is selected from one or more of the amino acid sequences shown as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7.

[0011] In some embodiments of the present application, the method further comprises: in the presence of hydrogen donor dehydrogenase, hydrogen donor and oxidized coenzyme, using the hydrogen donor dehydrogenase and the hydrogen donor to reduce the oxidized coenzyme to obtain the reduced coenzyme.

[0012] In some preferred embodiments of the present application, the oxidized coenzyme and the reduced coenzyme are selected from one or more of the following groups:

[0013] (1) NADP + and NADPH;

[0014] (2) NAD + and NADH;

[0015] and / or, the hydrogen donor and hydrogen donor dehydrogenase are selected from one or more of the following groups:

[0016] (1) glucose and glucose dehydrogenase;

[0017] (2) isopropyl alcohol and hydrogen donor alcohol dehydrogenase;

[0018] (3) formic acid or formate and formate dehydrogenase.

[0019] In the present application, the hydrogen donor dehydrogenase refers to a dehydrogenase which reduces the oxidized coenzyme by dehydrogenating the hydrogen donor to obtain the reduced coenzyme. The hydrogen donor alcohol dehydrogenase refers to an alcohol dehydrogenase which reduces the oxidized coenzyme by dehydrogenating the isopropyl alcohol to obtain the reduced coenzyme.

[0020] In some embodiments of the present application, the reaction system further comprises one or more of a cosolvent and a buffer.

[0021] In some preferred embodiments of the present application, the cosolvent is selected from one or more of dimethyl sulfoxide, methanol, ethanol, isopropanol, acetonitrile, toluene, and acetone; and / or, the buffer is a PBS buffer, a Tris buffer, or a HEPES buffer.

[0022] In some preferred embodiments of the present application, the cosolvent is dimethyl sulfoxide; and the buffer is a PBS buffer.

[0023] In some embodiments of the present application, the amino acid sequence of the glucose dehydrogenase is as shown in SEQ ID The 3-chloropropiophenone is added at a mass concentration of 50-200 mg / mL, as shown in NO: 15; and / or, the 3-chloropropiophenone is added at a mass concentration of 50-200 mg / mL.

[0024] In specific embodiments of the present application, the 3-chloropropiophenone is added at a mass concentration of 100 mg / mL.

[0025] In some embodiments of the present application, the NAD + The ratio of the added molar amount of NAD

[0026] The ratio of the added mass of the alcohol dehydrogenase to the added mass of 3-chloropropiophenone is (0.5-5):10, calculated based on the mass of the wet bacteria used; and / or, the ratio of the added molar amount of the alcohol dehydrogenase to the added molar amount of 3-chloropropiophenone is 1:(50-200).

[0027] The ratio of the added mass of the alcohol dehydrogenase to the added mass of 3-chloropropiophenone is (0.5-5):10, calculated based on the mass of the wet bacteria used; and / or, the ratio of the added molar amount of the alcohol dehydrogenase to the added molar amount of 3-chloropropiophenone is 1:(50-200).

[0028] The ratio of the added molar amount of glucose to the added molar amount of 3-chloropropiophenone is (1-5):1.

[0029] The ratio of the added mass of the alcohol dehydrogenase to the added mass of 3-chloropropiophenone is (0.5-5):10, calculated based on the mass of the wet bacteria used; and / or, the ratio of the added molar amount of the alcohol dehydrogenase to the added molar amount of 3-chloropropiophenone is 1:(50-200).

[0030] In specific embodiments of the present application, the NAD + The ratio of the added molar amount of NAD

[0031] The ratio of the mass of the alcohol dehydrogenase to the mass of the added 3-chloropropiophenone is 3:10, calculated based on the mass of the wet cells used;

[0032] The ratio of the mass of the glucose dehydrogenase to the mass of the added 3-chloropropiophenone is 1:10, calculated based on the mass of the wet cells used;

[0033] The ratio of the molar amount of the added glucose to the molar amount of the added 3-chloropropiophenone is 3:1.

[0034] The volume of the added dimethyl sulfoxide accounts for 5% of the total volume of the reaction system.

[0035] In some embodiments of the present application, the reaction conditions of the method comprise one or more of the following: the reaction temperature is 25-40℃; the pH of the reaction system is 6.0-9.0.

[0036] In specific embodiments of the present application, the reaction conditions of the method comprise one or more of the following: the reaction temperature is 37℃; the pH of the reaction system is 7.5.

[0037] The second aspect of the present application provides a biological material comprising one or more nucleic acids as shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14.

[0038] The biological material is a nucleic acid, a recombinant expression vector, or a transformant.

[0039] The recombinant expression vector comprises the nucleic acid.

[0040] The transformant comprises the nucleic acid or the recombinant expression vector.

[0041] In some preferred embodiments of the present application, the recombinant expression vector comprises a pET28a plasmid backbone or a pET21a plasmid backbone; and the host cell of the transformant is a eukaryotic cell or a prokaryotic cell.

[0042] In some preferred embodiments of the present application, the host cell of the transformant is a bacterium.

[0043] In specific embodiments of the present application, the host cell of the transformant is Escherichia coli or Bacillus subtilis.

[0044] The third aspect of the present application provides an application of an alcohol dehydrogenase in the preparation of (S)-3-chlorophenylpropanol, wherein the amino acid sequence of the alcohol dehydrogenase is selected from one or more of the amino acid sequences shown as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7.

[0045] The fourth aspect of the present application provides a composition comprising an alcohol dehydrogenase, wherein the amino acid sequence of the alcohol dehydrogenase is selected from two or more of the amino acid sequences shown as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7.

[0046] or, a reaction system as defined in the method according to the first aspect of the present application;

[0047] or, a mixture obtained by reaction of a reaction system as defined in the method according to the first aspect of the present application.

[0048] On the basis of common general knowledge in the art, the above-mentioned preferred conditions can be combined in any manner, thereby obtaining preferred embodiments of the present application.

[0049] The reagents and raw materials used in the present application are commercially available.

[0050] Thanks to the above technical solutions, the present application achieves the following technical effects:

[0051] The method for preparing (S)-3-chlorophenylpropanol provided by the present application can convert 3-chlorophenylpropanone with a high concentration (100 mg / mL) into alcohol dehydrogenase of (S)-3-chlorophenylpropanol, and the conversion rate can reach 100% within 6 h, and the ee value can be as high as 99.88%. The conversion rate is high, the stereoselectivity is high, the reaction conditions are simple, the cost is low, and the method is conducive to industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0052] Figure 1 Reaction scheme for the enzymatic production of (S)-3-chlorophenyl- ethanol from 3-chlorophenylpyruvic acid as substrate Figure. Wherein, ADH represents alcohol dehydrogenase, and GDH represents glucose dehydrogenase. DETAILED DESCRIPTION

[0053] The present application will be further described by way of examples, but the present application is not limited in the scope of the examples. The experimental methods in the following examples are not specified, and the methods are selected according to conventional methods and conditions, or according to the instructions of the products.

[0054] Unless otherwise indicated, various materials and reagents used in the examples of the present application are those commonly available in the art and known to those skilled in the art.

[0055] TB liquid medium: 10 g / L of proteose peptone, 18 g / L of yeast powder, 0.4% (v / v) of glycerol, 2.31 g / L of KH2PO4(anhydrous), 16.43 g / L of K2HPO4(trihydrate), wherein g / L refers to the ratio of the added mass (g) of a component to the total volume (L) of the TB liquid medium; (v / v) refers to the ratio of the added volume (L) of a component to the total volume (L) of the TB liquid medium, and the same applies hereinafter.

[0056] LB liquid medium: 10 g / L of proteose peptone, 5 g / L of yeast powder, 10 g / L of NaCl.

[0057] LB agar plate: 10 g / L of proteose peptone, 5 g / L of yeast powder, 10 g / L of NaCl, 18 g / L of agar, pH 7.2 (adjusted with 1 mol / L NaOH).

[0058] 3-chloropropiophenone and 3-chloropropenol of S and R configurations were purchased from Shanghai Biotechmed Pharmaceutical Technology Co., Ltd., the item number of 3-chloropropiophenone was BD86193, the item number of (S)-3-chloropropenol was BD11276, and the item number of (R)-3-chloropropenol was BD6772.

[0059] The detection method used in the examples is as follows:

[0060] 1. HPLC detection method for conversion rate of substrate (3-chloropropiophenone)

[0061] Chromatographic column: ZORBAX Eclipse plus C18 (3.5 μm, 150 x 4.6 mm); mobile phase: 0.1% TFA aqueous solution: 0.1% TFA acetonitrile solution = 50:50 (by volume); detection wavelength: 211 nm; flow rate: 1.0 mL / min; injection volume: 5 μL; column temperature: 35 °C; running time: 10 min.

[0062] Preparation method of reference solution: 15 mg to 50 mg of reference of substrate and product were weighed into a volumetric flask, dissolved and diluted to the mark with methanol, shaken well, and filtered for injection.

[0063] Retention time of control: 3-chloropropiophenone 5.936 min, 3-chloropropyl alcohol 4.264 min.

[0064] The calculation formula of conversion rate is:

[0065] A = (A1-A2) / (A1+A2) x 100% 底物A represents the peak area of 3-chloropropiophenone 产物 A represents the peak area of 3-chloropropiophenone

[0066] 2. UPLC detection method of ee value of product ((S)-3-chloropropyl alcohol)

[0067] Column: CHIRALPAK OD-H (5 μm, 250 x 4.6 mm); mobile phase: n-hexane: isopropanol = 98:2 (volume ratio); detection wavelength: 210 nm; flow rate: 1.0 mL / min; injection volume: 10 μL; column temperature: 25 °C; run time: 40 min.

[0068] Preparation method of reference solution: 10 mg of reference substance of S configuration and R configuration product was weighed into a 50 mL volumetric flask, dissolved and diluted with ethanol to the mark, shaken well, and filtered for injection.

[0069] Reference retention time: (S)-3-chloropropyl alcohol 23.359 min, (R)-3-chloropropyl alcohol 27.535 min.

[0070] The calculation formula of ee value is:

[0071] A represents the peak area of 3-chloropropiophenone S A represents the peak area of (S)-3-chloropropyl alcohol R A represents the peak area of (R)-3-chloropropyl alcohol.

[0072] Example 1. Acquisition of enzyme gene and preparation of crude enzyme solution

[0073] 1.1 Acquisition and synthesis of enzyme gene

[0074] The amino acid sequences of alcohol dehydrogenases E1-E4 were retrieved from the NCBI database, and the amino acid sequences of comparative alcohol dehydrogenases E5-E7 were obtained from published literature. Genes encoding alcohol dehydrogenases E1-E7 and glucose dehydrogenase E8 for cofactor regeneration were synthesized by Shengong Bioengineering (Shanghai) Co., Ltd. through nucleotide sequence optimization, and were cloned into the NdeI and HindIII enzyme cutting sites of the pET28a vector, respectively, to obtain various recombinant plasmids. The sources and sequences of all enzymes are shown in Table 1.

[0075] Table 1. Sources and sequences of enzymes

[0076]

[0077]

[0078] 1.2 Transformation of enzyme gene

[0079] The synthesized recombinant plasmids were transformed into Escherichia coli BL21(DE3) competent cells, and then the recombinant Escherichia coli bacterial suspensions were plated on LB agar plates containing 50 μg / mL kanamycin and incubated upside down at 37°C for 16 h until single colonies of recombinant Escherichia coli formed on the LB plates.

[0080] 1.3 Expression of enzyme genes

[0081] Single colonies of recombinant *E. coli* from LB agar plates were inoculated into LB liquid medium containing 50 μg / mL kanamycin and incubated at 37°C for 4 h. Then, they were transferred at a 1% (v / v) inoculation rate to TB liquid medium containing 50 μg / mL kanamycin and incubated at 37°C and 250 rpm until OD500 was reached. 600 At a concentration of approximately 0.6-0.8, add IPTG to a final concentration of 0.1 mM to induce enzyme expression. Induce at 25°C and 250 rpm for 16 h, then centrifuge at 4000 rpm for 20 min, discard the supernatant, and collect the wet cells.

[0082] Add 50 mM pH 7.0 phosphate buffer at a ratio of 1:10 (1 g of bacterial cells to 10 mL of buffer) to suspend the bacterial cells separately. Hybridize using a high-pressure homogenizer (4℃, 600 bar), centrifuge at 12000 rpm for 5 min, discard the precipitate, and obtain the supernatant, which is the crude enzyme solution for each enzyme. Store at -20℃ for later use. For each enzyme, each 1 mL of crude enzyme solution (mother liquor) contains the equivalent of 100 mg of wet bacterial cells.

[0083] Example 2 Screening of alcohol dehydrogenases

[0084] The crude enzyme solution prepared according to the method described in Example 1 was used to construct reaction systems for the preparation of (S)-3-chlorophenylpropanol catalyzed by alcohol dehydrogenases from different sources, as shown in Table 2 below. The concentration added refers to the concentration of the component before the reaction starts after all of it has been added to the reaction system (the total volume of the reaction system is calculated as 10 mL).

[0085] Table 2 Enzyme Catalytic Reaction System

[0086] Component Addition amount Addition concentration 3-chloropropiophenone 1 g 100 mg / mL (593 mM) Dimethyl sulfoxide 0.5 mL 5% (v / v) Glucose 3.2g 320 mg / mL (1,776 mM) 50 mM NAD + ]] 1 mL 5 mM 200 mM PBS buffer (pH 7.0) 2.5 mL 50 mM Glucose dehydrogenase E8 crude enzyme solution 1 mL 10 mg / mL Alcohol dehydrogenase crude enzyme solution 3 mL 30 mg / mL Deionized water Supplemented to 10 mL /

[0087] The reaction equation for enzyme-catalyzed reactions is as follows: Figure 1 As shown, the steps are as follows: Take a reaction vessel and add all components except the crude enzyme solution according to the amounts shown in Table 2. After adjusting the pH to 7.5, add glucose dehydrogenase E8 and crude enzyme solutions of any one of the alcohol dehydrogenases E1-E7 to prepare a 10 mL reaction system. Place the reaction systems containing different alcohol dehydrogenases at 37°C and 220 rpm, and control the pH at 7.5 throughout the reaction process.

[0088] At 3 and 6 hours of reaction, 1 mL of the reaction solution was taken and extracted with 2 mL of ethyl acetate. The upper organic phase was analyzed, and the conversion rates of 3-chlorophenylacetone at 3 and 6 hours of reaction, as well as the ee value of (S)-3-chlorophenylpropanol at 6 hours of reaction, were calculated. The results are shown in Table 3. The conversion rates of E1-E4 at both 3 and 6 hours of reaction were higher than those of the control enzymes E5-E7, and the conversion rate at 6 hours was 100%. The ee values ​​of E1 and E4 at 6 hours of reaction were higher, at 99.88% and 99.85%, respectively, which were higher than those of the control enzymes E5-E7.

[0089] Table 3 Results of alcohol dehydrogenase catalyzed reactions from different sources

[0090] Enzyme number 3h conversion rate 6h conversion rate 6h ee value Configuration E1 62.4％ 100％ 99.88％ S E2 76.4％ 100％ 99.77％ S E3 66.9％ 100％ 99.36％ S E4 64.0％ 100％ 99.85％ S Comparative enzyme E5 15.2％ 35.7％ 99.14％ S Comparative enzyme E6 45.3％ 78.3％ 99.82％ S Comparative enzyme E7 39.4％ 69.3％ 99.57％ S

[0091] sequence:

[0092] SEQ ID NO:1 (Alcohol dehydrogenase E1)

[0093] MTIALDNVVAVVTGAAGGIGRELVKALKAANAVVIATDMAANAEIEGADHYLQHDVTSEADWQAVAALAQEKYGRIDALVNNAGISVVTKFDETPLTEFHRVNAVNVNVDSIIIGTQVMLPLLREGGKVRK GGASVVNFSSVGGLRGAAFNAAYCTSKAAVKMLSKCLGAEFAALGYNVRVNSVHPGGIDTPMLGSIMDKYVELGAVPSRAVAVRGMEARHPIGRMGRPDEMGGGVVYLCSEAASFVTCTEFVMDGGFSQV

[0094] SEQ ID NO:2 (Alcohol dehydrogenase E1)

[0095] ATGACCATTGCCCTGGATAATGTGGTTGCCGTTGTTACCGGCGCAGCCGGCGGTATTGGTCGTGAACTGGTGAAAGCCCTGAAAGCAGCAAATGCCGTGGTTATTGCCACCGATATGGCAGCCAATGCCGAAATTGAAGGTGCAGATCATTATCTGCAGCATGATGTGACCAGCGAAGCAGATTGGCAGGCAGTTGCCGCCCTGGCACAGGAAAAATATGGCCGTATTGATGCACTGGTTAATAATGCCGGCATTAGCGTGGTGACCAAATTTGATGAAACCCCGCTGACCGAATTTCATCGTGTTAATGCCGTTAATGTTGATAGCATTATTATCGGCACCCAGGTTATGCTGCCGCTGCTGCGTGAAGGCGGTAAAGTTCGTAAAGGCGGCGCAAGCGTGGTGAATTTTAGCAGCGTGGGTGGTCTGCGTGGTGCCGCCTTTAATGCCGCATATTGTACCAGCAAAGCCGCCGTTAAAATGCTGAGTAAATGTCTGGGCGCCGAATTTGCCGCCCTGGGTTATAATGTTCGCGTGAATAGCGTTCATCCGGGCGGTATTGATACCCCGATGCTGGGTAGTATTATGGATAAATATGTTGAACTGGGCGCAGTTCCGAGTCGCGCAGTTGCCGTGCGTGGCATGGAAGCCCGTCATCCGATTGGTCGCATGGGCCGCCCGGATGAAATGGGTGGTGGCGTTGTTTATCTGTGTAGCGAAGCCGCCAGTTTTGTTACCTGTACCGAATTTGTGATGGATGGCGGCTTTAGTCAGGTGTAA

[0096] SEQ ID NO:3 (Alcohol dehydrogenase E2)

[0097] MTIALDNVVAVVTGAAGGIGRELVKALKAANATVIATDMAEEADIEGADVYMRHDVTSELDWEALAGLIYKDYGRLDALVNNAGISIVEKFEDTSLEAFHRVNAVNVDSIIIGTQILLPLLIEGGKSRKGGASVVNFSSVGGLRGAPFNAAYCTSKAAVKMLSKCLGAEFAALGYNVRVNSVHPGGVDTEMLGSILDRYVAMGAAPSREVAQAGMEARHPIGRQGRPEEMGGGVVYLCSEAASFVTCTEFVIDGGFSQV

[0098] SEQ ID NO:4 (Alcohol dehydrogenase E2)

[0099] ATGACCATTGCACTGGATAATGTGGTGGCAGTTGTTACCGGCGCAGCAGGCGGTATTGGCCGTGAACTGGTTAAAGCACTGAAAGCCGCAAATGCCACCGTTATTGCAACCGATATGGCCGAAGAAGCAGATATTGAAGGTGCAGATGTGTATATGCGCCATGATGTGACCAGTGAACTGGATTGGGAAGCCCTGGCCGGTCTGATCTATAAAGATTATGGTCGTCTGGATGCCCTGGTGAATAATGCAGGCATTAGCATTGTTGAAAAATTTGAAGATACCAGCCTGGAAGCATTTCATCGTGTTAATGCAGTTAATGTTGATAGTATTATCATCGGTACACAGATTCTGCTGCCGCTGCTGATTGAAGGTGGTAAAAGCCGTAAAGGCGGTGCAAGTGTGGTTAATTTTAGCAGCGTTGGCGGCCTGCGTGGCGCCCCTTTTAATGCCGCCTATTGCACCAGTAAAGCCGCCGTGAAAATGCTGAGCAAATGTCTGGGCGCAGAATTTGCCGCACTGGGTTATAATGTTCGTGTTAATAGTGTTCATCCGGGCGGTGTGGATACCGAAATGCTGGGTAGTATTCTGGATCGTTATGTGGCAATGGGTGCCGCACCGAGTCGCGAAGTGGCACAGGCCGGTATGGAAGCACGCCATCCGATTGGCCGCCAGGGCCGTCCTGAAGAAATGGGTGGCGGTGTGGTGTATCTGTGCAGCGAAGCAGCCAGTTTTGTTACCTGCACCGAATTTGTTATTGATGGCGGCTTTAGCCAGGTGTAA

[0100] SEQ ID NO:5 (Alcohol dehydrogenase E3)

[0101] MTIALDNVVAVVTGAAGGIGRELVKALKAAHATVIATDMAPDAQIEGADLYLQHDVTSEADWEALAGIIHRDYGRLDALVNNAGISIVTKFDETPL SEFHRVNAVNVDSIIIGTQVLLPLLRAGGQARGGGASIVNFSSVGGLR GAAFNAAYCTSKAAVKMLSKCLGAEFAALGYNIRVNSVHPGGINTGMLSSIMDRYVELGAVPSREVAMQGIVANHPIGRMGRPDEMSGGVVYLCSDAASFVTCSEFVMDGGFSQV

[0102] SEQ ID NO:6 (Alcohol dehydrogenase E3)

[0103] ATGACCATTGCACTGGATAATGTTGTTGCCGTGGTGACCGGTGCCGCCGGCGGTATTGGTCGCGAACTGGTGAAAGCACTGAAAGCCGCCCATGCAACCGTGATTGCCACCGATATGGCCCCGGATGCACAGATTGAAGGCGCCGATCTGTATCTGCAGCATGATGTTACCAGTGAAGCCGATTGGGAAGCACTGGCCGGCATTATTCATCGCGATTATGGTCGTCTGGATGCACTGGTTAATAATGCAGGCATTAGCATTGTTACCAAATTTGATGAAACCCCGCTGAGTGAATTTCATCGTGTTAATGCCGTGAATGTGGATAGCATTATTATTGGCACCCAGGTGCTGCTGCCGCTGCTGCGTGCAGGCGGTCAGGCTCGTGGTGGTGGCGCTAGTATTGTTAATTTTAGTAGTGTTGGTGGTCTGCGCGGTGCCGCATTCAATGCCGCATATTGCACCAGCAAAGCCGCCGTGAAAATGCTGAGTAAATGTCTGGGCGCAGAATTTGCCGCACTGGGTTATAATATTCGTGTTAATAGTGTGCATCCGGGTGGCATTAATACCGGTATGCTGAGTAGTATTATGGATCGTTATGTTGAACTGGGTGCCGTGCCGAGTCGCGAAGTGGCCATGCAGGGTATTGTTGCCAATCATCCGATTGGCCGCATGGGTCGCCCGGATGAAATGAGCGGTGGCGTTGTGTATCTGTGTAGCGATGCCGCCAGTTTTGTTACCTGCAGTGAATTTGTTATGGATGGCGGTTTTAGTCAGGTGTAA

[0104] SEQ ID NO:7 (Alcohol dehydrogenase E4)

[0105] MTIAVEGCIALVTGATGGIGREIVKAMKAAGATVIATDLAYTCDDTDADHYFRHDVTDQDDWDDIGAFVSETYGRLDALVNNAGISIVTRFEDTPLAEWHRVNSVNVDSIVIGTQSLLSLLQAGGKARKGGASVVNFSSVGGLRGAAFNAVYCTSKAAVRMLTKCMGAEFAALGYNIRVNSVHPGGIETPMLGG IMDRYVELGAVPSREVAEQGTIARHP IGRLGRPDEMAGGVVFLCSEASSF MTCDELVMDGGFSQV

[0106] SEQ ID NO:8 (Alcohol dehydrogenase E4)

[0107] ATGACCATTGCAGTTGAAGGCTGCATTGCACTGGTTACCGGCGCCACCGGCGGTATTGGTCGTGAAATTGTTAAAGCCATGAAAGCAGCAGGTGCCACCGTTATTGCAACCGATCTGGCCTATACCTGTGATGATACCGATGCCGATCATTATTTTCGTCATGATGTTACCGATCAGGATGATTGGGATGATATTGGTGCATTTGTTAGTGAAACCTATGGTCGCCTGGATGCACTGGTGAATAATGCAGGCATTAGCATTGTGACCCGTTTTGAAGATACCCCGCTGGCCGAATGGCATCGCGTTAATAGCGTTAATGTGGATAGCATTGTGATTGGTACACAGAGCCTGCTGAGTCTGCTGCAGGCAGGTGGTAAAGCCCGCAAAGGCGGCGCCAGCGTGGTTAATTTTAGCAGTGTTGGTGGCCTGCGCGGCGCCGCTTTTAATGCCGTTTATTGTACCAGCAAAGCAGCCGTGCGTATGCTGACCAAATGTATGGGTGCCGAATTTGCAGCACTGGGCTATAATATTCGTGTTAATAGTGTTCATCCGGGCGGCATTGAAACCCCGATGCTGGGTGGTATTATGGATCGTTATGTTGAACTGGGCGCCGTGCCGAGCCGTGAAGTTGCAGAACAGGGTACAATTGCCCGCCATCCGATTGGTCGTCTGGGCCGCCCGGATGAAATGGCCGGCGGTGTGGTGTTTCTGTGTAGTGAAGCCAGCAGCTTTATGACCTGTGATGAACTGGTTATGGATGGTGGCTTTAGCCAGGTGTAA

[0108] SEQ ID NO:9 (Comparative alcohol dehydrogenase E5)

[0109] MSVFVSGANGFIAQHIVDLLLKEDYKVIGSARSQEKAENLTEAFGNNPKFSMEVVPDISKLDAFDHVFQKHGKDIKIVLHTASPFCFDITDSERDLLIPAVNGVKGILHSIKKYAADSVERVVLTSSYAAVFDMAKENDKSLTFNEESWNPATWESCQSDPVNAYCGSKKFAEKAAWEFLEENRDSVKFELTAVNPVYVFGPQMFDKDVKKHLNTSCELVNSLMHLSPEDKIPELFGGYIDVRDVAKAHLVAFQKRETIGQRLIVSEARFTMQDVLDILNEDFPVLKGNIPVGKPGSGATHNTLGATLDNKKSKKLLGFKFRNLKETIDDTASQILKFEGRI

[0110] SEQ ID NO:10 (Comparative alcohol dehydrogenase E5)

[0111]

[0112] SEQ ID NO:11 (Comparative alcohol dehydrogenase E6)

[0113] MTIALNNVVAVVTGAAGGIGRELVKAMKAANAIVIATDMAPSADVEGADHYLQHDVTSEAGWKAVAALAQEKYGRVDALVHNAGISIVTKFEDTP LSDFHRVNTVNVDSIIIGTQVLLPLLKEGGKARAGGASVVNFSSVGGLRG AAFNAAYCTSKAAVKMLSKCLGAEFAALGYNIRVNSVHPGGIDTPMLGSIMDKYVELGAAPSREVAQAAMEMRHPIGRMGRPAEMGGGVVYLCSDAASFVTCTEFVMDGGFSQV

[0114] SEQ ID NO:12 (Comparative alcohol dehydrogenase E6)

[0115] ATGACCATCGCCCTGAATAATGTTGTGGCCGTTGTTACCGGCGCCGCAGGTGGTATTGGCAGAGAACTGGTGAAAGCAATGAAAGCAGCCAATGCAATTGTGATTGCCACCGATATGGCACCGAGTGCCGATGTTGAAGGTGCCGATCATTATCTGCAGCATGATGTGACCAGTGAAGCCGGTTGGAAAGCCGTTGCCGCCTTAGCTCAGGAAAAATATGGTCGTGTTGATGCACTGGTGCATAATGCAGGCATTAGCATTGTGACCAAATTTGAAGATACCCCGCTGAGTGATTTTCATCGCGTTAATACCGTTAACGTTGATAGCATTATCATCGGTACCCAGGTGCTGCTGCCGCTGTTAAAAGAAGGCGGTAAAGCCCGTGCCGGTGGCGCAAGCGTGGTGAACTTTAGCAGCGTGGGCGGCCTGCGTGGTGCAGCATTCAATGCAGCATATTGTACCAGTAAAGCAGCAGTTAAAATGCTGAGTAAGTGTCTGGGCGCAGAATTTGCAGCCCTGGGTTATAATATTCGTGTTAATAGCGTGCACCCGGGTGGCATTGATACCCCTATGCTGGGTAGCATTATGGATAAATATGTGGAACTGGGCGCAGCCCCGAGCAGAGAGGTGGCACAGGCAGCAATGGAAATGCGCCATCCGATTGGCCGTATGGGTCGCCCTGCTGAAATGGGTGGCGGTGTTGTTTATCTGTGCAGTGATGCAGCCAGTTTTGTTACCTGTACCGAATTTGTGATGGATGGCGGCTTTAGCCAGGTGTAA

[0116] SEQ ID NO:13 (Comparative alcohol dehydrogenase E7)

[0117] MTIALNNVVAVVTGAAGGIGRELVKAMKAANAIVIATDMAPSADVEGADHYLQHDVTSEAGWKAVAALAQEKYGRVDALVHNAGISIVTKFEDTPLSDFHRVNTVNVDSIIIGTQVLLPLLKEGGKARAGGASVVNFSSVAGLRGAAFNAAYCTSKAAVKMLSKCLGAEFAALGYNIRVNSVHPGGIDTPMLGS LMDKYVELGAAPSREVAQAAMEMRHP IGRMGRPAEMGGGVVYLCSDA ASFVTCTEFVMDGGFSQV

[0118] SEQ ID NO:14 (Comparative alcohol dehydrogenase E7)

[0119] ATGACCATCGCTCTGAACAACGTTGTTGCTGTTGTTACCGGTGCTGCTGGTGGTATCGGTCGTGAACTGGTTAAAGCTATGAAAGCTGCTAACGCTATCGTTATCGCTACCGACATGGCTCCGTCTGCTGACGTTGAAGGTGCTGACCACTACCTGCAGCACGACGTTACCTCTGAAGCTGGTTGGAAAGCTGTTGCTGCTCTGGCTCAGGAAAAATACGGTCGTGTTGACGCTCTGGTTCACAACGCTGGTATCTCTATCGTTACCAAATTCGAAGACACCCCGCTGTCTGACTTCCACCGTGTTAACACCGTTAACGTTGACTCTATCATCATCGGTACCCAGGTTCTGCTGCCGCTGCTGAAAGAAGGTGGTAAAGCTCGTGCTGGTGGTGCTTCTGTTGTTAACTTCTCTTCTGTTGCTGGTCTGCGTGGTGCTGCTTTCAACGCTGCTTACTGCACCTCTAAAGCTGCTGTTAAAATGCTGTCTAAATGCCTGGGTGCTGAATTCGCTGCTCTGGGTTACAACATCCGTGTTAACTCTGTTCACCCGGGTGGTATCGACACCCCGATGCTGGGTTCTCTGATGGACAAATACGTTGAACTGGGTGCTGCTCCGTCTCGTGAAGTTGCTCAGGCTGCTATGGAAATGCGTCACCCGATCGGTCGTATGGGTCGTCCGGCTGAAATGGGTGGTGGTGTTGTTTACCTGTGCTCTGACGCTGCTTCTTTCGTTACCTGCACCGAATTCGTTATGGACGGTGGTTTCTCTCAGGTTTAA

[0120] SEQ ID NO:15 (Glucose dehydrogenase E8)

[0121] MYPDLKGKVVAITGAASGLGKAMAIRFGKEQAKVVINYYSNKQDPNEVKEEVIKAGGEAVVVQGDVTKEEDVKNIVQTAIKEFGTLDIMINNAGLENPVPSHEMPLKDWDKVIGTNLTGAFLGSREAIKYFVENDIKGNVINMSSVHEVIPWPLFVHYAASKGGIKLMTETLALEYAPKGIRVNNIGPGAINTPINAEKFADPKQKADVESMIPMGYIGEPEEIAAVAAWLASKEASYVTGITLFADGGMTQYPSFQAGRG

[0122] SEQ ID NO:16 (Glucose dehydrogenase E8)

[0123] ATGTATCCGGATTTAAAAGGAAAAGTCGTCGCTATTACAGGAGCTGCTTCAGGGCTCGGAAAGGCGATGGCCATTCGCTTCGGCAAGGAGCAGGCAAAAGTGGTTATCAACTATTATAGTAATAAACAAGATCCGAACGAGGTAAAAGAAGAGGTCATCAAGGCGGGCGGTGAAGCTGTTGTCGTCCAAGGAGATG TCACGAAAGAGGAAGATGTAAAAAATATCGTGCAAACGGCAATTAAGGAGTTCGGCACACTCGATATTATGATTAATAATGCCGGTCTTGAAAATCCTGTGCCATCTCACGAAATGCCGCTCAAGGATTGGGATAAAGTCATCGGCACGAACTTAACGGGTGCCTTTTTAGGAAGCCGTGAAGCGATTAAATATTTC GTAGAAAACGATATCAAGGGAAATGTCATTAACATGTCCAGTGTGCACGAAGTGATTCCTTGGCCGTTATTTGTCCACTATGCGGCAAGTAAAGGCGGGATAAAGCTGATGACAGAAACATTAGCGTTGGAATACGCGCCGAAGGGCATTCGCGTCAATAATAATTGGGCCAGGTGCGATCAACACGCCAATCAATG CTGAAAAATTCGCTGACCCTAAACAGAAAGCTGATGTAGAAAGCATGATTCCAATGGGATATATCGGCGAACCGGAGGAGATCGCCGCAGTAGCAGCCTGGCTTGCTTCGAAGGAAGCCAGCTACGTCACAGGCATCACGTTATTCGCGGACGGCGGTATGACACAATATCCTTCATTCCAGGCAGGCCGCGTTAA

[0124] In the above embodiments, the concentration of each component in the reaction system refers to the concentration of that component before the reaction begins, after all of it has been added to the reaction system, i.e., the added concentration. The added concentration is calculated based on the total volume of the reaction system when it is fully prepared.

Claims

1. A process for the preparation of (S)-3-chlorophenylalanol, characterized in that, The method comprises: in a reaction system comprising an alcohol dehydrogenase, 3-chloropropiophenone and a reduced coenzyme, using the alcohol dehydrogenase to catalyze 3-chloropropiophenone, thereby preparing (S)-3-chloropropyl alcohol; the amino acid sequence of the alcohol dehydrogenase is selected from one or more of the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO:

7.

2. The method of claim 1, wherein, The method further comprises: in the presence of a hydrogen donor dehydrogenase, a hydrogen donor and an oxidized coenzyme, using the hydrogen donor dehydrogenase and the hydrogen donor to reduce the oxidized coenzyme to obtain the reduced coenzyme.

3. The method of claim 2, wherein, The oxidized coenzyme and the reduced coenzyme are selected from one or more of the following groups: (1) NADP + and NADPH; (2) NAD + and NADH; And / or, the hydrogen donor and the hydrogen donor dehydrogenase are selected from one or more of the following groups: (1) glucose and glucose dehydrogenase; (2) isopropyl alcohol and hydrogen donor alcohol dehydrogenase; (3) formic acid or formate and formic acid dehydrogenase.

4. The method of claim 3, wherein, The reaction system further comprises one or more of a cosolvent and a buffer; Preferably, the cosolvent is selected from one or more of dimethyl sulfoxide, methanol, ethanol, isopropyl alcohol, acetonitrile, toluene, acetone, preferably dimethyl sulfoxide; and / or the buffer is a PBS buffer, a Tris buffer or a HEPES buffer, preferably a PBS buffer.

5. The method of claim 3 or 4, wherein, The amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO: 15; and / or the added mass concentration of 3-chloropropiophenone is 50-200 mg / mL.

6. The method of any one of claims 3-5, wherein, NAD + with 3-chloropropiophenone in a ratio of 1 : (50-200) molar. And / or, the ratio of the added mass of the alcohol dehydrogenase to the added mass of 3-chloropropiophenone, calculated based on the mass of the wet bacteria used, is (0.5-5):10; And / or, the ratio of the added mass of the glucose dehydrogenase to the added mass of 3-chloropropiophenone, calculated based on the mass of the wet bacteria used, is (0.5-2):10; And / or, the ratio of the added molar amount of glucose to the added molar amount of 3-chloropropiophenone is (1-5):1; And / or, the added volume of dimethyl sulfoxide accounts for 1%-30% of the total volume of the reaction system.

7. The method of any one of claims 1-6, wherein, The reaction conditions of the method comprise one or more of the following: the reaction temperature is 25-40°C; the pH of the reaction system is 6.0-9.

0.

8. A biomaterial, characterized by, The biological material comprises one or more nucleic acids with a nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14; The biological material is a nucleic acid, a recombinant expression vector or a transformant. The recombinant expression vector comprises the nucleic acid; The transformant comprises the nucleic acid or the recombinant expression vector.

9. Use of an alcohol dehydrogenase for the preparation of (S)-3-chlorophenylpropanol, characterized in that The amino acid sequence of the alcohol dehydrogenase is selected from one or more of the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO:

7.

10. A composition characterized in that, The composition comprises an alcohol dehydrogenase, the amino acid sequence of which is selected from two or more of the amino acid sequences shown as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7; or, a reaction system as defined in any of the methods of claims 1-7; or, a mixture obtained by reaction from a reaction system as defined in any of the methods of claims 1-7.

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