CN110923277B

CN110923277B - Method for preparing S-3-dimethylamino-1- (2-thienyl) -1-propanol by biocatalysis

Info

Publication number: CN110923277B
Application number: CN201911380042.1A
Authority: CN
Inventors: 丁雪峰; 李佳松
Original assignee: Nanjing Lang'en Biological Science & Technology Co ltd
Current assignee: Nanjing Lang'en Biological Science & Technology Co ltd
Priority date: 2019-12-27
Filing date: 2019-12-27
Publication date: 2021-05-11
Anticipated expiration: 2039-12-27
Also published as: CN110923277A

Abstract

The invention discloses a method for preparing S-3-dimethylamino-1- (2-thienyl) -1-propanol by biocatalysis, which belongs to the technical field of biology, and is used for converting 3-dimethylamino-1- (2-thienyl) -1-acetone hydrochloride into S-3-dimethylamino-1- (2-thienyl) -1-propanol, has higher alcohol dehydrogenase activity and has one or more mutations of K49R, A68T, E101D, F147E, T152A, S169V and A235S. The whole system of the invention uses single enzyme for catalysis, uses alcohol for coenzyme circulation, has the substrate concentration of 100g/L-160g/L, the chiral purity of more than 99 percent, the substrate dosage/coenzyme dosage of 6000:1 and high coenzyme circulation frequency, and effectively reduces the production cost of S-3-dimethylamino-1- (2-thienyl) -1-propanol.

Description

Method for preparing S-3-dimethylamino-1- (2-thienyl) -1-propanol by biocatalysis

Technical Field

The invention relates to a method for preparing S-3-dimethylamino-1- (2-thienyl) -1-propanol by biocatalysis, belonging to the technical field of biology.

Background

Ketoreductases are a versatile catalyst that selectively reduce aldehydes or ketones enantiomers to the corresponding alcohols; (R) -specific ketoreductases have different properties from (S) -specific ketoreductases, and these catalysts are used more and more frequently in the industrial synthesis of optically active alcohols; optical activity is a prerequisite for the selective action of many pharmaceutically and pesticidally active compounds, in some cases one enantiomer having beneficial pharmaceutical activity and the other enantiomer having genotoxic action; therefore, in the synthesis of active compounds for pharmaceutical and agricultural chemicals, it is necessary to synthesize optically active alcohols using a catalyst having the required stereospecificity.

Duloxetine is a pharmaceutically active compound intended for use in the field of indications for depression and urinary incontinence; s-3-dimethylamino-1- (2-thienyl) -1-propanol is an important chiral alcohol and is a constituent unit in duloxetine synthesis; several literature and patents describe synthetic routes to duloxetine; the disadvantage of these synthetic routes is that the synthesis first yields a racemic alcohol mixture, which requires subsequent resolution of the racemate by salt formation with an optically active counter ion, conversion of the racemate to a mixture of diastereomers, followed by physical separation of the diastereomers; this leads to high process costs due to repeated separation of solids and liquids, and increased use of starting compounds due to the addition of optically active salts for separation; the stereospecific reduction of 3-methylamino-1- (2-thienyl) -propanone of the present invention provides a cheaper route to duloxetine.

Chinese patent CN103421854A discloses a biological preparation method of (S) -3- (dimethylamino) -1- (thiophene-2-yl) -1-propanol, which uses 3- (dimethylamino) -1- (thiophene-2-yl) -1-acetone or its salt as a substrate, and makes the substrate undergo asymmetric reduction reaction in the presence of a biocatalyst, a cofactor and a hydrogen donor to generate (S) -3- (dimethylamino) -1- (thiophene-2-yl) -1-propanol, the biocatalyst is a combination of Ketoreductase (KRED) and glucose dehydrogenase, and the hydrogen donor is glucose; the glucose dehydrogenase cycling system used in this patent is well known in the industry, the key ketoreductase sequence is not published in the patent, and the system uses a pH of 7.0 at which dimethyl-3-keto-3- (2-thienyl) -1-propylamine will undergo elimination in actual production, resulting in impurities such as 1- (thien-2-yl) prop-2-en-1-one), and subsequent enzymatic reduction will produce more additional byproducts such as 1- (thien-2-yl) prop-1-one, 1- (thien-2-yl) prop-1-ol, and 1- (thien-2-yl) prop-2-ol.

U.S. Pat. No.4,84, 26178 discloses engineered ketoreductase enzymes with improved properties, and also provides polynucleotides encoding the engineered ketoreductase enzymes, host cells capable of expressing the engineered ketoreductase enzymes, and methods of using the engineered ketoreductase enzymes to synthesize a variety of chiral compounds; engineered ketoreductase polypeptides are optimized for catalyzing the conversion of N, N-dimethyl-3-keto-3- (2-thienyl) -1-ketopropylamine to (S) -N, N-dimethyl-3-hydroxy-3- (2-thienyl) -1-propylamine; according to the scheme, isopropanol is used as a coenzyme circulation mode, but the reaction is effectively carried out only by forming negative pressure through suction filtration to remove acetone serving as a reaction byproduct, and the operation difficulty and the cost are increased during production amplification.

Disclosure of Invention

The invention mainly aims to provide a method for preparing S-3-dimethylamino-1- (2-thienyl) -1-propanol by biocatalysis.

The purpose of the invention can be achieved by adopting the following technical scheme:

a biocatalytic method for the preparation of S-3-dimethylamino-1- (2-thienyl) -1-propanol by converting 3-dimethylamino-1- (2-thienyl) -1-propanone hydrochloride to S-3-dimethylamino-1- (2-thienyl) -1-propanol using a ketoreductase mutant derived from a wild-type ketoreductase of Lactobacillus parachusseri, having a higher alcohol dehydrogenase activity than the wild-type sequence, having more than 90% similarity to SEQ ID No.8 and having one or more of the following characteristics: K49R, A68T, E101D, F147E, T152A, S169V and A235S, and the sequence of the ketoreductase mutant is SEQ ID NO. 4.

The ketoreductase activity of the sequence SEQ ID NO.4 is at least 2-10 times enhanced compared to the activity of the wild-type ketoreductase.

A polynucleotide encoding a polypeptide recombined with a ketoreductase having the sequence SEQ ID No. 4.

A polynucleotide whose sequence is SEQ ID NO. 3.

A recombinant plasmid comprising an expression vector linked to a polynucleotide having the sequence of SEQ ID No. 3.

A host cell comprising said recombinant plasmid.

A host cell, which is an Escherichia coli.

A host cell wherein the codons of said recombinant plasmid have been optimized for expression in said host cell.

The invention has the beneficial technical effects that:

the wild-type ketoreductase derived from Lactobacillus paracuchner can convert 3-dimethylamino-1- (2-thienyl) -1-propanone hydrochloride into S-3-dimethylamino-1- (2-thienyl) -1-propanol, has higher alcohol dehydrogenase activity compared with a wild-type sequence, and has one or more mutations of K49R, A68T, E101D, F147E, T152A, S169V and A235S. The ketoreductase mutant of the invention has obvious high specific enzyme activity, is improved by 2-10 times compared with wild ketoreductase, and can biologically catalyze 3-dimethylamino-1- (2-thienyl) -1-acetone hydrochloride to convert into S-3-dimethylamino-1- (2-thienyl) -1-propanol; the reaction condition is mild, the requirement on equipment is low, high temperature or cooling is not needed in the production process, the energy consumption is low, and the enzyme catalysis has high efficiency and specific selectivity, so that the key medical intermediate S-3-dimethylamino-1- (2-thienyl) -1-propanol for producing the antidepressant drug duloxetine by the method has no by-product and is convenient to purify; in addition, most of the solvent in the reaction is water, the discharge of three wastes is low, and the preparation process is green and environment-friendly.

The patent provides a preparation method of an antidepressant drug duloxetine intermediate S-3-dimethylamino-1- (2-thienyl) -1-propanol, the whole system uses single enzyme for catalysis, alcohols are used for coenzyme circulation, the concentration of a substrate is as high as 100g/L-160g/L, the chiral purity is more than 99%, the dosage of the substrate/the dosage of the coenzyme is as high as 6000:1, the circulation frequency of the coenzyme is high, and the production cost of the S-3-dimethylamino-1- (2-thienyl) -1-propanol is effectively reduced.

The technical scheme for realizing the purpose of the invention is as follows: the wild ketoreductase naturally existing in the microorganism Lactobacillus paracoccus has good organic solvent tolerance and alcohol dehydrogenase activity, and has the potential of converting 3-dimethylamino-1- (2-thienyl) -1-acetone hydrochloride into S-3-dimethylamino-1- (2-thienyl) -1-propanol. The inventors of the present disclosure have found that ketoreductases comprising a mutation at a position exhibit increased catalytic activity compared to the wild-type ketoreductase enzyme produced by Lactobacillus parachusseri (SEQ ID NO: 7). "wild-type ketoreductase", "wild-type KRED enzyme" and "wild-type KRED ketoreductase" refer to a polypeptide encoded by a polypeptide derived from Lactobacillus parabruchner having the sequence of SEQ ID NO: 7 amino acid sequence ketoreductase. The enzyme can convert 3-dimethylamino-1- (2-thienyl) -1-acetone hydrochloride into S-3-dimethylamino-1- (2-thienyl) -1-propanol. "wild-type" refers to the same form of material or substance as found in nature. For example, a wild-type protein or nucleic acid sequence is the original sequence form that can be isolated from nature and exists in an organism without artificial modification. "increased catalytic activity" refers to a ketoreductase that exhibits an increased rate of conversion of a substrate (e.g., 3-dimethylamino-1- (2-thienyl) -1-propanone hydrochloride or 3-dimethylamino-1- (2-thienyl) -1-propanone) to a product (e.g., S-3-dimethylamino-1- (2-thienyl) -1-propanol) as compared to the wild-type ketoreductase, as measured in an in vitro or in vivo assay.

The present invention provides a ketoreductase mutant derived from a wild-type ketoreductase of Lactobacillus parachusseri, which exhibits a ketoreductase that increases the rate of conversion of a substrate (e.g., 3-dimethylamino-1- (2-thienyl) -1-propanone hydrochloride) to a product (e.g., S-3-dimethylamino-1- (2-thienyl) -1-propanol). The ketoreductase mutant shows stronger catalytic activity compared with the wild-type ketoreductase of SEQ ID NO. 7. Ketoreductase mutants and polynucleotides encoding such mutants can be prepared using methods commonly used by those skilled in the art. Mutants can be obtained by in vitro recombination, polynucleotide mutagenesis, DNA shuffling, error-prone PCR and directed evolution methods etc. encoding the enzyme.

The ketoreductase mutant has one or more mutations selected from the following characteristics: K49R, a68T, E101D, F147E, T152A, S169V, a 235S.

The ketoreductase mutants described above, preferably from the sequence SEQ ID No.4, the full-length mutated ketoreductase enzyme is not essential for maintaining the catalytic activity of the enzyme. Accordingly, truncated analogs and catalytically active fragments of ketoreductase mutants are contemplated. For example, in some embodiments, several amino acids may be deleted from the C-terminus or N-terminus. Any particular truncated analog or fragment can be used in a corresponding assay to assess catalytic activity. Likewise, additional amino acid residues may be added to one or both termini without affecting catalytic activity. The additional sequences may be functional or non-functional. For example, the additional amino acid sequence may be used to aid in purification, as a marker, or to perform some other function. Thus, the ketoreductase mutants of the present disclosure may be in the form of fusion proteins, in which the ketoreductase mutants (or fragments thereof) are fused to other proteins, such as by way of example and not limitation, a solubilizing tag (e.g., SUMO protein), a purification tag (e.g., a metal-binding His tag), and a bacterial localization signal (e.g., a secretion signal).

The present invention provides a ketoreductase mutant which has at least 2-10 times enhanced ketoreductase activity as compared to wild-type ketoreductase.

The ketoreductase mutant coding sequence described above, which is preferably selected from SEQ ID NO.3, has been sequence optimized for expression in E.coli. In some embodiments, the polynucleotide comprises codons optimized for expression in a particular type of host cell. The codon usage and preferences for each different type of microorganism are known as are optimized codons for the expression of a particular amino acid in these microorganisms. The present invention provides a recombinant plasmid, and in some embodiments, the control sequence includes a promoter, a leader sequence, a polyadenylation sequence, a propeptide sequence, a signal peptide sequence, a transcription terminator, and the like. For bacterial host cells, suitable promoters for directing transcription of the coding sequence include, but are not limited to, the genes selected from bacteriophage T5, bacteriophage T7, bacteriophage lambda, E.coli lacUV5 operon, E.coli trp operon, E.coli tac operon, and the like.

Drawings

FIG. 1 is an expression plasmid map of Lpa-3;

FIG. 2 shows TLC patterns of 3 hours of biotransformation reactions of Lpa-1, Lpa-2, Lpa-3 and Lpa-CK, which are sequentially a mixture of substrate and product standards from left to right in lanes 1-5, samples of example 7, example 8, example 9 and example 10, with the upper band representing the substrate and the lower band representing the product.

Detailed Description

In order to make the technical solutions of the present invention more clear and definite for those skilled in the art, the present invention is further described in detail below with reference to the examples and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

As shown in fig. 1, the enzyme, the biocatalytic method for preparing the intermediate of duloxetine as an antidepressant, and the application thereof provided in this example are realized by the following examples.

Example 1:

the secondary structure and codon preference of the gene are adjusted by a whole-gene synthesis method so as to realize high expression in escherichia coli.

The Primer Premier (http:// Primer3.ut. ee /) and OPTIMIZER (http:// genes. urv. es/OPTIMIZER /) were used for design, and the Tm difference was kept within 3 ℃ and the Primer length was kept within 60base, and the obtained primers were dissolved in double distilled water and added to the following reaction system so that the final concentration of each Primer was 30nM and the final concentration of the head and tail primers was 0.6. mu.M.

The prepared PCR reaction system is placed in a Bori XP cycler gene amplification instrument and amplified according to the following procedures: 30s at 98 ℃, 45s at 55 ℃, 120s at 72 ℃ and 35 x.

The DNA fragment obtained by PCR was purified by gel cutting and cloned into the NdeI/XhoI site of pET30a by homologous recombination.

Single clones were picked for sequencing. The DNA sequence successfully sequenced is SEQ ID NO.1 and is named as Lpa-1, and the corresponding amino acid sequence is SEQ ID NO. 2.

Example 2:

2mM dNT Pmix(2mM each dNTP)	5μl
		10×Pfu buffer	5μl
Pfu DNA polymerase(10U/μl)	0.5μl
		ddH2O	The total volume of the reaction system was adjusted to 50. mu.l

Single clones were picked for sequencing. The DNA sequence successfully sequenced is SEQ ID NO.3 and is named as Lpa-2, and the corresponding amino acid sequence is SEQ ID NO. 4.

Example 3:

2mM dNTP mix(2mM each dNTP)	5μl
		10×Pfu buffer	5μl
Pfu DNA polymerase(10U/μl)	0.5μl
		ddH₂O	The total volume of the reaction system was adjusted to 50. mu.l

Single clones were picked for sequencing. The DNA sequence successfully sequenced is SEQ ID NO.5 and is named as Lpa-3, and the corresponding amino acid sequence is SEQ ID NO. 6.

Example 4:

synthesis of reference protein Lpa-CK gene sequence:

according to the sequence which is shown in WP-084973575 and is derived from Lactobacillus parabruchner, a Shanghai Czeri organism is entrusted to carry out whole-gene synthesis on the coding sequence of the protein, and the coding sequence is cloned into pET30a, so that a control protein expression plasmid Lpa-CK is obtained, and the corresponding amino acid sequence is SEQ ID NO. 7.

Example 5:

shake flask expression test:

coli single colonies containing the expression vector were picked and inoculated into 10ml of autoclaved medium: 10g/L tryptone, 5g/L yeast extract, 3.55g/L disodium hydrogen phosphate, 3.4g/L potassium dihydrogen phosphate, 2.68g/L ammonium chloride, 0.71g/L sodium sulfate, 0.493g/L magnesium sulfate heptahydrate, 0.027g/L ferric chloride hexahydrate, 5g/L glycerol, 0.8g/L glucose, and kanamycin to 50 mg/L. The culture was carried out at 30 ℃ and 250rpm overnight. Taking a 1L triangular flask the next day, and carrying out the following steps: 100 into 100ml of autoclaved medium: 10g/L tryptone, 5g/L yeast extract, 3.55g/L disodium hydrogen phosphate, 3.4g/L potassium dihydrogen phosphate, 2.68g/L ammonium chloride, 0.71g/L sodium sulfate, 0.493g/L magnesium sulfate heptahydrate, 0.027g/L ferric chloride hexahydrate, 5g/L glycerol, 0.3g/L glucose, and kanamycin to 50 mg/L. The cells were cultured at 30 ℃ until the OD 5-6 of the cells became zero, and the cells were immediately placed in a flask in a shaker at 25 ℃ and cultured at 250rpm for 1 hour. IPTG was added to a final concentration of 0.1mM and incubation was continued at 25 ℃ for 16 hours at 250 rpm. After completion of the culture, the culture was centrifuged at 12000g at 4 ℃ for 20 minutes to collect wet cells. Then the bacterial pellet is washed twice with distilled water, and the bacterial is collected and preserved at-70 ℃. Meanwhile, 2g of the thalli are added into 6ml of pure water for ultrasonic disruption, SDS-PAGE detection is carried out, and the crude enzyme liquid is stored at the temperature of minus 20 ℃.

Example 6:

fed-batch fermentation:

the fed-batch fermentation was carried out in a computer-controlled bioreactor (Shanghai Seisaku) with a reactor capacity of 15L and a working volume of 8L, using 24g/L yeast extract, 12g/L peptone, 0.4% glucose, 2.31g/L catalase phosphate and 12.54g/L dipotassium hydrogen phosphate as the medium, pH 7.0. 200ml of culture was prepared for the primary inoculum and inoculated at OD 2.0. Throughout the fermentation, the temperature was maintained at 37 ℃, the dissolved oxygen concentration during fermentation was automatically controlled at 30% by the agitation rate (rpm) and aeration supply cascade, while the pH of the medium was maintained at 7.0 by 50% (v/v) orthophosphoric acid and 30% (v/v) aqueous ammonia. During the fermentation, when a large amount of dissolved oxygen rises, feeding is started. The feed solution contained 9% w/v peptone, 9% w/v yeast extract, 14% w/v glycerol. When OD600 was about 35.0 (wet weight about 60g/L), induction was carried out with 0.2mM IPTG for 16 hours. Taking 2g of thallus, adding 6ml of pure water, carrying out ultrasonic disruption, carrying out SDS-PAGE detection, and storing the crude enzyme liquid at-20 ℃.

Example 7:

and (3) carrying out biotransformation reaction:

380ml of 0.1M triethanolamine buffer solution, 620ml of isopropanol and 188g of 3-dimethylamino-1- (2-thienyl) -1-acetone hydrochloride are sequentially added into a 3L three-neck flask, and uniformly mixed to pre-dissolve a substrate; and taking 800ml of the reaction system, adding 25mg/LNADP and 50ml of Lpa-1 crude enzyme solution, uniformly mixing, adjusting the temperature of a water bath to 37 ℃, and stirring for reaction overnight. And an intermediate sample was taken for storage at 3 hours.

Example 8:

and (3) carrying out biotransformation reaction:

380ml of 0.1M triethanolamine buffer solution, 620ml of isopropanol and 188g of 3-dimethylamino-1- (2-thienyl) -1-acetone hydrochloride are sequentially added into a 3L three-neck flask, and uniformly mixed to pre-dissolve a substrate; and taking 800ml of the reaction system, adding 25mg/LNADP and 50ml of Lpa-2 crude enzyme solution, uniformly mixing, adjusting the temperature of a water bath to 37 ℃, and stirring for reaction overnight. And an intermediate sample was taken for storage at 3 hours.

Example 9:

and (3) carrying out biotransformation reaction:

380ml of 0.1M triethanolamine buffer solution, 620ml of isopropanol and 188g of 3-dimethylamino-1- (2-thienyl) -1-acetone hydrochloride are sequentially added into a 3L three-neck flask, and uniformly mixed to pre-dissolve a substrate; and (3) adding 25mg/LNADP and 50ml of Lpa-3 crude enzyme solution into 800ml of the reaction system, uniformly mixing, adjusting a water bath, and stirring at 37 ℃ for reaction overnight. And an intermediate sample was taken for storage at 3 hours.

Example 10:

control enzyme Lpa-CK biotransformation reaction:

380ml of 0.1M triethanolamine buffer solution, 620ml of isopropanol and 188g of 3-dimethylamino-1- (2-thienyl) -1-acetone hydrochloride are sequentially added into a 3L three-neck flask, and uniformly mixed to pre-dissolve a substrate; taking 800ml of the reaction system, adding 25mg/L NADP and 50ml Lpa-CK crude enzyme solution, mixing uniformly, adjusting water bath to 37 ℃, and stirring for reaction overnight. And an intermediate sample was taken for storage at 3 hours.

Example 11:

thin-layer chromatography analysis:

extracting a small amount of reaction liquid with ethyl acetate according to a ratio of 1:10, spotting 1ul on a silica gel plate, heating and drying to obtain the color of the potassium permanganate, wherein a developing agent is dichloromethane/methanol (10: 1). The results are shown in FIG. 2.

Example 12:

and (3) enzyme activity detection:

taking 6 5ml centrifuge tubes, respectively marking 1-6, respectively adding 3mM NADPH solution 0ul, 40ul, 80ul, 100ul, 120ul and 160ul, then supplementing 0.1M phosphate buffer solution with pH7.0 to 3ml each tube, mixing uniformly, detecting at 340nm and recording the absorbance value; obtaining a standard curve Y ═ k × X of NADPH according to the above measured values, wherein Y is the value of absorbance, X is the concentration (mM) of NADPH, and R2 of the curve is more than 99.5%; diluting the enzyme solution with pure water by a certain dilution ratio (reference dilution ratio: 600-1000 times), wherein the dilution ratio is suitable for changing the light absorption value per minute by 0.02-0.04; 5ml of centrifuge tube is taken, the samples are added into the centrifuge tube according to the following proportion, the mixture is quickly mixed, and the mixture is immediately poured into a cuvette.

Detection reagent	Dosage of
		Isopropanol (I-propanol)	500ul
2％NADP	100uL
		100mMPBS(pH7.0)	2.35mL
Diluted enzyme solution	50uL

Detecting the change of absorbance at 340nm, recording a value every 1min, and keeping the change rate basically the same every minute, wherein the absorbance at 0min is S0, and the absorbance at 3min is S3;

the enzyme activity calculation formula is as follows:

enzyme activity (U/ml) [ (S0-S3) × 3ml × N ]/[ kXtime (t/min) × enzyme addition (ml) ]

Wherein N is the dilution multiple of the enzyme solution.

The detection results are as follows:

sample to be tested	Mutation point	Enzyme activity U/ml
			Lpa-1	K49R，A68T，F147E	560
Lpa-2	A68T，E101D，S169V，A235S	1120
			Lpa-3	K49R，F147E，T152A，S169V	1550
Lpa-CK	－	160

Thus, the ketoreductase mutant with enhanced activity provided by the invention has the ketoreductase activity which is at least enhanced by 2-10 times compared with the activity of the wild-type ketoreductase. Compared with wild ketoreductase, the method increases the conversion rate of the substrate 3-dimethylamino-1- (2-thienyl) -1-acetone hydrochloride to the product S-3-dimethylamino-1- (2-thienyl) -1-propanol, further increases the substrate concentration per unit volume, and is beneficial to improving the production efficiency during industrial amplification.

The above description is only for the purpose of illustrating the present invention and is not intended to limit the scope of the present invention, and any person skilled in the art can substitute or change the technical solution of the present invention and its conception within the scope of the present invention.

Sequence listing

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50 55 60

Ser Asp Glu Thr Gly Trp Thr Glu Leu Phe Asp Thr Thr Glu Asn Ala

65 70 75 80

Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser

85 90 95

Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser

100 105 110

Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg

115 120 125

Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile

130 135 140

Glu Gly Phe Val Gly Asp Pro Ala Leu Gly Ala Tyr Asn Ala Ser Lys

145 150 155 160

Gly Ala Val Arg Ile Met Ser Lys Val Ala Ala Leu Asp Cys Ala Leu

165 170 175

Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys

180 185 190

Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln

195 200 205

Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala

210 215 220

Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly

225 230 235 240

Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln

245 250

<210> 5

<211> 759

<212> DNA

<213> Artificial sequence ()

<400> 5

atgaccgacc gtctgaaagg taaagttgct atcgttaccg gtggtaccct gggtatcggt 60

ctggctatcg ctgacaaatt cgttgaagaa ggtgctaaag ttgttatcac cggtcgtcac 120

gctgacgttg gtgaaaaagc tgctcgttct atcggtggtc cggacgttat ccgtttcgtt 180

cagcacgacg cttctgacga agctggttgg accgaactgt tcgacaccac cgaaaacgct 240

ttcggtccgg ttaccaccgt tgttaacaac gctggtatcg ctgtttctaa atctgttgaa 300

gaaaccacca ccgaagaatg gcgtaaactg ctgtctgtta acctggacgg tgttttcttc 360

ggtacccgtc tgggtatcca gcgtatgaaa aacaaaggtc tgggtgcttc tatcatcaac 420

atgtcttcta tcgaaggtga agttggtgac ccggctctgg gtgcttacaa cgcttctaaa 480

ggtgctgttc gtatcatgtc taaagttgct gctctggact gcgctctgaa agactacgac 540

gttcgtgtta acaccgttca cccgggttac atcaaaaccc cgctggttga cgacctggaa 600

ggtgctgaag aaatgatgtc tcagcgtacc aaaaccccga tgggtcacat cggtgaaccg 660

aacgacatcg cttggatctg cgtttacctg gcttctgacg aagctaaatt cgctaccggt 720

gctgaattcg ttgttgacgg tggttacacc gctcagtaa 759

<210> 6

<211> 252

<212> PRT

<213> Artificial sequence ()

<400> 6

Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr

1 5 10 15

Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala

20 25 30

Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala

35 40 45

Arg Ser Ile Gly Gly Pro Asp Val Ile Arg Phe Val Gln His Asp Ala

50 55 60

Ser Asp Glu Ala Gly Trp Thr Glu Leu Phe Asp Thr Thr Glu Asn Ala

65 70 75 80

Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser

85 90 95

Lys Ser Val Glu Glu Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser

100 105 110

Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg

115 120 125

Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile

130 135 140

Glu Gly Glu Val Gly Asp Pro Ala Leu Gly Ala Tyr Asn Ala Ser Lys

145 150 155 160

Gly Ala Val Arg Ile Met Ser Lys Val Ala Ala Leu Asp Cys Ala Leu

165 170 175

Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys

180 185 190

Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln

195 200 205

Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala

210 215 220

Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ala Lys Phe Ala Thr Gly

225 230 235 240

Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln

245 250

<210> 7

<211> 252

<212> PRT

<213> Lactobacillus parabuchneri

<400> 7

Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr

1 5 10 15

Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala

20 25 30

Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala

35 40 45

Lys Ser Ile Gly Gly Pro Asp Val Ile Arg Phe Val Gln His Asp Ala

50 55 60

Ser Asp Glu Ala Gly Trp Thr Glu Leu Phe Asp Thr Thr Glu Asn Ala

65 70 75 80

Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser

85 90 95

Lys Ser Val Glu Glu Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser

100 105 110

Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg

115 120 125

Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile

130 135 140

Glu Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys

145 150 155 160

Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu

165 170 175

Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys

180 185 190

Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln

195 200 205

Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala

210 215 220

Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ala Lys Phe Ala Thr Gly

225 230 235 240

Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln

245 250

Claims

1. A method for preparing S-3-dimethylamino-1- (2-thienyl) -1-propanol by biocatalysis is characterized in that 3-dimethylamino-1- (2-thienyl) -1-acetone hydrochloride is converted into S-3-dimethylamino-1- (2-thienyl) -1-propanol, a ketoreductase mutant which is derived from wild ketoreductase of Lactobacillus parabuchner and has higher alcohol dehydrogenase activity compared with the wild sequence is adopted, the sequence of the ketoreductase mutant is SEQ ID NO.4, and the ketoreductase activity of the sequence of SEQ ID NO.4 is at least enhanced by 2-10 times compared with the activity of the wild ketoreductase.

2. A polynucleotide encoding a ketoreductase recombinant polypeptide having the sequence of SEQ id No. 4.

3. A polynucleotide according to claim 2, having the sequence shown in SEQ ID No. 3.

4. A recombinant plasmid comprising a ketoreductase mutant encoding a polynucleotide having the sequence of SEQ ID No. 3.

5. A host cell comprising the recombinant plasmid of claim 4.

6. A host cell according to claim 5, wherein the cell is an E.coli cell.

7. A host cell according to claim 6, wherein the codons of said recombinant plasmid have been optimized for expression in said host cell.