CN114277007A - Alcohol dehydrogenase mutant and application thereof - Google Patents

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to an alcohol dehydrogenase mutant and application thereof, wherein the mutant is obtained by mutating 214 th glutamic acid, 215 th threonine and 237 th serine of alcohol dehydrogenase with an amino acid sequence of SEQ ID No. 2; or the amino acid sequence of the amino acid sequence is phenylalanine at position 161, serine at position 196 and glutamic acid at position 214 of the alcohol dehydrogenase of SEQ ID No. 2. The alcohol dehydrogenase mutant has excellent catalytic activity and stereoselectivity, and can efficiently catalyze and prepare a series of chiral diaryl alcohols with R-and S-configurations and para-substituent groups; the alcohol dehydrogenase mutant is coupled with glucose dehydrogenase or formate dehydrogenase, and can be used for synthesizing chiral diaryl alcohol intermediates of various antihistamine drugs; the method for preparing the diaryl chiral alcohol by the asymmetric catalytic reduction of the alcohol dehydrogenase has the advantages of simple and convenient operation, high substrate concentration, complete reaction and high product purity, and has strong industrial application prospect.

Description

Alcohol dehydrogenase mutant and application thereof

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to an alcohol dehydrogenase mutant and application thereof, which are used for catalyzing synthesis of para-substituted diaryl chiral alcohol.

Background

The chiral diaryl alcohol is an important chiral compound and can be used for synthesizing various medicaments such as betahistine, rotroxamine and the like, so the chiral diaryl alcohol has wide application in the field of medicines.

At present, the method for producing chiral diaryl alcohol is mainly a chemical asymmetric synthesis method, and the method takes latent chiral diaryl ketone as a raw material and trans-RuCl₂[(R)-xylbinap][(R)-daipen]、(S)-[Ru(BINAP)Cl₂]₂(NE3), (S, S) -6-CHOONa and the like are used as catalysts or chiral BINAL-H and the like are used as chiral reducing agents, and asymmetric reduction reaction is carried out under certain conditions (high pressure) to obtain chiral diaryl alcohol (specifically, the references "C.Y.Chen, et al, org.Lett.,2003,5,5039-.

However, the catalyst or chiral reducing agent used in the chemical asymmetric synthesis method is expensive, high pressure condition is required for the reaction, and the optical purity of the synthesized product is low, which is not beneficial to industrial production, and can not meet the requirement of the medicine on the optical purity.

The enzyme asymmetric reduction method is a method for obtaining chiral diaryl alcohol by taking latent chiral diaryl ketone as a raw material and taking enzyme as a catalyst to carry out asymmetric reduction reaction. Compared with the chemical asymmetric synthesis method, the enzymatic asymmetric reduction method has the advantages of mild action conditions, low cost and high optical purity of the synthesized product, and meets the targets of industrial development such as sustainable development, green chemistry, environment-friendly manufacturing and the like. Therefore, the method for producing the chiral diaryl alcohol by using the enzyme asymmetric reduction method has important significance for realizing large-scale industrial production of the chiral diaryl alcohol and large-scale application of the chiral diaryl alcohol in the field of medicines.

The change of different functional groups in the structure of the medicine can change the physicochemical properties, charge density and the like of the whole molecule, and corresponding properties can be increased or toxic and side effects or adverse reactions can be weakened for the medicine according to the characteristics of the substituent groups. Different substituents have their own characteristics, and drug modification and optimization are generally achieved by adding or replacing substituents at different positions.

The existing enzyme for producing chiral diaryl Alcohol by asymmetrically reducing latent chiral diaryl ketone is mainly Alcohol Dehydrogenase (ADH), but when different latent chiral diaryl ketones are reduced, the catalytic activity and stereoselectivity difference are large, and large-scale industrial production of all chiral para-substituted diaryl alcohols and large-scale application of the chiral para-substituted diaryl alcohols in the field of medicine cannot be realized, so that the Alcohol dehydrogenase capable of asymmetrically reducing latent chiral diaryl ketones with different para-substituents to produce chiral diaryl alcohols is required to be continuously developed.

Disclosure of Invention

The invention aims to provide an alcohol dehydrogenase mutant and application thereof, wherein the mutant has excellent catalytic activity and stereoselectivity for 2-pyridyl [4- (trifluoromethyl) phenyl ] ketone and (4-methylphenyl) (2-pyridyl) ketone.

The first aspect of the present invention provides a mutant of alcohol dehydrogenase, which is obtained by mutating the 214 th glutamic acid, the 215 th threonine and the 237 th serine of alcohol dehydrogenase having an amino acid sequence of SEQ ID No. 2; or

The mutant is obtained by mutating phenylalanine 161, serine 196 and glutamic acid 214 of alcohol dehydrogenase with the amino acid sequence of SEQ ID No. 2.

Further, the mutant is obtained by replacing glutamic acid at position 214 with isoleucine, threonine at position 215 with serine, and serine at position 237 with alanine in alcohol dehydrogenase whose amino acid sequence is shown in SEQ ID No.2, and is named as M1.

Further, the mutant is obtained by replacing phenylalanine at position 161, serine at position 196 and glutamic acid at position 214 of alcohol dehydrogenase having an amino acid sequence shown as SEQ ID No.2 with valine, glycine and amino acid sequence M2.

The second aspect of the present invention provides a gene encoding a mutant of the above-mentioned alcohol dehydrogenase.

In a third aspect of the present invention, there is provided a vector carrying the above gene.

Further, the vector is pET28a (+) plasmid, pET28b (+) plasmid or pET20b (+) plasmid, preferably pET28a (+) plasmid.

The fourth aspect of the present invention provides a host cell (recombinant bacterium) containing the above-mentioned vector.

Further, the host cell is in recombinant Escherichia coli; specifically, the starting bacterium is e.coli BL21(DE 3).

Further, the construction method of the recombinant Escherichia coli comprises the following steps: cloning the gene (nucleic acid molecule) encoding the mutant into a vector (pET28a (+) plasmid) to obtain a recombinant vector; and transforming the obtained recombinant vector into escherichia coli to obtain the recombinant escherichia coli.

The mutant of the alcohol dehydrogenase can be obtained by separating and purifying the recombinant escherichia coli obtained by culturing.

Specifically, the method for producing the mutant of the alcohol dehydrogenase by using the recombinant bacterium comprises the following steps: inoculating the recombinant bacteria into LB culture medium containing 40-60 mug/mL kanamycin sulfate, culturing at 30-40 ℃ and 100-₆₀₀When the content of the recombinant alcohol dehydrogenase reaches 0.5-1.0, 0.05-1.0mM isopropyl-beta-D-hexa-generation galactofuranoside (IPTG) is added for induction, the induction temperature is 16-30 ℃, and the mutant for efficiently expressing the recombinant alcohol dehydrogenase can be obtained after 5-10 hours of induction.

The fourth aspect of the invention provides the application of the mutant, the gene, the vector or the host cell of the alcohol dehydrogenase in catalyzing the synthesis of the para-substituted diaryl chiral alcohol.

Further, the diaryl chiral alcohol is 2-pyridyl [4- (trifluoromethyl) phenyl ] methanol or (4-methylphenyl) (2-pyridyl) methanol; the substrates are 2-pyridyl [4- (trifluoromethyl) phenyl ] methanone and (4-methylphenyl) (2-pyridyl) methanone, respectively.

The difference of substrate substituent groups can change the size or the property of one side of the substrate, the existence of steric hindrance can be caused by the overlarge substrate substituent groups, so that the substrate cannot form a catalytic conformation, and the change of the property can influence the binding acting force between the substrate and the enzyme, such as the increase of hydrophobic acting force, so that the km value is reduced, and the catalytic efficiency is improved.

The fifth aspect of the invention provides a method for synthesizing a diaryl chiral alcohol, which comprises the following steps: and adding the mutant of the alcohol dehydrogenase into a reaction system containing a latent chiral carbonyl compound for reaction to obtain the diaryl chiral alcohol.

Further, the latent chiral carbonyl compound is 2-pyridyl [4- (trifluoromethyl) phenyl ] methanone or (4-methylphenyl) (2-pyridyl) methanone.

Further, the concentration of the latent chiral carbonyl compound in the reaction system is 10-100 mM; the addition amount of the mutant of the alcohol dehydrogenase is 1-10kU/L by taking the reaction system as reference.

Further, the reaction system also comprises a coenzyme circulating system, and the coenzyme circulating system contains one of the following combinations: glucose dehydrogenase GDH and D-glucose; phosphite dehydrogenase (FTDH) and phosphite; formate Dehydrogenase (FDH) and formate; lactate Dehydrogenase (LDH) and lactate; or glycerol dehydrogenase and glycerol.

Specifically, the synthesis method comprises the following steps: construction of reaction System, 2-pyridyl [4- (trifluoromethyl) phenyl group]The concentration of methanone or (4-methylphenyl) (2-pyridyl) methanone is 10-100mM, the dosage of the above alcohol dehydrogenation mutant is 1-10kU/L, and NADP⁺The usage amount of (nicotinamide adenine dinucleotide phosphate) is 0.1-1.0mM, and a coenzyme circulating system is added, wherein the coenzyme circulating system contains glucose dehydrogenase GDH and D-glucose, the usage amount of the glucose dehydrogenase GDH is 1-10kU/L, the usage amount of the D-glucose is 20-1000mM, and the concentration of a phosphate buffer solution is 0.1-0.2M; at 30-35 ℃ and pH6-8Reacting for 1-24h under the condition, after the asymmetric reduction reaction is finished, extracting chiral 2-pyridyl [4- (trifluoromethyl) phenyl group from the reaction liquid according to an organic solvent extraction method]Methanol or (4-methylphenyl) (2-pyridyl) methanol.

Further, the coenzyme cycle system may also be phosphite/phosphite dehydrogenase (FTDH), formate/Formate Dehydrogenase (FDH), lactate/Lactate Dehydrogenase (LDH), or glycerol/glycerol dehydrogenase.

Further, the chromatographic analysis method of the (R) -and (S) -products is as follows: and (3) adding 500 mu L of ethyl acetate into 100 mu L of reaction liquid, shaking for 1-2min, centrifuging at 12000rpm for 2-5min, taking the supernatant into a centrifuge tube, adding 500 mu L of chromatographic pure ethanol when the organic matter is completely volatilized naturally, and carrying out chiral liquid chromatography to analyze the conversion rate and the ee value. The chromatographic conditions were as follows: daicel Chiralcel AD-H (5 μm, 250 mm. times.4.6 mm) liquid chromatography column with n-hexane as mobile phase: isopropanol (90:10, v/v), flow rate 1mL/min, column temperature 30 ℃, ultraviolet detection wavelength 254nm, sample volume 10 uL, (S) -and (R) - (4-methylphenyl) (2-pyridyl) methanol retention times of 12.6min and 14.7min, respectively, 2-pyridyl [4- (trifluoromethyl) phenyl ] methanol retention times of 9.4min and 12.1min, respectively.

Compared with the prior art, the technical scheme of the invention has the following advantages:

(1) the alcohol dehydrogenase mutation obtained by the invention has higher activity on various carbonyl compounds, and can catalyze and reduce various aliphatic or aryl substituted ketone substrates, especially biaryl ketone substrates with larger steric hindrance. The method has the advantages that the wild type alcohol dehydrogenase (KPADH, derived from Kluyveromyces polyspora) is subjected to molecular modification through a combined mutation means, so that the stereoselectivity of the enzyme is improved, and the enzyme has higher industrial application value;

(2) compared with a wild-type alcohol dehydrogenase KPADH, the alcohol dehydrogenase mutants M1 and M2 of the invention have improved stereoselectivity for substrates 2-pyridyl [4- (trifluoromethyl) phenyl ] methanone and (4-methylphenyl) (2-pyridyl) methanone, and the ee values of products 2-pyridyl [4- (trifluoromethyl) phenyl ] methanol and (4-methylphenyl) (2-pyridyl) methanol are improved from 49.4% (S) and 82.6% (R) of the wild type to 99.9% (S) (M1) and 99.9% (R) (M2); the alcohol dehydrogenase mutant obtained by the invention is particularly suitable for asymmetric reduction of para-substituted diaryl ketone, and has good industrial application prospect.

Drawings

FIG. 1 is a full plasmid PCR nucleic acid electrophoresis of alcohol dehydrogenase mutants M1 and M2;

FIG. 2 is a gradient elution protein electrophoresis diagram of an alcohol dehydrogenase mutant;

FIG. 3 is a chiral chromatogram of the product of catalytic reduction of 2-pyridyl [4- (trifluoromethyl) phenyl ] methanone by alcohol dehydrogenase mutant M2;

FIG. 4 is a chiral chromatogram of a product obtained by catalytic reduction of (4-methylphenyl) (2-pyridyl) methanone by using an alcohol dehydrogenase mutant M2.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

In the following examples, the activity of alcohol dehydrogenase and the optical purity of the product were determined as follows:

the total reaction system was 200 μ L, including: 1.0mM NADPH (reduced nicotinamide adenine dinucleotide phosphate), 1.0mM substrate 2-pyridyl [4- (trifluoromethyl) phenyl ] methanone or (4-methylphenyl) (2-pyridyl) methanone, sodium phosphate buffer (PBS, 100mM, pH7.0), mixing well, incubating at 30 deg.C for 2min, adding appropriate amount of enzyme solution, and detecting the change of light absorption value at 340 nm.

The calculation formula of the enzyme activity is as follows:

enzyme activity (U) ═ EW × V × 10³/(6220×1)；

Wherein EW is the change of absorbance at 340nm within 1 min; v is the volume of the reaction solution, and the unit is mL; 6220 molar extinction coefficient of NADPH, in L/(mol. cm); 1 is the path length in cm. 1 activity unit (U) corresponds to the amount of enzyme required to catalytically oxidize 1. mu. mol NADPH per minute under the above conditions.

Method for measuring optical purity ee:

AS: molar concentration of (S) -product obtained by liquid chromatography; AR: molar concentration of (R) -product obtained by liquid chromatography;

example 1: construction of alcohol dehydrogenase mutant Gene and recombinant expression transformant

Site-directed mutagenesis is carried out on residues at 214, 215 and 237 (161, 196 and 237 amino acids) by adopting a whole plasmid PCR method to construct an iterative combinatorial mutant. The primers were designed as follows (all described in the 5 '-3' direction, underlined for mutation sites):

m1 (using pET28a-KPADH recombinant plasmid as template)

E214I-F：AAGAAACTAAATATTACTTGTGAAATT

E214I-R：AATTTCACAAGTAATATTTAGTTTCTT

T215S-F：AAACTAAATGAAAGCTGTGAAATTATC

E215S-R：GATAATTTCACAGCTTTCATTTAGTTT

S237A-F：AAGACTCACTTCGCACAATTCATTGAT

S237A-R：ATCAATGAATTGTGCGAAGTGAGTCTT

M2 (using pET28a-KPADH recombinant plasmid as template)

F161V-F TATGAAAATGTCGTTACTGCTTATTGT

F161V-R ACAATAAGCAGTAACGACATTTTCATA

S196G-F ACTATCCACCCAGGTTTCGTTTTCGGA

S196G-R TCCGAAAACGAAACCTGGGTGGATAGT

E214G-F AAGAAACTAAATGGTACTTGTGAAATT

E214G-R AATTTCACAAGTACCATTTAGTTTCTT

The PCR reaction system is as follows: the PCR reaction system (50. mu.L) included 1.0. mu.L of KOD enzyme (2.5U/. mu.L), 1.0. mu.L of template (5-50ng), 4.0. mu.L of dNTP, 5.0. mu.L of 10 × reaction buffer, 1.0. mu.L of each of upstream and downstream primers, ddH₂O make up to 50. mu.L.

The PCR amplification procedure was: (1) denaturation at 94 ℃ for 3min, (2) denaturation at 94 ℃ for 30sec, (3) annealing at 54 ℃ for 30sec, (4) extension at 72 ℃ for 150sec, repeating steps (2) - (4) for 10-15 cycles, finally extension at 72 ℃ for 10min, and storing the PCR amplification product at 4 ℃.

After the PCR was completed, DpnI (methylated template digestive enzyme) restriction enzyme was added to the reaction mixture and incubated at 37 ℃ for 1h, followed by CaCl₂Thermal transformation method 10. mu.L of digested PCR reaction solution was transferred into 50. mu.L of E.coli BL21(DE3) competent cells, and uniformly spread on LB agar plate containing 50. mu.g/mL kanamycin sulfate, and cultured in inversion at 37 ℃ for 12 hours.

Example 2: expression and purification of alcohol dehydrogenase and its mutant

The recombinant E.coli carrying the stereoselectivity-improving mutant was inoculated at 2% of the transfer amount into LB medium containing kanamycin sulfate (50. mu.g/mL), cultured at 37 ℃ with shaking at 200rpm, and the absorbance OD of the culture solution₆₀₀When the concentration reaches 0.8, 0.2mM isopropyl-beta-D-hexa-generation galactofuranoside (IPTG) is added for induction, the induction temperature is 25 ℃, after 8 hours of induction, the thalli of the mutant of the high-efficiency expression recombinant alcohol dehydrogenase is obtained by centrifugation at 8000rpm for 10min, the collected thalli are suspended in potassium phosphate buffer solution (100mM, pH 6.0) and are broken by ultrasound.

The column used for purification was a nickel affinity column, HisTrap FF crude, and affinity chromatography was performed using a histidine tag on the recombinant protein. Firstly, the nickel column is balanced by using the solution A, the crude enzyme solution is loaded, the solution A is continuously used for eluting a penetration peak, after the balance, the solution B (20mM sodium phosphate, 500mM NaCl, 1000mM imidazole, pH 7.4) is used for gradient elution, and the recombinant protein combined on the nickel column is eluted, so that the recombinant alcohol dehydrogenase mutant is obtained. The purified protein was analyzed by viability assay (NADPH was a coenzyme) and SDS-PAGE. After nickel column purification, a single band is shown at about 45kDa, and the impurity protein is less, which indicates that the column purification effect is better. The purified alcohol dehydrogenase protein was then replaced into Tris-HCl (100mM, pH7.0) buffer using a Hi-Trap desaling Desalting column (GE Healthcare).

Example 3: case of alcohol dehydrogenase mutant for reducing potentially chiral carbonyl compound

Examples of the latent chiral carbonyl compounds to be investigated include 4-chlorophenyl-pyridin-2-yl-methanone ((4-chlorophenyl) - (pyridine-2-yl) -methanone, CPMK), (4-methylphenyl) (2-pyridyl) methanone (2- (4-methylbenzol) pyridine), 2-pyridyl [4- (trifluoromethyl) phenyl ] methanone (2-pyridyl [4- (trifluoromethyl) phenyl ] methanone). As can be seen from Table 1, the ee values of the reduction products of M1 and M2 for the substrate substrates 2-pyridyl [4- (trifluoromethyl) phenyl ] methanone and (4-methylphenyl) (2-pyridyl) methanone were 97% or more. The obvious improvement of the stereoselectivity is due to the introduction of the mutation sites, the binding force of the enzyme and the substrate is enhanced, and the substrate can be favorable for the stability and the catalysis of the substrate.

TABLE 1

Example 4: kinetic and stereoselective analysis of alcohol dehydrogenase mutants

And (3) measuring the activity of the KPADH under different substrate concentrations and coenzyme concentrations, making a double reciprocal curve according to the reciprocal of the activity and the substrate concentrations, and calculating kinetic parameters.

As is clear from Table 2, KPADH is a substrate for 2-pyridyl [4- (trifluoromethyl) phenyl ] group]K of methanone_cat/K_mIs 2.41s^-1·mM^-1The reduced product configuration was S configuration, and ee value was 49.4% (S). Synthesis of (S) -, (R) -2-pyridinyl [4- (trifluoromethyl) phenyl ] by mutants M1, M2]The stereoselectivity of methanol is improved to more than 98 percent, and the ee values of the products are respectively 99.0 percent (S) (M1) and 98.7 percent (R) (M2).

From Table 3, it can be seen that k of KPAADH to the substrate (4-methylphenyl) (2-pyridyl) methanone_cat/K_mIs 4.58s^-1·mM^-1The reduced product configuration was S configuration, and ee value was 82.6% (S). The mutant M1, M2 synthesizes (S) -, (R) - (4-methylphenyl) (2-pyridyl) methanol stereoselectivity is improved to reach more than 97%, and ee values of products are respectively 99.9% (S) (M1) and 97.7% (R) (M2).

TABLE 2 kinetic parameters and stereoselectivity of alcohol dehydrogenase mutants for the substrate 2-pyridyl [4- (trifluoromethyl) phenyl ] methanone

TABLE 3 kinetic parameters and stereoselectivity of alcohol dehydrogenase mutants for the substrate (4-methylphenyl) (2-pyridyl) methanone

Example 5: asymmetric reduction of alcohol dehydrogenase mutant to prepare (S) - (4-methylphenyl) (2-pyridyl) methanol with high optical purity

A 20mL biocatalytic system was established: 100mM potassium phosphate buffer (pH7.0), 10g/L of the alcohol dehydrogenase mutant M1 cell obtained in example 2, (4-methylphenyl) (2-pyridyl) methanone 50mM was added. The reaction was carried out at 30 ℃ and 200rpm for 24h, with a constant pH of 7.0. The results of the conversion analysis during the reaction are shown in Table 4. When the substrate concentration is 50mM, the alcohol dehydrogenase mutants M1 can reach more than 99% of substrate conversion rate in 24h, and the reduction products are all (S) - (4-methylphenyl) (2-pyridyl) methanol, wherein the ee value of the wild type KPADH reduction product is only 82.6%, and the ee value of the mutant M1 reduction product can reach 99%. And re-dissolving the obtained crude (S) - (4-methylphenyl) (2-pyridyl) methanol in ethanol, adding a corresponding pure product serving as a seed crystal, and recrystallizing at 4 ℃ to finally obtain the product with the optical purity of more than 99.9% ee.

TABLE 4 alcohol dehydrogenase mutant M1 catalyzes the asymmetric reduction of 50mM (4-methylphenyl) (2-pyridyl) methanone

Example 6: asymmetric reduction of alcohol dehydrogenase mutant to prepare (R) -2-pyridyl [4- (trifluoromethyl) phenyl ] methanol with high optical purity

A 20mL biocatalytic system was established: 100mM potassium phosphate buffer (pH7.0), 10g/L of the alcohol dehydrogenase mutant M2 cell obtained in example 2, 50mM of 2-pyridyl [4- (trifluoromethyl) phenyl ] methanone was added. The reaction was carried out at 30 ℃ and 200rpm for 24h, with a constant pH of 7.0. The results of the conversion analysis during the reaction are shown in Table 5. When the substrate concentration is 50mM, the alcohol dehydrogenase mutants M2 can reach more than 99% of substrate conversion rate in 24h, and the reduction products are (R) -2-pyridyl [4- (trifluoromethyl) phenyl ] methanol, wherein the ee value of the wild type KPADH reduction product is 49.4% (S), and the ee value of the mutant M2 reduction product can reach 98.7% (R). And re-dissolving the obtained (R) -2-pyridyl [4- (trifluoromethyl) phenyl ] methanol crude product in ethanol, adding a corresponding product pure product serving as a seed crystal, and recrystallizing at 4 ℃ to finally obtain the product with the optical purity of more than 99% ee.

TABLE 5 alcohol dehydrogenase mutant M2 catalyzes the asymmetric reduction of 50mM 2-pyridinyl [4- (trifluoromethyl) phenyl ] methanone

In conclusion, the alcohol dehydrogenase mutant has excellent catalytic activity and stereoselectivity, and can efficiently catalyze and prepare a series of chiral diaryl alcohols with R-and S-configurations and para-substituent groups; the alcohol dehydrogenase mutant is coupled with glucose dehydrogenase or formate dehydrogenase, and can be used for synthesizing chiral diaryl alcohol intermediates of various antihistamine drugs; the method for preparing the diaryl chiral alcohol by the asymmetric catalytic reduction of the alcohol dehydrogenase has the advantages of simple and convenient operation, high substrate concentration, complete reaction and high product purity, and has strong industrial application prospect.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.

Sequence listing

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<120> alcohol dehydrogenase mutant and application thereof

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Ser Gln Asp Lys Ala Asp Lys Leu Leu Lys Gln Tyr Asn Asn Pro Asn

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Leu Ser Tyr Glu Ile Val Pro Glu Ile Ala Asn Leu Asp Ala Phe Asp

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Asp Ile Phe Lys Lys His Gly Lys Glu Ile Lys Tyr Val Ile His Ala

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

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Ser Ile Met Thr Pro His Arg Gln Asn Asp Pro Thr Leu Thr Leu Asp

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Lys Phe Val Lys Glu Asn Ser Asp Ala Val Lys Phe Lys Leu Thr Thr

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Claims (10)

1. A mutant of alcohol dehydrogenase, characterized in that the mutant is obtained by mutating 214 th glutamic acid, 215 th threonine and 237 th serine of alcohol dehydrogenase with an amino acid sequence of SEQ ID No. 2; or

The amino acid sequence is obtained by mutating phenylalanine 161, serine 196 and glutamic acid 214 of alcohol dehydrogenase with SEQ ID No. 2.

2. The mutant of alcohol dehydrogenase according to claim 1, wherein the mutant is obtained by replacing glutamic acid at position 214 with isoleucine, threonine at position 215 with serine, and serine at position 237 with alanine in the alcohol dehydrogenase whose amino acid sequence is shown in SEQ ID No. 2.

3. The mutant of alcohol dehydrogenase according to claim 1, wherein the mutant is obtained by replacing phenylalanine at position 161, serine at position 196 and glutamic acid at position 214 of the alcohol dehydrogenase having an amino acid sequence shown in SEQ ID No.2 with valine, glycine and glycine.

4. A gene encoding a mutant of the alcohol dehydrogenase of any one of claims 1-3.

5. A vector carrying the gene of claim 4.

6. A host cell comprising the vector of claim 5.

7. Use of a mutant of an alcohol dehydrogenase according to any one of claims 1-3, a gene according to claim 4, a vector according to claim 5 or a host cell according to claim 6 for catalyzing the synthesis of a para-substituted bisaryl chiral alcohol.

8. Use according to claim 7, wherein the bisaryl chiral alcohol is 2-pyridyl [4- (trifluoromethyl) phenyl ] methanol or (4-methylphenyl) (2-pyridyl) methanol.

9. A method for synthesizing diaryl chiral alcohol is characterized by comprising the following steps: adding the mutant of alcohol dehydrogenase described in any one of claims 1-3 into a reaction system containing a latent chiral carbonyl compound to perform a reaction to obtain the diaryl chiral alcohol; the latent chiral diaryl ketone is 2-pyridyl [4- (trifluoromethyl) phenyl ] ketone or (4-methylphenyl) (2-pyridyl) ketone.

10. The synthetic method according to claim 9 wherein the concentration of the potentially chiral carbonyl compound in the reaction system is 10-100 mM; the addition amount of the alcohol dehydrogenase mutant is 1-10 kU/L.

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