Method for preparing (S)-3-methylamino-1-(2-thienyl)-1-propanol through asymmetric reduction of alcohol dehydrogenase

Abstract

The invention discloses a method for preparing a duloxetine intermediate (S)-3-methylamino-1-(2-thienyl)-1-propyl alcohol by virtue of asymmetric reduction of alcohol dehydrogenase. The alcohol dehydrogenase is derived from Kluyveromyces marxianus, the amino acid sequence of the alcohol dehydrogenase is as shown in SEQ ID NO. 1, and the nucleotide sequence for coding the alcohol dehydrogenase is as shown in SEQ ID NO. 2. The alcohol dehydrogenase can catalyze 3-methylamino-1-(2-thienyl)-1-acetone to generate a duloxetine intermediate (S)-3-methylamino-1-(2-thienyl)-1-propanol through asymmetric reduction, in an optimized enzyme catalysis system, the concentration of a reaction substrate is 30 g / L, the reaction conversion rate reaches 99.5%, the chiral purity of a product is 99.7% ee, and the alcohol dehydrogenase has good industrial application value. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the field of biocatalytic methods and technical applications, and relates to the application of an alcohol dehydrogenase in the preparation of a duloxetine intermediate, and in particular to the application of an alcohol dehydrogenase in the preparation of (S)-3-methylamino-1-(2-thienyl)-1-propanol. Background Art

[0002] Duloxetine, chemical name (S)-N-methyl-3-(1-naphthyloxy)-3-(2-thienyl)-1-propylamine, trade name Cymbalta, is a dual inhibitor of 5-hydroxytryptamine and norepinephrine reuptake developed by Eli Lilly and Company of the United States. Its hydrochloride is clinically used to treat depression, stress urinary incontinence, chronic pain associated with depression, and diabetic peripheral neuropathy pain. Of the two isomers of duloxetine, only the (S)-type has the above-mentioned pharmacological activity, and its structural formula is as follows:

[0003]

[0004] (S)-3-methylamino-1-(2-thienyl)-1-propanol is a key intermediate in the synthesis of duloxetine. Currently, there are two main methods for the preparation of (S)-3-methylamino-1-(2-thienyl)-1-propanol: asymmetric synthesis and chemical resolution.

[0005] Chemical asymmetric synthesis methods, such as Angew Chem Int Ed. 2005, 44: 1687-1689, Tetrahedron Lett. 1990, 31: 7101-7104, WO2010003942, EP1510517, etc., use chiral ruthenium or rhodium metal catalysts to catalyze reduction reactions to obtain (S)-3-methylamino-1-(2-thienyl)-1-propanol. Since ruthenium or rhodium metal catalysts are required for catalysis and the reaction requires pressurized hydrogen, its economic efficiency, safety and environmental friendliness cannot meet the production needs.

[0006] Chemical resolution methods, such as the chemical resolution methods described in Tetrahedron Asymm. 2003, 14: 1631-1636 and WO2004005307, which use (S)-mandelic acid and (-)-2,3:4,6-di-O-isopropylidene-2-keto-L-gulonic acid as resolution agents, respectively, cannot be used as the main production method for (S)-3-methylamino-1-(2-thienyl)-1-propanol due to low yield and poor economic efficiency.

[0007] Compared with chemical synthesis, enzyme-catalyzed synthesis has high chemical, regional and stereoselectivity, mild reaction conditions, easy post-treatment and low energy consumption. It is an environmentally friendly synthesis method and also a major development trend in chemical production and related fields today.

[0008] At present, there are no literature reports on the enzymatic resolution and synthesis of (S)-3-methylamino-1-(2-thienyl)-1-propanol, but there are many reports on the synthesis of (S)-3-methylamino-1-(2-thienyl)-1-propanol by asymmetric reduction catalysis of alcohol dehydrogenase (ketoreductase, carbonyl reductase). For example, CN110229796 discloses a ketoreductase derived from Rhodococcus ruber, with a feed concentration of 5-10 g / L, a conversion rate of 95-99%, and a product ee value of >99%; CN105039361 discloses a ketoreductase derived from Rhodosporidium toruloides carbonyl reductase, feed concentration 50g / L, conversion rate 75%, product ee value>99%; CN103740738 discloses a carbonyl reductase derived from Chryseobacterium sp.CA49, feed concentration 20g / L, conversion rate 99%, product ee value>99%; US9228213 discloses a ketoreductase derived from Lactobacillus kefiri and its mutants, feed concentration 146g / L, conversion rate>99%, ee value>99%. The enzyme catalytic efficiency reported in the first three of the above reports is not ideal. The enzyme disclosed in US9228213 has a higher catalytic efficiency, but the imported enzyme is expensive and has a high cost of use, and the reaction process requires negative pressure control, and the addition of co-substrate isopropanol and coenzyme NADP. + It needs to react completely with fresh enzyme solution, the process control is difficult and has high requirements for bioreactor equipment.

[0009] In view of the important application value of (S)-3-methylamino-1-(2-thienyl)-1-propanol, it is necessary to break through the existing patent restrictions and technical deficiencies, explore and screen alcohol dehydrogenases with good catalytic performance, and construct an efficient and easy-to-control catalytic process, which is of great significance to improving the industrialization of duloxetine enzymatic process.

[0010] Alcohol dehydrogenase (ADH), also known as ketoreductase (KRED) or carbonyl reductase (CRED), is an efficient and selective biocatalyst that can reduce carbonyl compounds to chiral alcohols in a stereoselective manner. (R)-specific alcohol dehydrogenases have different properties from (S)-specific alcohol dehydrogenases, and these catalysts are increasingly used in the industrial synthesis of optically active alcohols. The alcohol dehydrogenase screened from Kluyveromyces marxianus can be used to catalyze the reaction of 3-methylamino-1-(2-thienyl)-1-propanone to produce duloxetine intermediate (S)-3-methylamino-1-(2-thienyl)-1-propanol. Summary of the invention

[0011] The purpose of the present invention is to provide a method for preparing (S)-3-methylamino-1-(2-thienyl)-1-propanol from 3-methylamino-1-(2-thienyl)-1-propanone by catalyzing alcohol dehydrogenase. The alcohol dehydrogenase is derived from Kluyveromyces marxianus. The method is simple to operate, green and environmentally friendly, and has good industrial application prospects.

[0012] In order to achieve the above object, the present invention adopts the following technical solutions:

[0013] In a first aspect, the present invention provides a method for preparing (S)-3-methylamino-1-(2-thienyl)-1-propanol, using alcohol dehydrogenase to catalyze the reaction of 3-methylamino-1-(2-thienyl)-1-acetone to generate a duloxetine intermediate (S)-3-methylamino-1-(2-thienyl)-1-propanol, and the reaction formula is as follows:

[0014]

[0015] In the second aspect, the enzyme-catalyzed preparation method described in the present invention is specifically, in the reaction medium, adding coenzyme, cosubstrate, coenzyme regeneration enzyme, 3-methylamino-1-(2-thienyl)-1-propanone, and then adding alcohol dehydrogenase to react to generate duloxetine intermediate (S)-3-methylamino-1-(2-thienyl)-1-propanol.

[0016] In a third aspect, in one embodiment of the present invention, the method specifically comprises: reacting the substrate 3-methylamino-1-(2-thienyl)-1-propanone, alcohol dehydrogenase, NADP + , GDH, glucose and buffer are put into the reaction container for catalytic reaction. The substrate concentration is 30g / L, the buffer is 0.1M pH8.5 triethanolamine buffer, the reaction temperature is 40°C, the co-substrate glucose is 60g / L, the concentration of glucose dehydrogenase (based on the amount of bacteria) is 3g / L, the NADP +The concentration of the mixture was 0.75 g / L, and the alcohol dehydrogenase was added at a final concentration of 30 g / L (based on the amount of bacteria) for shaking reaction.

[0017] The alcohol dehydrogenase is derived from Kluyveromyces marxianus.

[0018] The amino acid sequence of the alcohol dehydrogenase is shown in SEQ ID NO.1.

[0019] The alcohol dehydrogenase is prepared by the following method:

[0020] (1) synthesizing a nucleotide sequence encoding the alcohol dehydrogenase and constructing an engineered bacterium;

[0021] (2) Induce and culture the engineered bacteria, collect the bacteria by centrifugation, and then prepare the required enzyme preparation.

[0022] Wherein, the nucleotide sequence is shown as SEQ ID NO.2.

[0023] The reaction medium is selected from phosphate buffer, triethanolamine buffer, Tris-HCl buffer or boric acid-borax buffer.

[0024] The reaction pH is 7.0-9.0.

[0025] The reaction temperature is 25-60°C.

[0026] The substrate concentration is 10-40 g / L.

[0027] The alcohol dehydrogenase (calculated by bacterial cell amount) is added in an amount of 20-40 g / L.

[0028] The coenzyme is NAD(P) + , the addition amount is 0.5-1g / L.

[0029] The cosubstrate is glucose with a concentration of 40-80 g / L.

[0030] The coenzyme regeneration enzyme is glucose dehydrogenase, and the addition amount of glucose dehydrogenase (calculated by bacterial cell amount) is 2-5 g / L.

[0031] In a fourth aspect, the present invention provides a method for producing the alcohol dehydrogenase, which comprises using pET21a as a vector and E. coli BL21 as a host to express the alcohol dehydrogenase gene, then centrifuging the induced expression fermentation broth to remove the supernatant, collecting the bacteria, and then preparing the desired enzyme preparation such as enzyme solution.

[0032] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0033] (1) The present invention constructs a genetically engineered Escherichia coli containing the alcohol dehydrogenase KmADH gene from Kluyveromyces marxianus, obtains the alcohol dehydrogenase by induction culture, and applies it to catalyze the asymmetric reduction of 3-methylamino-1-(2-thienyl)-1-propanone to prepare (S)-3-methylamino-1-(2-thienyl)-1-propanol. Under optimized enzyme reaction conditions, the substrate concentration is 30 g / L, the reaction conversion rate reaches 99.5%, and the product ee value is 99.7%;

[0034] (2) Alcohol dehydrogenase KmADH from Kluyveromyces marxianus catalyzes the 3-methylamino

[0035] -1-(2-thienyl)-1-propanone to produce (S)-3-methylamino-1-(2-thienyl)-1-propanol has not been reported in the literature so far, and the enzyme has relatively high enzyme activity and selectivity;

[0036] The invention has the advantages of simple reaction operation, mild reaction conditions, good stereoselectivity, high product optical purity, etc., and has good industrial application potential. DETAILED DESCRIPTION

[0037] The present invention is further defined in the following examples. It should be understood that these examples, while indicating preferred embodiments of the present invention, are given by way of illustration only and do not limit the present invention as claimed.

[0038] Technical solution: The present invention first screens out an effective alcohol dehydrogenase KmADH from Kluyveromyces marxianus, constructs enzyme-producing recombinant genetically engineered Escherichia coli, and prepares alcohol dehydrogenase enzyme solution from the recombinant Escherichia coli expressing alcohol dehydrogenase. Under optimized conditions, the catalytic substrate is 3-methylamino-1-(2-thienyl)-1-propanone, which is asymmetric reduced to generate (S)-3-methylamino-1-(2-thienyl)-1-propanol.

[0039] Experimental materials and reagents: 1. All the materials used in the present invention are common commercial products and can be purchased on the market. 2. Culture medium: LB liquid culture medium: yeast powder 5g / L, peptone 10g / L, sodium chloride 10g / L, pH 6.8-7.0; fermentation medium: sucrose 5g / L, peptone 15g / L, sodium dihydrogen phosphate 1g / L, iron ion 1mM, pH 7.0.

[0040] Detection and analysis methods, 1) Substrate and product HPLC detection method: chromatographic column ZORBAX Eclipse XDB-C18, 4.6mm×150mm×5μm, column temperature 30℃, flow rate 1mL / min, detection wavelength 246nm, mobile phase A: 0.1% (V / V) phosphoric acid; mobile phase B: acetonitrile, mobile phase gradient see Table 1.

[0041] Table 1. HPLC analysis mobile phase gradient

[0042] T(min) A(％) B(％) 0 95 5 3 95 5 10 40 60 13 40 60 13.01 95 5 20 95 5

[0043] 2) Product chiral HPLC detection conditions: CHIRALPAK ○R IC column, 4.6 mm × 250 mm × 5 μm, UV detection wavelength 241 nm, mobile phase of n-hexane: isopropanol: ethanolamine (90:10:0.1, V / V), flow rate 1.0 mL / min.

[0044] Example 1: Construction of recombinant engineering bacteria

[0045] 1. Construction of alcohol dehydrogenase KmADH recombinant engineering bacteria

[0046] The gene of alcohol dehydrogenase KmADH from Kluyveromyces marxianus was synthesized, and NdeI and XhoI sites were introduced at both ends of the gene, respectively, and cloned into the pET21a vector to obtain the recombinant vector pET21a-KmADH; its amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence encoding the amino acid is shown in SEQ ID NO.2. The constructed recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cells by chemical transformation method, coated on LB plates containing 50 μg / mL ampicillin resistance, cultured at 37°C for 8 to 12 hours, and single clone cells were picked to obtain the recombinant genetic engineering bacteria BL21 (DE3) / pET21a-KmADH that can be induced to express alcohol dehydrogenase.

[0047] 2. Construction of Glucose Dehydrogenase BsGDH Engineering Bacteria

[0048] Bacillus subtilis glucose dehydrogenase BsGDH (NCBI ID: NC_000964.3) was obtained by total gene synthesis and ligated to the pET21a vector by double restriction digestion to obtain the recombinant plasmid pET21a-BsGDH, whose amino acid sequence is shown in SEQ ID NO.3, and the nucleotide sequence encoding the amino acid is shown in SEQ ID NO.4. The recombinant plasmid was introduced into Escherichia coli BL21 (DE3) to obtain recombinant glucose dehydrogenase Escherichia coli BL21 (DE3) / pET21a-BsGDH.

[0049] Example 2: Induced expression of recombinant engineered bacteria and enzyme solution preparation

[0050] Seed liquid culture: The engineered bacteria of alcohol dehydrogenase KmADH and glucose dehydrogenase BsGDH were inoculated into LB liquid culture medium containing 50 μg / mL ampicillin, and cultured at 37° C. for 12 to 16 h.

[0051] Induced expression: The recombinant engineered bacteria seed solution was inoculated into a fresh fermentation medium containing a final concentration of 50 μg / mL ampicillin at 1% (v / v) and cultured at 37°C, 200 rpm for 2-3 h. 600 When the pH value was between 0.6 and 0.8, the inducer IPTG was added, the final concentration of IPTG was 0.2 mM, and the culture was carried out at 25°C for 20 h.

[0052] Preparation of enzyme solution: After the induction culture is completed, the fermentation broth is centrifuged at high speed to obtain the whole cell body of the genetically engineered bacteria. The body is suspended in 0.1M triethanolamine buffer at pH 8.5 (the body concentration is 200g / L), and then the bacterial suspension is broken by ultrasonic or homogenizer to obtain the enzyme solution.

[0053] Example 3: Enzyme activity and selectivity verification of alcohol dehydrogenase KmADH

[0054] The alcohol dehydrogenase KmADH and glucose dehydrogenase BsGDH enzyme solutions prepared in Example 2 were used as catalysts, and 3-methylamino-1-(2-thienyl)-1-propanone was used as a substrate.

[0055] The composition of the catalytic system and the reaction conditions are as follows: 1 mL of the reaction system contains 0.01 g of 3-methylamino-1-(2-thienyl)-1-propanone, 0.02 g of glucose, and 0.5 mg of NADP. + , 0.1mL alcohol dehydrogenase KmADH enzyme solution (bacterial volume 0.02g), 0.02mL glucose dehydrogenase BsGDH enzyme solution (bacterial volume 0.004g) and triethanolamine buffer (0.1M, pH 8.5). The system was placed on a constant temperature mixer at 40°C, 1000rpm. After reacting for 6h, the conversion rate of the substrate 3-methylamino-1-(2-thienyl)-1-propanone was detected to be 99.5%, and the product ee value was 99.7%.

[0056] Example 4 Alcohol dehydrogenase KmADH catalyzed reaction in different reaction media

[0057] The reaction system in Example 3 was used, with different reaction media and the same other conditions, and the reaction was carried out for 1 hour; the relative enzyme activity of alcohol dehydrogenase KmADH in different reaction media was determined, and the reaction media used were 0.1M pH 8.0 phosphate buffer, triethanolamine buffer, Tris-HCl buffer and boric acid-borax buffer. The reaction results are shown in Table 2: When triethanolamine buffer was used as the reaction medium, the activity of alcohol dehydrogenase KmADH in catalyzing 3-methylamino-1-(2-thienyl)-1-propanone was the highest.

[0058] Table 2 Results of alcohol dehydrogenase KmADH catalytic reaction for 1 h in different reaction media

[0059]

[0060]

[0061] Example 5: Optimal pH value of alcohol dehydrogenase KmADH

[0062] The reaction system in Example 3 was used, with different reaction pH values, but the other conditions were the same, and the reaction was carried out for 1 hour; the relative enzyme activity of alcohol dehydrogenase KmADH at different pH gradients (7.0-9.0) was determined, and the buffer used was 0.1 M triethanolamine buffer. The reaction results are shown in Table 3: When the pH was 8.5, the relative activity of alcohol dehydrogenase KmADH in catalyzing 3-methylamino-1-(2-thienyl)-1-propanone was the highest.

[0063] Table 3 Results of alcohol dehydrogenase KmADH catalytic reaction for 1 h at different reaction pH

[0064] Reaction pH Relative enzyme activity (%) Product ee (%) 7 36.4 99.7 7.5 56 99.9 8 93.8 99.6 8.5 100 99.7 9 33.8 99.8

[0065] Example 6: Optimum temperature of alcohol dehydrogenase KmADH

[0066] The reaction system in Example 3 was used, with different reaction temperatures and other conditions being the same, and the reaction was carried out for 1 h. The relative enzyme activity of alcohol dehydrogenase KmADH was determined at different temperature gradients (25-60°C). The reaction results are shown in Table 4: when the reaction temperature was 40°C, the relative activity of alcohol dehydrogenase KmADH in catalyzing 3-methylamino-1-(2-thienyl)-1-propanone was the highest.

[0067] Table 4 Results of alcohol dehydrogenase KmADH catalytic reaction for 1 h at different reaction temperatures

[0068]

[0069]

[0070] Example 7 Alcohol dehydrogenase KmADH catalytic reaction at different substrate concentrations

[0071] The reaction system in Example 3 was used, with different substrate concentrations, 1h reaction, and the others were the same; the relative enzyme activity of alcohol dehydrogenase KmADH at different substrate concentrations was determined, and the substrate concentrations were 10-40 g / L. The reaction results are shown in Table 5: When the substrate concentration was 30 g / L, the reduction effect of alcohol dehydrogenase KmADH was the best.

[0072] Table 5 Results of alcohol dehydrogenase KmADH catalytic reaction for 1 h at different substrate concentrations

[0073] Substrate concentration (g / L) Relative enzyme activity (%) Product ee (%) 10 100 99.6 20 100 99.9 30 100 99.7 40 90.4 99.8

[0074] Example 8 Catalytic reaction under different alcohol dehydrogenase KmADH addition amounts

[0075] The reaction system in Example 3 was used, with different amounts of alcohol dehydrogenase added, and the other conditions were the same, and the reaction was carried out for 1 hour; the ability to catalyze the substrate at different alcohol dehydrogenase concentrations was measured, and the amount of alcohol dehydrogenase added (based on the amount of bacteria) was 20-40 g / L. The reaction results are shown in Table 6: When the amount of alcohol dehydrogenase added was 30 g / L (based on the amount of bacteria), the effect of catalyzing 3-methylamino-1-(2-thienyl)-1-propanone was the best.

[0076] Table 6 Results of 1h catalytic reaction under different alcohol dehydrogenase KmADH addition amounts

[0077] Amount of alcohol dehydrogenase added (g / L based on bacterial volume) Relative enzyme activity (%) Product ee (%) 20 89.1 99.5 30 100 99.7 40 99.4 99.1

[0078] Example 9 Alcohol dehydrogenase KmADH catalytic reaction under different coenzymes and their added amounts

[0079] The reaction system in Example 3 was used, with different coenzymes and addition amounts, and the other aspects were the same, and the reaction was carried out for 1 h. The relative enzyme activity of alcohol dehydrogenase KmADH was determined under different coenzymes and different coenzyme addition amounts. The coenzymes used were NAD + and NADP + The addition amount is 0.5-1g / L. The reaction results are shown in Table 7: + As a coenzyme, the relative activity of alcohol dehydrogenase KmADH catalyzing 3-methylamino-1-(2-thienyl)-1-propanone was the highest when the addition amount was 0.75g / L.

[0080] Table 7 Results of 1 h catalytic reaction of alcohol dehydrogenase KmADH under different coenzymes and their added amounts

[0081]

[0082] Example 10 Alcohol dehydrogenase KmADH catalytic reaction under different glucose addition amounts

[0083] The reaction system in Example 3 was used, with different amounts of glucose added, and the other parts were the same, and the reaction was carried out for 1 hour; the relative enzyme activity of alcohol dehydrogenase KmADH at different amounts of glucose added was measured, and the addition amounts were 40-80 g / L, respectively. The reaction results are shown in Table 8: When the amount of glucose added was 60 g / L, the relative activity of alcohol dehydrogenase KmADH catalyzing 3-methylamino-1-(2-thienyl)-1-propanone was the highest.

[0084] Table 8 Results of alcohol dehydrogenase KmADH catalytic reaction for 1 h at different glucose addition amounts

[0085] Glucose addition amount (g / L) Relative enzyme activity (%) Product ee (%) 40 93.6 99.5 50 97.1 99.2 60 100 99.7 70 99.8 99.3 80 99.9 99.4

[0086] Example 11 Alcohol dehydrogenase KmADH catalytic reaction at different GDH addition amounts

[0087] The reaction system in Example 3 was used, with different amounts of GDH added, and the others were the same, and the reaction was carried out for 1 hour; the relative enzyme activity of alcohol dehydrogenase KmADH was determined at different amounts of glucose dehydrogenase GDH added (based on the amount of bacteria), and the addition amounts were 2-5 g / L. The reaction results are shown in Table 9: When the addition amount of glucose dehydrogenase (based on the amount of bacteria) was 3 g / L, the relative activity of alcohol dehydrogenase catalyzing 3-methylamino-1-(2-thienyl)-1-acetone was the highest.

[0088] Table 9 Results of alcohol dehydrogenase KmADH catalytic reaction for 1 h at different GDH addition amounts

[0089]

[0090]

[0091] Example 12: Asymmetric reduction of 3-methylamino-1-(2-thienyl)-1-propanone by alcohol dehydrogenase KmADH to generate duloxetine intermediate (S)-3-methylamino-1-(2-thienyl)-1-propanol

[0092] The composition of the catalytic system and the reaction conditions are as follows: 50 mL of the reaction system, 7.5 mL of alcohol dehydrogenase KmADH enzyme solution (1.5 g of bacterial cell), 3 g of glucose, 1.5 g of 3-methylamino-1-(2-thienyl)-1-propanone, 0.75 mL of glucose dehydrogenase BsGDH enzyme solution (0.15 g of bacterial cell) and 7.5 mg of NADP were added to the reaction bottle. + , add 0.1M triethanolamine buffer with pH 8.5 to 50mL. The reaction was carried out at 40°C, pH 8.5 was controlled during the process, and the rotation speed was 200rpm; after 24 hours of reaction, samples were taken for HPLC analysis, and the conversion rate of the substrate 3-methylamino-1-(2-thienyl)-1-propanone was 99.5%, and the product ee value was 99.7%.

[0093] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the examples without departing from the principles and spirit of the invention, the scope of the invention being defined by the appended claims and their equivalents.

[0094]

[0095]

[0096]

Claims

1. A method for preparing (S)-3-methylamino-1-(2-thienyl)-1-propanol, characterized in that: Alcohol dehydrogenase is used to catalyze the reaction of 3-methylamino-1-(2-thienyl)-1-propanone to generate the duloxetine intermediate (S)-3-methylamino-1-(2-thienyl)-1-propanol. The reaction formula is as follows: The alcohol dehydrogenase is derived from Kluyveromyces marxianus.

2. A method for preparing (S)-3-methylamino-1-(2-thienyl)-1-propanol, characterized in that: In a reaction medium, coenzymes, cosubstrates, coenzyme regenerating enzymes, 3-methylamino-1-(2-thienyl)-1-propanone are added, and then alcohol dehydrogenase is added to react to generate a duloxetine intermediate (S)-3-methylamino-1-(2-thienyl)-1-propanol, wherein the alcohol dehydrogenase is derived from Kluyveromyces marxianus.

3. The preparation method according to claim 1 or 2, characterized in that: The amino acid sequence of the alcohol dehydrogenase is shown in SEQ ID NO.

1.

4. The preparation method according to claim 1 or 2, characterized in that: The alcohol dehydrogenase is prepared by the following method: (1) synthesizing a nucleotide sequence encoding the alcohol dehydrogenase and constructing an engineered bacterium; (2) Inducing and culturing the engineered bacteria, collecting the bacteria by centrifugation, and then preparing the desired enzyme preparation; The nucleotide sequence is shown as SEQ ID NO.

2.

5. The preparation method according to claim 1 or 2, characterized in that: The reaction medium is selected from phosphate buffer, triethanolamine buffer, Tris-HCl buffer or boric acid-borax buffer.

6. The preparation method according to claim 1 or 2, characterized in that: The pH of the reaction is 7.0-9.0, the reaction temperature is 25-60° C., and the substrate concentration is 10-40 g / L.

7. The preparation method according to claim 1 or 2, characterized in that: The alcohol dehydrogenase (calculated by bacterial cell amount) is added in an amount of 20-40 g / L.

8. The preparation method according to claim 1 or 2, characterized in that: The coenzyme is NAD(P) + , the addition amount is 0.5-1g / L.

9. The preparation method according to claim 1 or 2, characterized in that: The co-substrate is glucose, and the addition amount is 40-80g / L.

10. The preparation method according to claim 1 or 2, characterized in that: The coenzyme regeneration enzyme is glucose dehydrogenase, and the addition amount of glucose dehydrogenase (calculated by bacterial cell amount) is 2-5 g / L.

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