CN114457125A

CN114457125A - Method for synthesizing (R) -1- [3,5-bis (trifluoromethyl) phenyl ] ethylamine by coupling transaminase and alcohol dehydrogenase

Info

Publication number: CN114457125A
Application number: CN202111485627.7A
Authority: CN
Inventors: 欧志敏; 卢媛; 李景华; 程朋朋; 王金美; 张楚玥
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2021-12-07
Filing date: 2021-12-07
Publication date: 2022-05-10
Anticipated expiration: 2041-12-07
Also published as: CN114457125B

Abstract

The invention discloses a method for synthesizing (R) -1- [3,5-bis (trifluoromethyl) phenyl by coupling transaminase and alcohol dehydrogenase]Method for preparing ethylamine by taking R-omega-transaminase and alcohol dehydrogenase as double-enzyme catalysts and 3, 5-bistrifluoroethylene as catalystMethylacetophenone as amino acceptor, isopropylamine hydrochloride as amine donor, pyridoxal phosphate and NAD⁺Adding cosolvent as auxiliary factor, using buffer solution with pH value of 6-10 as reaction medium to form reaction system, stirring at 30-50 deg.C and 150-]Ethylamine; when the substrate concentration is 166mM, the yield is improved from 82.96% to 99.9%, and the amination efficiency of the substrate BPO is obviously improved, so that the process for preparing R-BPA by efficiently aminating BPO is established, and a reference is provided for later industrial application.

Description

Method for synthesizing (R) -1- [3,5-bis (trifluoromethyl) phenyl ] ethylamine by coupling transaminase and alcohol dehydrogenase

(I) the technical field

The invention relates to a preparation method of (R) -1- [3,5-bis (trifluoromethyl) phenyl ] ethylamine, in particular to a preparation method of (R) -1- [3,5-bis (trifluoromethyl) phenyl ] ethylamine (R-BPA) by using a R-omega-transaminase (ATA117) and Alcohol Dehydrogenase (ADH) dual-enzyme coupling system and using the system in asymmetric amination reaction of 3,5-bis (trifluoromethyl) acetophenone (BPO).

(II) background of the invention

(R) -1- [3,5-bis (trifluoromethyl) phenyl](R) -1- [3,5-bis (trifluoromethylphenyl) phenyl ethyl amine]ethaneamine, R-BPA), Cas number 127733-47-5, molecular formula C₁₀ H₉ F₆N, molecular weight 257.18. R-BPA is a good candidate drug-the chiral intermediate of selective tetrodotoxin sensitive (TTX-S) blockers, TTX-S has a variety of therapeutic uses in the treatment of pain. Local anesthetic lidocaine and volatile anesthetic halothane act with poor selectivity and low potency (IC) against tetrodotoxin-insensitive (TTX-R) and TTX-S sodium channels₅₀Values in the range 50. mu.M-10 mM). These anesthetics at higher systemic concentrations may cause destructive side effects such as paralysis and cardiac arrest. However, systemic administration of lidocaine at low concentrations is effective for the treatment of chronic pain (NPL 11). In rats, mechanical allodynia behavior was significantly reduced if very low doses of TTX were applied to DRG of the damaged segment of the L5 spinal nerve (NPL 12). This result suggests that the TTX-S subtype of sodium channel plays an important role in the maintenance of allodynic behaviour in animal models of neuropathic pain.

R-BPA is an important chiral amine drug intermediate. Chiral amines are indispensable pharmaceutical building blocks in active pharmaceutical ingredients, agrochemicals and bioactive natural products. Transaminases (TAs) have been used as extremely attractive biocatalysts for the synthesis of optically pure chiral amines due to their advantages of excellent stereoselectivity, high yield, broad substrate specificity, environmental friendliness, and the absence of redox cofactor recycling. The enzyme catalyzes the transfer of an amino group from an amino donor to a carbonyl group by a ping-pong reaction mechanism using pyridoxal 5' -phosphate (PLP) as a cofactor. Compared to chemical catalysts in the synthesis of chiral amino compounds, TAs are one of the most promising enzymes, which can provide a more compact reductive amination pathway. TAs can be classified into two types, α -transaminase (α -TAs) which transfers an amino group at the α -position of an amino acid and ω -transaminase (ω -TAs) which acts on an amino group at any other position than the α -position, depending on the position of the transferred amino group. The substrates of omega-TAs do not necessarily require carboxyl groups compared to alpha-TAs, which makes them more versatile than alpha-TAs in biocatalytic conversion. Biocatalytic asymmetric amination is becoming an attractive alternative to the traditional chemical methods for the synthesis of chiral amines.

At present, the methods for preparing chiral amine mainly comprise a chemical method, a biological resolution method and a biological asymmetric synthesis method. The synthesis of chiral amine by chemical method usually needs expensive metal catalyst and special solvent, and has high production cost, low enantioselectivity and certain environmental pollution. The maximum theoretical yield of the high-purity chiral amine prepared by the chiral resolution method of the biocatalyst is only 50%. Therefore, both methods cannot meet the requirements of practical production. The first choice strategy for producing chiral amine by a biological method is asymmetric synthesis, the theoretical yield of the chiral amine reaches 100 percent, the high yield of the chiral amine has wide application prospect in the field of production industry, and related research is widely regarded.

In practice, due to unfavorable thermodynamic equilibrium, it is often necessary to superstoichiometrically add isopropylamine and remove the corresponding acetone by-product in situ to shift the reaction equilibrium in the desired direction. In order to maximize the productivity of TAs, it is important to find efficient methods for promoting biotransformation. Process control strategies have been developed to remove products in situ, with much attention being paid to the cascade of additional biochemical reactions that utilize byproducts.

omega-TAs catalyze transamination reactions between an amino donor and an amino acceptor substrate, enabling catalytic asymmetric ammonification on the carbonyl group of a prochiral substrate under mild conditions, without the need for additional cofactor recycling. The omega-TAs are used for producing the chiral amine with optical purity, so that the high-pressure, high-temperature, high-energy consumption and high pollution of the traditional chemical method can be avoided, and the method is an advanced biological preparation which accords with the green production concept. Based on this, the present inventors have conducted studies on the application of omega-TAs to biocatalysis.

Disclosure of the invention

The invention aims to provide a method for synthesizing (R) -1- [3,5-bis (trifluoromethyl) phenyl ] ethylamine by coupling transaminase and alcohol dehydrogenase, which is implemented by screening a substrate spectrum of R-omega-TAs to obtain a corresponding substrate with good yield and enantiomer excess (e.e.%), and further researching the influence of a series of reaction factors (cosolvent, temperature, pH, ADH, ion species and the like) on asymmetric amination. Ethanol was substituted for dimethyl sulfoxide (DMSO) conventionally used. In addition, in the transamination reaction using Isopropylamine (IPA) as amine donor, adding a proper amount of Saccharomyces cerevisiae Alcohol Dehydrogenase (ADH) has a significant effect of promoting the yield. Under the optimal reaction condition, the R-omega-TAs/ADH coupling system catalyzes the asymmetric amination of the 3,5-bis (trifluoromethyl) acetophenone, and the catalytic reaction efficiency is obviously improved, so that the process for preparing the R-BPA by efficiently aminating the BPO through the double-enzyme-level connected system is established, and the reference is provided for the later-stage industrial application.

The technical scheme adopted by the invention is as follows:

the invention provides a method for synthesizing (R) -1- [3,5-bis (trifluoromethyl) phenyl by coupling R-omega-transaminase (R-omega-TAs) and Alcohol Dehydrogenase (ADH)]A process for ethylamine (R-BPA), the process being carried out as follows: taking R-omega-transaminase and alcohol dehydrogenase as double-enzyme catalysts, 3,5-bis (trifluoromethyl) acetophenone (BPO) as an amino acceptor, isopropyl amine hydrochloride (IPA) as an amine donor, and pyridoxal phosphate (PLP) and NAD (NAD)⁺Adding cosolvent as cofactor, using buffer solution with pH6-10 as reaction medium to form reaction system, mixing and stirring in shaking table at 30-50 deg.C (preferably 40 deg.C) and 150-200rpm (preferably 180rpm) for 12-36h (preferably 24h), separating and purifying reaction solution to obtain (R) -1- [3,5-bis (trifluoromethyl) phenyl]Ethylamine; the cosolvent is methanol, ethanol, Tween 80, Tween 20, span 80, glycerol or dimethyl sulfoxide (DMSO).

Further, the cosolvent is preferably ethanol.

Further, in the reaction system, the adding amount of the R-omega-transaminase is 1-2U/mL (preferably 1.35U/mL) based on the volume of the reaction system; the adding amount of the alcohol dehydrogenase is 1-2U/mL (preferably 1.35U/mL) based on the volume of the reaction system; the adding amount of the 3,5-bis (trifluoromethyl) acetophenone is 25-170mmol/L (preferably 166mmol/L) based on the volume of the reaction system; the addition amount of the isopropylamine hydrochloride is 125-850mmol/L (preferably 832mmol/L) based on the volume of the reaction system; the addition amount of the pyridoxal phosphate is 0.5-1.5mmol/L (preferably 1mmol/L) based on the volume of the reaction system; the NAD⁺The adding amount is 0.5-1.5mmol/L (preferably 1mmol/L) based on the volume of the reaction system; the adding amount of the cosolvent is 10-30% (preferably 15%) by volume of the reaction system.

Further, the R-omega-transaminase is pure enzyme extracted by carrying out ultrasonic disruption on wet thalli obtained by fermenting and culturing engineering bacteria containing an R-omega-transaminase gene, the nucleotide sequence of the R-omega-transaminase gene is shown in SEQ ID NO.1, and the pure enzyme is added in the form of aqueous solution, wherein the preferable concentration is 15-20 mg/mL; the engineering bacteria are constructed by inserting an R-omega-transaminase gene (JA717225.1, ATA117) between BamHI and XhoI restriction sites of a pETduet-1 expression vector and transforming escherichia coli BL21(DE 3).

Further, the pure enzyme of the R-omega-transaminase is prepared by the following steps:

(1) and (3) induction culture: inoculating the engineered bacterium containing the R-omega-transaminase gene into 50mL LB liquid culture medium containing 30-80 mug/mL (preferably 50 mug/mL) ampicillin, and culturing at 37 ℃ and 180rpm overnight to obtain a seed solution; then inoculating the seed solution into a fresh LB liquid culture medium containing 30-80. mu.g/mL (preferably 50. mu.g/mL) ampicillin in an amount of 1-5% (preferably 3%) by volume, and culturing at 37 deg.C and 180rpm to OD₆₀₀0.6-0.9 (preferably 0.8), adding isopropyl-beta-thiogalactoside (IPTG) with final concentration of 0.5-1.5mM (preferably 1mM), transferring to 16-26 deg.C (preferably 18 deg.C), inducing expression for 10-20 hr (preferably 15 hr), centrifuging (preferably placing the culture solution at 4 deg.C, 8000rpm for 10min), collecting cell precipitate, washing with 0.9% (v/w) physiological saline (preferably 2 times), and collecting wet thallusA cell; (2) crude enzyme solution: re-suspending the wet bacterial cells (preferably 50 g/L) harvested in the step (1) in 100mM sodium phosphate buffer solution (pH 7.0), carrying out ultrasonic disruption for 5 minutes (power 400W, working for 3 seconds, and disruption for 7 seconds), collecting the disruption solution, centrifuging (preferably 8000rpm, and centrifuging at 4 ℃ for 10 minutes), and taking the supernatant to obtain a crude transaminase solution; (3) pure enzyme: mixing the transaminase crude enzyme solution with a chromatography medium according to the volume ratio of 8:1 (preferably placed in ice cubes, shaken slowly for 1h in a 40rpm shaker) and then loaded onto a Beyogold^TMA His-tag chromatographic column, wherein the column is washed by non-denatured washing liquid for 5 times to remove impure protein, and the volume of the column is eluted by 1 to 2 times; eluting 6-10 times (preferably 8 times) by using non-denatured eluent, eluting 1-2 column volumes each time, collecting eluent containing target protein, performing ultrafiltration concentration, and freeze-drying to obtain pure enzyme of the R-omega-transaminase; the non-denaturing washing solution is 50mM sodium phosphate buffer, pH8.0, containing 300mM NaCl and 2mM imidazole; the non-denaturing eluent was a 50mM sodium phosphate buffer, pH8.0, containing 300mM NaCl and 50mM imidazole.

Further, the alcohol dehydrogenase is pure enzyme obtained by carrying out ultrasonic disruption extraction on wet bacteria obtained by fermenting and culturing engineering bacteria containing alcohol dehydrogenase genes, and the nucleotide sequence of the alcohol dehydrogenase genes is shown as SEQ ID No. 2; the pure enzyme is added in the form of an aqueous solution, preferably at a concentration of 15-20 mg/mL. The engineering bacteria are constructed by inserting an ethanol dehydrogenase gene (NP-014555.1, ADH) between BamHI and XhoI restriction sites of a pET-28a (+) expression vector and transforming Escherichia coli BL21(DE 3).

Further, the pure enzyme of the alcohol dehydrogenase is prepared by the following method: (1) and (3) induction culture: inoculating the ethanol dehydrogenase gene engineering bacteria to LB liquid culture medium containing 30-80 mug/mL (preferably 50 mug/mL) Kan antibiotics, and placing the LB liquid culture medium in a shaker at 37 ℃ and 180rpm for overnight culture to obtain seed liquid; inoculating to LB liquid medium containing 30-80. mu.g/mL (preferably 50. mu.g/mL) Kan at a volume concentration of 1-5% (preferably 3%), and further culturing at 37 deg.C and 180rpm to OD₆₀₀0.6-0.9 (preferably 0.7), adding IPTG with final concentration of 0.5-1.5mM (preferably 1mM), inducing expression at 23 deg.C and 180rpm for 10-20 hr (preferably 15 hr), and centrifuging (preferably subpackaging the culture solution into centrifuge tube)Centrifuging at 4 deg.C and 8000rpm for 10min), collecting cell precipitate, washing with 0.9% physiological saline (preferably 3 times), centrifuging (preferably 4 deg.C and 8000rpm for 10min), and collecting wet cells; (2) crude enzyme solution: suspending the harvested wet cells in a 100mM sodium phosphate buffer solution with pH of 7.0 in an amount of 50g/L, carrying out ultrasonic treatment on the mixture for 5 minutes under an ice bath condition, carrying out work for 3 seconds at a power of 400W, crushing for 7 seconds, collecting the crushed liquid after ultrasonic treatment, centrifuging for 10 minutes at a temperature of 4 ℃ at 8000rpm, and taking the supernatant to obtain a crude enzyme solution of the alcohol dehydrogenase; (3) pure enzyme solution: mixing the crude enzyme solution of the alcohol dehydrogenase with a chromatography medium according to the volume ratio of 8:1 (preferably placed in ice cubes, shaken slowly for 1h in a 40rpm shaker) and then loaded onto a Beyogold^TMHis-tag chromatographic column, washing the column with non-denaturing washing liquid for 5 times to remove impurity protein, and eluting 1-2 column volumes each time; eluting for 6-10 times by using non-denatured eluent, eluting for 1-2 column volumes each time, collecting eluent containing target protein, and performing ultrafiltration concentration and freeze-drying to obtain pure enzyme of the R-omega-transaminase; the non-denaturing washing solution is 50mM sodium phosphate buffer, pH8.0, containing 300mM NaCl and 2mM imidazole; the non-denaturing eluent was a 50mM sodium phosphate buffer, pH8.0, containing 300mM NaCl and 50mM imidazole.

The ultrafiltration concentration of the invention refers to that crude enzyme liquid is filled into an ultrafiltration tube (10000MWCO) for ultrafiltration concentration treatment, and the intercepted liquid is taken as concentrated solution; the freeze-drying is to pre-freeze the concentrated solution in a refrigerator with the temperature of 80 ℃ below zero for 8h and then put the pre-frozen concentrated solution into a freeze dryer to be dried in vacuum with the temperature of 65 ℃ below zero for 12 h.

Further, the reaction system also comprises metal ions and reagents, and then the reaction system comprises: taking R-omega-transaminase and alcohol dehydrogenase as double-enzyme catalysts, 3,5-bis (trifluoromethyl) acetophenone (BPO) as an amino acceptor, isopropylamine hydrochloride (IPA) as an amine donor, and pyridoxal phosphate (PLP) and NAD (NAD)⁺Adding cosolvent, metal ions and reagent as auxiliary factors, and forming a reaction system by using a buffer solution with the pH value of 6-10 as a reaction medium; the metal ion and the reagent are Ca²⁺、Mg²⁺、Cu²⁺、Fe²⁺、Zn²⁺、Mn²⁺、Na⁺、K⁺Or EDTA, preferably EDTA; the metal ions and the reagent are in a reaction systemWere added to the reaction solution at a final concentration of 10mM each.

Further, the reaction liquid separation and purification method comprises the following steps: after the reaction is finished, adding equal volume of ethyl acetate into the reaction solution for extraction, drying the extract by using anhydrous sodium sulfate, and volatilizing the ethyl acetate to obtain R-BPA.

Compared with the prior art, the invention has the following beneficial effects:

in practice, due to unfavorable thermodynamic equilibrium, it is often necessary to superstoichiometrically add isopropylamine and remove the corresponding acetone by-product in situ to shift the reaction equilibrium in the desired direction. In order to maximize the productivity of R- ω -transaminase, it is important to find an efficient method for accelerating biotransformation. According to the invention, transaminase ATA117 and yeast alcohol dehydrogenase ADH are used in a combined manner, and more green and environment-friendly ethanol is used for replacing a common cosolvent DMSO of an organic substrate, so that the solubility of the substrate BPO in an aqueous phase system is enhanced. The first step BPO amination reaction process is as follows: ATA117 takes isopropylamine as an amino donor and PLP as a coenzyme to catalyze the amination of BPO as a substrate to R-BPA, and the byproduct is acetone. The reaction process for removing the acetone as the byproduct in situ in the second step comprises the following steps: the ADH catalyzes and assists the substrate ethanol to generate acetaldehyde to realize the regeneration of coenzyme NADH, and sufficient NADH promotes the ADH to catalyze acetone to generate propanol. The second step reaction converts the byproduct acetone of the first step reaction into ethanol, and improves the amination reaction efficiency of the substrate BPO. The ethanol not only plays a role of an organic substrate cosolvent, but also is used as a substrate of the second step cascade reaction for in-situ regeneration of coenzyme NADH. According to the invention, the first-step substrate amination reaction catalyzed by ATA117 and the in-situ removal reaction of acetone as a second-step by-product catalyzed by ADH are cascaded, the catalytic yield of 82.96% (single enzyme) is improved to 99.9% (double-enzyme cascade) when the substrate concentration is 166mM, the amination efficiency of the substrate BPO is obviously improved, and thus the process for preparing R-BPA by efficiently aminating BPO is established, and the process control strategy provides reference for later-stage industrial application.

(IV) description of the drawings

FIG. 1 shows the SDS-PAGE (SDS-PAGE) of the purified transaminase ATA117(A) and dehydrogenase ADH (B) of the present invention on sodium dodecyl sulfate polyacrylamide gel.

FIG. 2 is a schematic diagram of the reaction mechanism of the cascade reaction constructed by the double enzymes for aminating BPO to generate R-BPA.

(V) detailed description of the preferred embodiments

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

the R- ω -transaminase (ATA117) gene used in the examples of the present invention was derived from NCBI library (https:// www.ncbi.nlm.nih.gov/nuccore/JA 717225.1).

The yeast Alcohol Dehydrogenase (ADH) gene used in the examples of the present invention was derived from the NCBI library (https:// www.ncbi.nlm.nih.gov/protein/NP-014555.1).

The Beyogold^TMThe His-tag Purification Resin protein Purification column is of a reduction-resistant chelate type and is purchased from Shanghai Bintian biotechnology limited company in China.

Composition of LB liquid medium: 5g/L of yeast extract, 10g/L of peptone, 10g/L of sodium chloride, water as solvent and pH 7.

LB solid medium composition: 5g/L of yeast extract, 10g/L of peptone, 10g/L of sodium chloride, 18g/L of agar, water as a solvent and pH 7.

Example 1: preparation of transaminase

1. Transaminase crude enzyme liquid

The R-omega-transaminase gene (gene accession number JA717225.1, nucleotide sequence shown in SEQ ID NO.1, and designated as transaminase gene ATA117) was inserted between the BamHI and XhoI restriction sites of the pETduet-1 expression vector, transformed into E.coli DH 5. alpha. competence, the recombinant plasmid pETduet-ATA117 was extracted and E.coli BL21(DE3) was transformed. The obtained transformant was inoculated into 50mL of LB liquid medium containing 50. mu.g/mL of ampicillin (Amp), and cultured overnight at 37 ℃ and 180rpm to obtain a seed solution of recombinant Escherichia coli. Inoculating the seed solution into 0.15L of fresh LB liquid medium containing 50. mu.g/mL ampicillin at a volume concentration of 3%, and performing fermentation culture at 37 deg.C and 180rpm to an Optical Density (OD) at 600nm₆₀₀) At 0.8, isopropyl-. beta. -thiogalactoside (IPTG) was added to a final concentration of 1mM to induce gene expression, and the culture was then transferred to a shaker at 180rpm at 18 ℃ to induce gene expressionThe expression is induced for 15 h. The culture solution was dispensed into 50mL centrifuge tubes and centrifuged at 8000rpm for 10min at 4 ℃ to collect the over-expressed cell pellet, which was washed 3 times with 0.9% (v/w) physiological saline. 0.8g of the harvested wet cells were resuspended in 100mM sodium phosphate buffer (pH 7.0) in an amount of 50g/L and the mixture was sonicated for 5 minutes (power 400W, working for 3 seconds, disruption for 7 seconds). Collecting the crushed solution in a 50mL centrifuge tube, centrifuging for 10min at 8000rpm and 4 ℃, taking the supernatant, loading into an ultrafiltration tube (10000MWCO) for ultrafiltration concentration treatment, and intercepting the liquid to obtain 8mL of the concentrated transaminase crude enzyme solution.

The nucleotide sequence of the R-omega-aminotransferase gene is shown as SEQ ID NO. 1:

atggcgttctcagcggacacccctgaaatcgtttacacccacgacaccggtctggactatatcacctact ctgactacgaactggacccggctaacccgctggctggtggtgctgcttggatcgaaggtgctttcgttcc gccgtctgaagctcgtatccctatcttcgaccagggtttttatacttctgacgctacctacaccaccttc cacgtttggaacggtaacgctttccgtctgggggaccacatcgaacgtctgttctctaatgcggaatcta ttcgtttgatcccgccgctgacccaggacgaagttaaagagatcgctctggaactggttgctaaaaccga actgcgtgaagcgatggttaccgttacgatcacccgtggttactcttctaccccattcgagcgtgacatc accaaacatcgtccgcaggtttacatgagcgctagcccgtaccagtggatcgtaccgtttgaccgcatcc gtgacggtgttcacctgatggttgctcagtcagttcgtcgtacaccgcgtagctctatcgacccgcaggt taaaaacttccagtggggtgacctgatccgtgcaattcaggaaacccacgctcgtggtttcgagttgccg ctgctgctggactgcgacaacctgctggctgaaggtccgggcttcaacgttgttgttatcaaagacggtg ttgttcgttctccgggtcgtgctgctctgccgggtatcacccgtaaaaccgttctggaaatcgctgaatc tctgggtcacgaagctatcctggctgacatcaccccggctgaactgtacgacgctgacgaagttctgggt tgctcaaccggtggtggtgtttggccgttcgtttctgttgacggtaactctatctctgacggtgttccgg gtccggttacccagtctatcatccgtcgttactgggaactgaacgttgaaccttcttctctgctgacccc ggtacagtac。

2. transaminase pure enzyme solution

(1) Chromatographic column pre-equilibration

2ml of 50% Beyogold^TMHis-tag Purification Resin was centrifuged at 8000rpm for 1min at 4 ℃ to discard the stock solution, to obtain 1ml of a gel. A3 mL affinity column containing 1mL of gel was loaded with non-denaturing lysis buffer (pH 8.0, 50mM sodium phosphate buffer and 300mM NaCl)Pre-balancing, centrifuging at 8000rpm at 4 deg.C for 1min, discarding liquid, repeating balancing twice, and discarding supernatant to obtain the chromatography column after pre-balancing.

(2) Sample loading and elution

Loading 8mL of transaminase crude enzyme solution prepared by the method in the step 1 (the ratio of the crude enzyme solution to the gel volume of the column in the step 1 is 8:1), loading the solution into the chromatographic column pre-balanced in the step (1), uniformly oscillating, placing the chromatographic column in an ice block, slowly shaking the solution in a shaking table at 40rpm for 1h, transferring the solution into a new chromatographic column, eluting the column by using a non-denaturing washing solution (pH 8.0, 50mM sodium phosphate buffer solution, 300mM NaCl and 2mM imidazole) depending on gravity drop to remove impure proteins, and washing the column for 5 times, wherein the dosage of each time is 1 column volume, and the elution speed is about 0.5 mL/min. The transaminase-target protein was then purified by elution with non-denaturing eluents (pH 8.0, 50mM sodium phosphate buffer, 300mM NaCl and 50mM imidazole) 8 times for 1 column volume per elution, and the eluates collected for each column volume were designated Lane 1-Lane 8 and monitored by 12% SDS-PAGE, as shown in FIG. 1, panel A. From SDS-PAGE, it can be seen that Lane 1-Lane 6 all contain target proteins, the molecular weight is about 35kDa, the content is reduced from small to large, and Lane7 and Lane8 do not elute corresponding proteins, so they are not shown. And finally, combining the eluates of Lane 1-Lane 6 to obtain the eluent containing the target protein, pre-freezing the eluent in a refrigerator at the temperature of-80 ℃ for 8 hours, and then putting the eluent into a freeze dryer for vacuum drying at the temperature of-65 ℃ for 12 hours to obtain 15mg of pure transaminase.

3. Enzyme activity detection

The transaminase activity assay system (2mL) consists of: 832mM isopropylamine hydrochloride (IPA, amine donor), 1mM pyridoxal 5' -phosphate (PLP, cofactor), 0.3mL dimethyl sulfoxide (DMSO, co-solvent), 166.56 mM 3, 5-bistrifluoromethylacetophenone (BPO, substrate), 1mL of a 15mg/mL aqueous transaminase pure solution prepared in step 2, made up to 2mL with 100mM Tris-HCl (pH 9.0) buffer. The reaction was carried out at 45 ℃ for 2h at 180rpm, and 1. mu.L of trifluoroacetate (TFA, 1%) was added to terminate the reaction. The peak area of (R) -1- [3,5-bis (trifluoromethyl) phenyl ] ethylamine ((R) -BPA) in the reaction solution was measured by Gas Chromatography (GC), and the content of (R) -BPA was obtained from a standard curve by the same method as in example 3.

Under the above reaction conditions, the enzyme activity unit (U) is defined as the amount of enzyme required to produce 1. mu. mol (R) -BPA per minute under standard assay conditions. The enzyme activity of the pure enzyme prepared in the step 2 is determined to be 0.18U/mg.

Example 2: preparation of alcohol dehydrogenase

1. Crude enzyme solution of alcohol dehydrogenase

An ethanol dehydrogenase gene (NP-014555.1, amino acid sequence shown in SEQ ID NO.2 and recorded as ADH) is inserted between BamHI and XhoI restriction sites of a pET-28a (+) expression vector, transformed into Escherichia coli DH5 alpha competent cells, pET28a-ADH recombinant plasmids are extracted and introduced into Escherichia coli BL21(DE3) competence, activated in a shaker at 37 ℃ and 180rpm for 1h, coated with LB plates containing 50. mu.g/mL Kan antibiotics, and then placed upside down in a shaker at 37 ℃ for standing and culturing overnight. Single colonies were picked up in 50mL LB liquid medium containing 50. mu.g/mL Kan antibiotic and cultured overnight in a shaker at 37 ℃ and 180rpm to obtain a seed solution. Transferring the inoculum size of 3% volume concentration into 0.15L LB liquid medium containing 50. mu.g/mL Kan, and further culturing at 37 deg.C and 180rpm to OD₆₀₀At 0.7, IPTG was added to the final concentration of 1mM, and the induction was carried out at 23 ℃ and 180rpm for 15 hours. After induction, the culture solution is subpackaged in a centrifuge tube and placed in a centrifuge with the temperature of 4 ℃ and the rpm of 8000 for 10 min. The collected cell pellets were washed with 10mL of 0.9% (v/w) physiological saline each time, 3 times in total. Wet cells were collected by centrifugation at 8000rpm for 10min at 4 ℃. 0.8g of the harvested wet cells were resuspended in a quantity of 50g/L in 100mM sodium phosphate buffer (pH 7.0) and the mixture was sonicated for 5 minutes under ice bath conditions (power 400W, working 3 seconds, disruption 7 seconds). Collecting the crushed liquid after ultrasonic treatment, centrifuging at 8000rpm and 4 deg.C for 10min, collecting supernatant, loading into ultrafiltration tube (10000MWCO), and ultrafiltering and concentrating (5000 Xg, 30min), wherein the trapped liquid is 8mL of the concentrated crude enzyme solution of alcohol dehydrogenase.

The amino acid sequence of the alcohol dehydrogenase is shown as SEQ ID NO. 2:

MSIPETQKGVIFYESHGKLEYKDIPVPKPKANELLINVKYSGVCHTDLHAWHGDWPLPVKLPLV GGHEGA

GVVVGMGENVKGWKIGDYAGIKWLNGSCMACEYCELGNESNCPHADLSGYTHDGSFQQYAT ADAVQAAHI

PQGTDLAQVAPILCAGITVYKALKSANLMAGHWVAISGAAGGLGSLAVQYAKAMGYRVLGID GGEGKEEL

FRSIGGEVFIDFTKEKDIVGAVLKATDGGAHGVINVSVSEAAIEASTRYVRANGTTVLVGMPAG AKCCSD

VFNQVVKSISIVGSYVGNRADTREALDFFARGLVKSPIKVVGLSTLPEIYEKME。

2. pure enzyme solution of alcohol dehydrogenase

(1) Chromatographic column pre-equilibration

The procedure of example 1 was repeated, and the column packing volume of the gel was 1 mL.

(2) And (2) loading 8mL of the crude alcohol dehydrogenase prepared in the step (1) (the volume ratio of the crude alcohol dehydrogenase to the gel loaded in the step (1) is 8:1), loading the crude alcohol dehydrogenase into the chromatographic column pre-equilibrated in the step (1), uniformly oscillating, placing the mixture into an ice block, slowly shaking the mixture in a shaking bed at 40rpm for 1h, transferring the mixture into a new chromatographic column, eluting the mixture by using a non-denaturing washing solution (pH 8.0, 50mM sodium phosphate buffer solution, 300mM NaCl and 2mM imidazole) depending on gravity reduction to remove impure proteins, washing the column for 5 times, wherein the dosage is 1 column volume each time, and the elution speed is about 0.5 mL/min. Then, the target protein of alcohol dehydrogenase was purified by eluting with a non-denaturing eluent (pH 8.0, 50mM sodium phosphate buffer, 300mM NaCl and 50mM imidazole) 8 times at 1 column volume per elution, and the eluates, designated Lane1 to Lane8, were collected for each column volume and monitored by 12% SDS-PAGE, and the results are shown in B of FIG. 1. And mixing the eluates of Lane 1-Lane 6 to obtain the eluent containing the target protein, pre-freezing the eluent in a refrigerator at the temperature of-80 ℃ for 8 hours, and then putting the eluent into a freeze dryer to carry out vacuum drying at the temperature of-65 ℃ for 12 hours to obtain the pure alcohol dehydrogenase of 18 mg.

3. Determination of enzyme Activity

The 5mL ethanol dehydrogenase enzyme activity determination system comprises: 2.5mL Glycine-sodium hydroxide buffer (pH 8.6) at a final concentration of 1X 10^-3mol/L NAD⁺And final concentration 5X 10^-3mol/L ethanol, in 25 ℃ water bath warm 20 minutes, adding in the same conditions warm 1mL 18mg/mL step 2 preparation of pure enzyme ethanol dehydrogenase aqueous solution. The absorbance at 340nm was then read every 1 minute for 5 consecutive minutes, timed immediately. The amount of enzyme required to increase the A340 by 0.001 per minute was one unit (U) at 25 ℃ and pH 8.6. The enzyme activity was determined to be 0.15U/mg.

Example 3: screening of transaminase substrate profiles

A 2mL system was constructed in 13 glass vials: 13 different substrates at a final concentration of 166mM (added after first solubilised with co-solvent ethanol), isopropylamine hydrochloride (IPA) at a final concentration of 832mM, pyridoxal phosphate (PLP) at a final concentration of 1mM, 15mg/mL of the pure enzyme aqueous transaminase solution prepared by the method of example 1 (total enzyme activity 2.7U), 15% ethanol (co-solvent) at a final volume concentration, made up to 2mL with 0.1M Tris-HCl buffer pH 9.

Placing the reaction system in a shaker at 45 ℃ and 180rpm for reaction for 24h, taking out reaction liquid, centrifuging at 4 ℃ and 8000rpm for 10min, collecting supernatant, adding equal volume of ethyl acetate for extraction, repeatedly extracting for three times, drying the collected extract with anhydrous sodium sulfate, volatilizing ethyl acetate at normal temperature, detecting the peak areas of substrates 3,5-bis (trifluoromethyl) acetophenone (BPO) and (R) -1- [3,5-bis (trifluoromethyl) phenyl ] ethylamine ((R) -BPA) and (S) -1- [3,5-bis (trifluoromethyl) phenyl ] ethylamine ((S) -BPA) by gas chromatography, calculating the content by adopting an internal standard method (adding dodecane as an internal standard substance), calculating the yield of (R) -BPA and the enantiomer excess value (eep) by adopting a formula (1) and a formula (2), the results are shown in Table 1.

Gas Chromatography (GC) detection conditions: chiral chromatography column CP7502(25m × 0.25mm × 0.25 μm); the sample inlet temperature is 250 ℃, the column temperature is 110 ℃, the detector is 250 ℃, the flow rate is 1mL/min, and the split ratio is 1: 15, sample size 1. mu.L.

The yield (X) and enantiomeric excess (eep) of the product (R) -BPA were calculated by the formula (1) and the formula (2):

in formula (1), Ms: the molecular weight of the substrate; and Mp: molecular weight of product (R) -BPA; q: the mass of substrate at the beginning of the reaction; p: quality of product (R) -BPA at the end of the reaction.

In the formula (2), C_R: the concentration of R-BPA; cs: concentration of S-BPA.

TABLE 1 screening of transaminase substrate profiles

Transaminase can catalyze amination of various aldehydes and ketones, so the substrate range is relatively wide, and engineering (R) -omega-TA (ATA117) has successfully realized industrial application of sitagliptin (an oral hypoglycemic drug) by using sitagliptin precursor ketone as a substrate. To examine the substrate specificity of the transaminase ATA117 to broaden the substrate spectrum of the transaminase ATA117, the reactivity of 13 different substrates (amino acceptors) was compared. As can be seen from Table 1, the yield of the reaction using methyl acetoacetate, ethyl acetoacetate, and 3, 5-bistrifluoromethylacetophenone as substrates was very good. However, the R-BPA enantiomer excess values corresponding to the three are slightly different. In the reaction taking 3, 5-bistrifluoromethylacetophenone as a substrate, the method has the advantages of considerable yield and better enantioselectivity, and is selected as a next experimental research object.

Example 4: effect of pH on transaminase Single-enzyme catalyzed amination reactions

In a 2mL reaction system, 166mM substrate BPO (added after previously dissolved in ethanol), 832mM amino donor IPA, 1mM cofactor pyridoxal phosphate (PLP), 1mL of a 15mg/mL aqueous solution of the transaminase pure enzyme prepared by the method of example 1 (total enzyme activity 2.7U), 15% ethanol as a cosolvent in a final volume concentration, and buffers of different pH were added to make up to 2 mL. The buffers were 0.1M pH6 sodium phosphate buffer, pH 7 sodium phosphate buffer, pH 8Tris-HCl, pH 9Tris-HCl and glycine-NaOH (pH 10), respectively. The reaction flask was placed at 45 ℃ and 180rpm for 24 h. After that, the mixture was centrifuged at 8000rpm for 10 minutes at 4 ℃ to collect the supernatant, and ethyl acetate of the same volume was added thereto for extraction, and after extraction was repeated three times, the collected extract was dried over anhydrous sodium sulfate and ethyl acetate was evaporated at normal temperature, and the yield of R-BPA and the enantiomeric excess were measured by GC method in example 3, and the results are shown in Table 2.

TABLE 2 influence of pH on transaminase monoenzymatically catalyzed amination reactions

The influence of the pH value of the buffer on the catalytic performance of the transaminase ATA117 is examined in the range of pH 6-pH 10, and the conversion rate is gradually increased along with the increase of the pH value in the range of pH 6-pH 9, and slightly reduced but still maintained at a higher level by the time of pH 10. From Table 2 it can be seen that the pH of the reaction medium may influence the activity of the enzyme, probably because a change in the pH of the reaction medium causes a change in the conformation of the active site of the enzyme. As can be seen from the trend of the yield, the enzyme is alkalophilic enzyme, the yield is controlled to be more than 80% when the pH value of the buffer solution is more than 8, and the products at the moment are all the target product R-BPA. Therefore, 0.1M Tris-HCl pH9 was determined as the optimal buffer for the system.

Example 5: effect of temperature on the Dual-enzyme coupling reaction

Double enzyme coupling group: a2 mL reaction system was charged with 166mM substrate BPO (previously dissolved in co-solvent ethanol), 832mM amino donor IPA, 1mM cofactor pyridoxal phosphate (PLP), and 1mM cofactor NAD⁺1mL of an alcohol dehydrogenase pure enzyme aqueous solution (total enzyme activity is 2.7U) prepared by the method of example 2 and 1mL of a transaminase pure enzyme aqueous solution (total enzyme activity is 2.7U) prepared by the method of example 1, wherein the volume final concentration of 15% ethanol is used as a cosolvent, and 0.1M of a pH 9Tris-HCl buffer solution is used for complementing the system to 2 mL.

Control group: the alcohol dehydrogenase in the coupled group was removed, the other was the same, and the effect of temperature on the two-enzyme catalyzed cascade reaction was studied.

The reaction flasks of the double enzyme coupling group and the control group were simultaneously placed at different temperatures of 30 ℃ to 55 ℃ and reacted at 180rpm for 24 hours. Then, the mixture was centrifuged at 8000rpm for 10 minutes at 4 ℃ to collect the supernatant, and ethyl acetate of the same volume was added thereto for extraction, and after extraction was repeated three times, the collected extract was dried over anhydrous sodium sulfate and ethyl acetate was volatilized at normal temperature, and the yield of R-BPA and the enantiomeric excess value were measured by GC method in example 3, and the results are shown in Table 3.

TABLE 3 Effect of temperature on the Cascade reaction of the two-enzyme System

Temperature significantly affects the catalytic activity of the enzyme, which changes with temperature. Due to denaturation of proteins, the enzyme activity is lost or inhibited at high temperatures. As can be seen from the table, the yield of the transaminase-single-enzyme catalyzed reaction (control) increases and then decreases in a certain temperature range (30-55 ℃), and reaches a maximum at 50 ℃. For a two-enzyme catalyzed cascade, temperature may affect the catalytic activity of both enzymes simultaneously. As can be seen from Table 3, the addition of the alcohol dehydrogenase ADH significantly accelerates the reaction in the forward direction in the range from 30 ℃ to 40 ℃. The superstoichiometric addition of isopropylamine results in the formation of the corresponding acetone by-product by transamination, while the addition of ADH catalyzes the reaction with ethanol as co-substrate to remove the corresponding acetone by-product in situ, to shift the reaction equilibrium forward, and to promote catalytic efficiency. The reaction mechanism is shown in FIG. 2. When the temperature was further increased to 50 ℃ the advantage of the cascade was no longer evident, and the yield was slightly increased compared to the control, at which point the ADH had little effect. Thus, the optimum temperature for the reaction is 40 ℃ for a two-enzyme catalyzed cascade system.

Example 6: effect of Metal ions and reagents on the Dual-enzyme coupling reaction

To investigate the effect of metal ions and reagents on the two-enzyme coupling reaction, Ca was added to a final concentration of 10mM each² ⁺、Mg²⁺、Cu²⁺、Fe²⁺、Zn²⁺、Mn²⁺、Na⁺、K⁺And EDTA into the reaction system.

2mL of reaction system: 166mM substrate BPO (previously solubilized with co-solvent ethanol), 832mM amino donor IPA, 1mM cofactor pyridoxal phosphate(PLP), final concentration 1mM cofactor NAD⁺1mL of an aqueous solution of 18mg/mL of the alcohol dehydrogenase pure enzyme prepared by the method of example 2 (total enzyme activity: 2.7U) and 1mL of an aqueous solution of 15mg/mL of the transaminase pure enzyme prepared by the method of example 1 (total enzyme activity: 2.7U) were added to the mixture to give metal ions and a reagent (Ca) each having a final concentration of 10mM²⁺、Mg²⁺、Cu²⁺、Fe²⁺、Zn²⁺、Mn²⁺、 Na⁺、K⁺And EDTA), 15% ethanol final volume as co-solvent, using 0.1M Tris-HCl buffer pH9 to make up 2 mL. After 24 hours of reaction at 40 ℃ and 180rpm, centrifugation is carried out at 4 ℃ and 8000rpm for 10 minutes, supernatant is collected and added with equal volume of ethyl acetate for extraction, after three times of extraction, collected extract is dried by anhydrous sodium sulfate, then ethyl acetate is volatilized at normal temperature, and R-BPA yield and enantiomer excess value are detected by GC method in example 3. The relative yield was calculated using the reaction without any metal ions and reagents as a control (100%).

TABLE 4 Effect of Metal ions and reagents on the Cascade reaction of two enzyme systems

This example examined the effect of high concentrations of metal ions and EDTA on the cascade of transaminase and alcohol dehydrogenase, and the results are shown in Table 4. As can be seen, the yields of most of the reaction groups were improved to some extent (1.2-1.6 times) by adding different kinds of metal ions and EDTA, with the most obvious effect (1.533 times) of adding 10mM of metal ion chelating agent EDTA. While 10mM of Mn was added²⁺And Fe³⁺Then has different degrees of inhibition effect on the reaction efficiency, wherein Fe is used³⁺The inhibitory effect of (a) is most pronounced. The cascade reaction system has slight dependence on metal ions.

Example 7: effect of Co-solvent on the Cascade reaction of two enzyme systems

This example evaluates the effect of various co-solvents, such as methanol, ethanol, tween 80, tween 20, span 80, glycerol and DMSO, on the two enzyme system cascade, taking into account the substrate solubility effect.

2mL of reaction system: substrate BPO at a final concentration of 166mM (dissolved with different co-solvents), amino donor IPA at a final concentration of 832mM, co-factor pyridoxal phosphate (PLP) at a final concentration of 1mM, co-factor NAD at a final concentration of 1mM⁺1mL of an aqueous solution of alcohol dehydrogenase-pure enzyme (total enzyme activity 2.7U) prepared by the method of example 2 and 1mL of an aqueous solution of transaminase-pure enzyme (total enzyme activity 2.7U) prepared by the method of example 1 at a concentration of 15% by volume of a final cosolvent, and 0.1M of a pH 9Tris-HCl buffer solution to make up for 2 mL.

After 24 hours of reaction at 40 ℃ and 180rpm, centrifuging at 4 ℃ and 8000rpm for 10min, collecting supernatant, adding equal volume of ethyl acetate into the supernatant for extraction, repeating the extraction for three times, drying the collected extract by using anhydrous sodium sulfate, volatilizing the ethyl acetate at normal temperature, and detecting the yield of R-BPA and the enantiomeric excess value by using a GC method in example 3. The relative yield was calculated using the reaction without any metal ions and reagents as a control (100%), and the results are shown in table 5.

TABLE 5 Effect of Co-solvent on the Cascade reaction of two-enzyme System

The solubility properties of different cosolvents for substrates are obviously different, and it can be seen from Table 5 that the types of cosolvents mainly have certain influence on the yield, but hardly have influence on the enantiomeric excess value of R-BPA, and the ee is_p(%) is controlled at a higher level (96% or more). Two groups of data of Tween 20 and Tween 80 show that the solubilization effects of Tween solvents on BPO are not very different, the effect is generally slightly better than span 80, and slightly worse than the yield of methanol as a cosolvent. Effect of DMSO, ethanol and Glycerol as Co-solvents on yield and ee_p(%) from both perspectives, it is suitable as a cosolvent for the asymmetric amination of BPO to R-BPA. Among them, ethanol is most effective. On the other hand, ethanol can also be used as an auxiliary substrate in the cascade reaction to promote the in-situ removal of acetone byproducts and promote the reaction. Ethanol was chosen as the co-solvent for the cascade reaction.

Example 8 Dual enzyme coupling reaction

A reaction flask was charged with 166mM substrate BPO (first solubilized with co-solvent ethanol), 832mM amino donor IPA, 1mM cofactor pyridoxal phosphate (PLP), and 1mM cofactor NAD⁺1mL of an aqueous solution of 18mg/mL of pure alcohol dehydrogenase prepared by the method of example 2 (total enzyme activity: 2.7U), 1mL of an aqueous solution of 15mg/mL of pure transaminase prepared by the method of example 1 (total enzyme activity: 2.7U), and 10mM EDTA, 15% ethanol in final volume concentration as a cosolvent, and 0.1M of a pH 9Tris-HCl buffer solution were used to make up 2mL of the reaction system. The system reacts for 24 hours at 40 ℃ and 180rpm, then is centrifuged for 10min at 4 ℃ and 8000rpm, the supernatant is collected and added with equal volume of ethyl acetate for extraction, after three times of extraction, the collected extract is dried by anhydrous sodium sulfate and then is volatilized at normal temperature to remove ethyl acetate, and the R-BPA yield is 99 percent and the enantiomeric excess value is 99.9 percent according to the detection of the GC method in the embodiment 3.

Sequence listing

<110> Zhejiang industrial university

<120> method for synthesizing (R) -1- [3,5-bis (trifluoromethyl) phenyl ] ethylamine by coupling transaminase and alcohol dehydrogenase

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Met Ser Ile Pro Glu Thr Gln Lys Gly Val Ile Phe Tyr Glu Ser His

1 5 10 15

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

20 25 30

Glu Leu Leu Ile Asn Val Lys Tyr Ser Gly Val Cys His Thr Asp Leu

35 40 45

His Ala Trp His Gly Asp Trp Pro Leu Pro Val Lys Leu Pro Leu Val

50 55 60

Gly Gly His Glu Gly Ala Gly Val Val Val Gly Met Gly Glu Asn Val

65 70 75 80

Lys Gly Trp Lys Ile Gly Asp Tyr Ala Gly Ile Lys Trp Leu Asn Gly

85 90 95

Ser Cys Met Ala Cys Glu Tyr Cys Glu Leu Gly Asn Glu Ser Asn Cys

100 105 110

Pro His Ala Asp Leu Ser Gly Tyr Thr His Asp Gly Ser Phe Gln Gln

115 120 125

Tyr Ala Thr Ala Asp Ala Val Gln Ala Ala His Ile Pro Gln Gly Thr

130 135 140

Asp Leu Ala Gln Val Ala Pro Ile Leu Cys Ala Gly Ile Thr Val Tyr

145 150 155 160

Lys Ala Leu Lys Ser Ala Asn Leu Met Ala Gly His Trp Val Ala Ile

165 170 175

Ser Gly Ala Ala Gly Gly Leu Gly Ser Leu Ala Val Gln Tyr Ala Lys

180 185 190

Ala Met Gly Tyr Arg Val Leu Gly Ile Asp Gly Gly Glu Gly Lys Glu

195 200 205

Glu Leu Phe Arg Ser Ile Gly Gly Glu Val Phe Ile Asp Phe Thr Lys

210 215 220

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

225 230 235 240

His Gly Val Ile Asn Val Ser Val Ser Glu Ala Ala Ile Glu Ala Ser

245 250 255

Thr Arg Tyr Val Arg Ala Asn Gly Thr Thr Val Leu Val Gly Met Pro

260 265 270

Ala Gly Ala Lys Cys Cys Ser Asp Val Phe Asn Gln Val Val Lys Ser

275 280 285

Ile Ser Ile Val Gly Ser Tyr Val Gly Asn Arg Ala Asp Thr Arg Glu

290 295 300

Ala Leu Asp Phe Phe Ala Arg Gly Leu Val Lys Ser Pro Ile Lys Val

305 310 315 320

Val Gly Leu Ser Thr Leu Pro Glu Ile Tyr Glu Lys Met Glu

325 330

Claims

1. R-omega-transaminase and alcohol dehydrogenase are coupled to synthesize (R) -1- [3,5-bis (trifluoromethyl) phenyl]A method for producing ethylamine, characterized in that the method is carried out as follows: taking R-omega-transaminase and alcohol dehydrogenase as double-enzyme catalysts, 3,5-bis (trifluoromethyl) acetophenone as an amino acceptor, isopropylamine hydrochloride as an amine donor, and pyridoxal phosphate and NAD⁺Adding cosolvent as auxiliary factor, using buffer solution with pH value of 6-10 as reaction medium to form reaction system, mixing and stirring in shaking table at 30-50 deg.C and 150-]Ethylamine; the cosolvent is methanol, ethanol, tween 80, tween 20, span 80, glycerol or dimethyl sulfoxide.

2. The method according to claim 1, wherein in the reaction system, the R- ω -transaminase is presentThe adding amount is 1-2U/mL based on the volume of the reaction system; the adding amount of the alcohol dehydrogenase is 1-2U/mL based on the volume of a reaction system; the adding amount of the 3,5-bis (trifluoromethyl) acetophenone is 25-170mmol/L based on the volume of a reaction system; the addition amount of the isopropylamine hydrochloride is 125-850mmol/L based on the volume of the reaction system; the addition amount of the pyridoxal phosphate is 0.5-1.5mmol/L based on the volume of the reaction system; the NAD⁺The adding amount is 0.5-1.5mmol/L calculated by the volume of the reaction system; the adding amount of the cosolvent is 10-30% by volume of the reaction system.

3. The method of claim 1, wherein the R- ω -transaminase is a pure enzyme obtained by subjecting a wet cell of an engineered bacterium containing an R- ω -transaminase gene, which is cultured by fermentation, to ultrasonic disruption, wherein the nucleotide sequence of the R- ω -transaminase gene is represented by SEQ ID No. 1; the pure enzyme is added in the form of an aqueous solution.

4. The method as set forth in claim 3, wherein the engineered bacterium is constructed by inserting the R- ω -transaminase gene between BamHI and XhoI restriction sites of pETduet-1 expression vector and transforming E.coli BL21(DE 3).

5. The method according to claim 3, wherein said pure R- ω -transaminase is prepared by the following steps:

(1) and (3) induction culture: inoculating the engineering bacteria containing the R-omega-transaminase gene into an LB liquid culture medium containing 30-80 mu g/mL ampicillin, and culturing at 37 ℃ and 180rpm overnight to obtain a seed solution; then inoculating the seed solution into a fresh LB liquid culture medium containing 30-80 mug/mL ampicillin according to the inoculation amount with the volume concentration of 1-5%, culturing at 37 ℃ and 180rpm until OD is reached₆₀₀0.6-0.9, adding isopropyl-beta-thiogalactoside with final concentration of 0.5-1.5mM, transferring to 16-26 deg.C for induced expression for 10-20h, centrifuging, collecting cell precipitate, washing with 0.9% physiological saline, and collecting wet thallus cell; (2) crude enzyme solution: resuspending the wet bacterial cells harvested in the step (1) in 100mM sodium phosphate buffer solution with pH 7.0, ultrasonically crushing for 5 minutes at 400W for 3 seconds, crushing for 7 seconds, and collectingCrushing the solution, centrifuging, and taking supernatant to obtain a crude transaminase solution; (3) pure enzyme: uniformly mixing the transaminase crude enzyme solution with a chromatography medium, loading the mixture to a BeyoGoldTMHis-tag chromatography column, washing the column with a non-denaturing washing solution for 5 times to remove foreign proteins, and eluting 1-2 column volumes each time; eluting for 6-10 times by using non-denatured eluent, eluting for 1-2 column volumes each time, collecting eluent containing target protein, performing ultrafiltration concentration, and freeze-drying to obtain pure enzyme of the R-omega-transaminase; the non-denaturing washing solution is 50mM sodium phosphate buffer, pH8.0, containing 300mM NaCl and 2mM imidazole; the non-denaturing eluent was a 50mM sodium phosphate buffer, pH8.0, containing 300mM NaCl and 50mM imidazole.

6. The method as claimed in claim 1, wherein the alcohol dehydrogenase is a pure enzyme obtained by ultrasonication and extraction of wet bacteria obtained by fermentation and culture of an engineering bacterium containing an alcohol dehydrogenase gene, and the nucleotide sequence of the alcohol dehydrogenase gene is shown as SEQ ID No. 2.

7. The method of claim 6, wherein the engineered bacterium is constructed by inserting an alcohol dehydrogenase gene between BamHI and XhoI restriction sites of pET-28a (+) expression vector and transforming E.coli BL21(DE 3).

8. The process as claimed in claim 6, wherein the pure alcohol dehydrogenase is prepared by the following method: (1) and (3) induction culture: inoculating the ethanol dehydrogenase gene engineering bacteria to an LB liquid culture medium containing 30-80 mu g/mL Kan antibiotics, and placing the LB liquid culture medium in a shaker at 37 ℃ and 180rpm for overnight culture to obtain a seed solution; inoculating to LB liquid medium containing 30-80. mu.g/mL Kan in an amount of 1-5% by volume, and further culturing at 37 deg.C and 180rpm to OD₆₀₀0.6-0.9, adding IPTG with final concentration of 0.5-1.5mM, inducing expression at 23 deg.C and 180rpm for 10-20h, centrifuging, collecting cell precipitate, washing with 0.9% physiological saline, centrifuging, and collecting wet cell; (2) crude enzyme solution: the harvested wet cells were resuspended in 100mM sodium phosphate buffer, pH 7.0, the mixture was sonicated under ice bath conditions for 5 minutes at 400W power for 3 seconds,crushing for 7 seconds, collecting the crushed liquid after ultrasonic treatment, centrifuging, and taking supernatant to obtain crude enzyme liquid of the alcohol dehydrogenase; (3) pure enzyme solution: mixing the crude enzyme solution of alcohol dehydrogenase with a chromatography medium according to the volume ratio of 8:1, and then loading the mixture to a Beyogold^TMHis-tag chromatographic column, washing the column with non-denaturing washing liquid for 5 times to remove impurity protein, and eluting 1-2 column volumes each time; eluting for 6-10 times by using non-denatured eluent, eluting for 1-2 column volumes each time, collecting eluent containing target protein, performing ultrafiltration concentration, and freeze-drying to obtain pure enzyme of the R-omega-transaminase; the non-denaturing washing solution is 50mM sodium phosphate buffer, pH8.0, containing 300mM NaCl and 2mM imidazole; the non-denaturing eluent was a 50mM sodium phosphate buffer, pH8.0, containing 300mM NaCl and 50mM imidazole.

9. The method of claim 1, wherein the reaction system further comprises a metal ion and a reagent, the metal ion and the reagent being Ca²⁺、Mg²⁺、Cu²⁺、Fe²⁺、Zn²⁺、Mn²⁺、Na⁺、K⁺Or EDTA.

10. The method according to claim 9, wherein the metal ion and the reagent are added to the reaction system to a final concentration of 10mM each.