CN114457125B

CN114457125B - Method for synthesizing (R) -1- [3,5-bis (trifluoromethyl) phenyl ] ethylamine by coupling transaminase and alcohol dehydrogenase

Info

Publication number: CN114457125B
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: 2024-04-09
Anticipated expiration: 2041-12-07
Also published as: CN114457125A

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 using R-omega-transaminase and alcohol dehydrogenase as double enzyme catalysts, 3, 5-bistrifluoromethyl acetophenone as amino acceptor, isopropylamine hydrochloride as amine donor, pyridoxal phosphate and NAD ⁺ Adding cosolvent as auxiliary factor, forming reaction system with pH6-10 buffer solution as reaction medium, stirring at 30-50deg.C and 150-200rpm to react to obtain (R) -1- [3,5-bis (trifluoromethyl) phenyl]Ethylamine; when the concentration of the substrate is 166mM, the yield is increased from 82.96% to 99.9%, and the amination efficiency of the substrate BPO is obviously improved, so that a process for preparing R-BPA by efficiently aminating the 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

Field of the art

The invention relates to the preparation of (R) -1- [3,5-bis (trifluoromethyl) phenyl ] ethylamine, in particular to the preparation of (R) -1- [3,5-bis (trifluoromethyl) phenyl ] ethylamine (R-BPA) by using a double enzyme coupling system of R-omega-aminotransferase (ATA 117) and Alcohol Dehydrogenase (ADH) and using the double enzyme coupling system for the asymmetric amination of 3,5-bis (trifluoromethyl) acetophenone (BPO).

(II) background art

(R) -1- [3,5-bis (trifluoromethyl) phenyl ]]Ethylamine ((R) -1- [3,5-Bis (trifluoromethyl) phenyl)]ethane, R-BPA), cas number 127733-47-5, molecular formula C ₁₀ H ₉ F ₆ N, molecular weight 257.18.R-BPA is a good candidate drug-a chiral intermediate of a selective tetrodotoxin-sensitive (TTX-S) blocker, TTX-S having a variety of therapeutic uses in pain treatment. Local anesthetic lidocaine and volatile anesthetic halothane act with poor selectivity and low potency on tetrodotoxin insensitive (TTX-R) and TTX-S sodium channels (IC) ₅₀ Values in the range 50. Mu.M-10 mM). These anesthetics may cause damaging side effects such as paralysis and asystole at higher systemic concentrations. However, systemic administration of lidocaine at low concentrations is effective for the treatment of chronic pain (NPL 11). In rats, mechanical allodynia behavior (NPL 12) was significantly reduced if very low doses of TTX were applied to DRG of the damaged segment of the L5 spinal nerve. This result suggests that the TTX-S subtype of sodium channel plays an important role in the maintenance of allodynia behavior in neuropathic pain animal models.

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

Currently, the methods for preparing chiral amines mainly comprise a chemical method, a biological resolution method and a biological asymmetric synthesis method. The chemical method for synthesizing chiral amine often needs to use expensive metal catalyst and special solvent, has higher production cost and lower enantioselectivity, and can cause certain environmental pollution. The maximum theoretical yield of the high-purity chiral amine prepared by the chiral resolution method through the biocatalyst is only 50%. Therefore, both of these methods cannot meet the actual production requirements. The preferred strategy for producing chiral amine by biological method is asymmetric synthesis, the theoretical yield is up to 100%, and the high yield has wide application prospect in the field of production industry, and related research is widely paid attention to.

In practice, due to unfavorable thermodynamic equilibrium, it is often necessary to superstoichiometric isopropylamine and remove the corresponding acetone by-product in situ in order to shift the reaction equilibrium in the desired direction. In order to maximize the productivity of TAs, it is important to find an effective method for promoting bioconversion. Process control strategies have been developed to remove products in situ, with cascading reactions utilizing additional biochemical reactions of byproducts attracting much attention.

omega-TAs catalyze the transamination reaction between an amino donor and an amino acceptor substrate, enabling the catalytic chemical combination of asymmetric ammonification on the prochiral substrate carbonyl under mild conditions without the need for additional cofactor recycling. The omega-TAs is used for producing the optically pure chiral amine, so that the high-temperature high-energy consumption 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 invention has been developed to apply omega-TAs to biocatalysis.

(III) summary 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 characterized in that after a substrate spectrum of R-omega-TAs is screened to obtain a corresponding substrate with excellent yield and enantiomer excess value (e.e.%) and a series of reaction factors (cosolvent, temperature, pH, ADH, ionic species and the like) are further studied to influence asymmetric amination reaction. Ethanol was used instead of dimethyl sulfoxide (DMSO) conventionally used. In addition, the addition of an appropriate amount of Saccharomyces cerevisiae Alcohol Dehydrogenase (ADH) in the transamination reaction with Isopropylamine (IPA) as an amine donor has a significant promoting effect on the improvement of the yield. Under the optimal reaction condition, the R-omega-TAs/ADH coupling system catalyzes the asymmetric amination of 3, 5-bistrifluoromethyl acetophenone, and the catalytic reaction efficiency is obviously improved, so that a process for preparing R-BPA by efficiently aminating BPO in a double-enzyme-level conjunct system is established, and a reference is provided for later 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-aminotransferase (R-omega-TAs) with Alcohol Dehydrogenase (ADH)]A process for ethylamine (R-BPA), the process being carried out as follows: r-omega-transaminase and alcohol dehydrogenase are used as double enzyme catalysts, 3, 5-bistrifluoromethyl acetophenone (BPO) is used as an amino acceptor, isopropyl amine hydrochloride (IPA) is used as an amine donor, pyridoxal phosphate (PLP) and NAD are used as an amino donor ⁺ Adding cosolvent as auxiliary factor, forming reaction system with pH6-10 buffer solution as reaction medium, mixing and stirring in shaking table of 30-50deg.C (preferably 40deg.C) and 150-200rpm (preferably 180 rpm) for reaction for 12-36 hr (preferably 24 hr), separating and purifying the 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 addition amount of R-omega-aminotransferase is 1-2U/mL (preferably 1.35U/mL) based on the volume of the reaction system; the addition amount of the alcohol dehydrogenase is 1-2U/mL (preferably 1.35U/mL) based on the volume of the reaction system; the addition amount of the 3,5-bis (trifluoromethyl) acetophenone is calculated by the volume of a reaction system25-170mmol/L (preferably 166 mmol/L); the addition amount of the isopropylamine hydrochloride is 125-850mmol/L (preferably 832 mmol/L) based on the volume of the reaction system; the adding amount of pyridoxal phosphate is 0.5-1.5mmol/L (preferably 1 mmol/L) based on the volume of the reaction system; the NAD ⁺ The addition amount is 0.5-1.5mmol/L (preferably 1 mmol/L) based on the volume of the reaction system; the addition amount of the cosolvent is 10% -30% (preferably 15%) based on the volume of the reaction system.

Further, the R-omega-aminotransferase is a pure enzyme extracted by ultrasonic crushing of wet thalli which are fermented and cultured by engineering bacteria containing the R-omega-aminotransferase gene, the nucleotide sequence of the R-omega-aminotransferase gene is shown as SEQ ID NO.1, and the pure enzyme is added in the form of an aqueous solution, and the preferable concentration is 15-20mg/mL; the engineering bacteria are constructed by inserting R-omega-tranexamiase genes (JA 717225.1, ATA 117) between BamHI and XhoI restriction sites of pETdure-1 expression vectors and transforming escherichia coli BL21 (DE 3).

Further, the pure enzyme of R-omega-aminotransferase is prepared as follows:

(1) Induction culture: inoculating engineering bacteria containing R-omega-aminotransferase gene into 50mL LB liquid medium containing 30-80 mug/mL (preferably 50 mug/mL) ampicillin, and culturing at 37 ℃ and 180rpm for overnight to obtain seed liquid; the seed solution is transferred to fresh LB liquid medium containing 30-80 mu g/mL (preferably 50 mu g/mL) ampicillin according to the inoculation amount with the volume concentration of 1% -5% (preferably 3%), and is cultivated at 37 ℃ and 180rpm until OD ₆₀₀ 0.6-0.9 (preferably 0.8), isopropyl-beta-thiogalactoside (IPTG) was added at a final concentration of 0.5-1.5mM (preferably 1 mM), transferred to 16-26 ℃ (preferably 18 ℃) to induce expression for 10-20h (preferably 15 h), centrifuged (preferably the culture broth was placed at 4 ℃, centrifuged at 8000rpm for 10 min), cell pellet was collected, washed with 0.9% (v/w) physiological saline (preferably 2 times), and wet bacterial cells were collected; (2) crude enzyme solution: resuspending the wet somatic cells harvested in the step (1) (preferably in an amount of 50 g/L) in 100mM sodium phosphate buffer (pH 7.0), performing ultrasonic crushing for 5 minutes (power 400W, working for 3 seconds and crushing for 7 seconds), collecting crushed liquid, centrifuging (preferably at 8000rpm and at 4 ℃ for 10 minutes), and taking supernatant to obtain transaminase crude enzyme liquid; (3) pure enzyme: the transaminase crude enzyme liquid and the chromatography medium are subjected to volumeRatio 8:1 mixing (preferably in ice cubes, shaking slowly in a 40rpm shaker for 1 h), and then applying to Beyogold ^TM His-tag chromatographic column is washed by non-denatured washing liquid for 5 times to remove the impurity protein, and the volume of the column is 1-2 times of elution; eluting with non-denaturing eluent for 6-10 times (preferably 8 times), eluting 1-2 column volumes each time, collecting eluent containing target protein, ultrafiltering, concentrating, and lyophilizing to obtain pure enzyme of R-omega-transaminase; the non-denaturing washing is pH8.0, 50mM sodium phosphate buffer containing 300mM NaCl and 2mM imidazole; the non-denaturing eluate is pH8.0, 50mM sodium phosphate buffer containing 300mM NaCl and 50mM imidazole.

Further, the alcohol dehydrogenase is pure enzyme obtained by ultrasonic crushing and extracting wet thalli 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 15-20mg/mL. The engineering bacteria are constructed by inserting an alcohol 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 as follows: (1) induction culture: inoculating ethanol dehydrogenase genetically engineered bacteria to LB liquid medium containing 30-80 mug/mL (preferably 50 mug/mL) Kan antibiotics, and placing in a shaking table at 37 ℃ and 180rpm for overnight culture to obtain seed liquid; transferring into LB liquid medium containing 30-80 μg/mL (preferably 50 μg/mL) Kan at 1-5% (preferably 3%) by volume, and culturing at 37deg.C and 180rpm until OD ₆₀₀ 0.6-0.9 (preferably 0.7), adding IPTG with a final concentration of 0.5-1.5mM (preferably 1 mM), inducing expression at 23deg.C and 180rpm for 10-20h (preferably 15 h), centrifuging (preferably by packing the culture solution in a centrifuge tube and placing in a centrifuge at 4deg.C and centrifuging at 8000rpm for 10 min), washing the collected cell precipitate with 0.9% physiological saline (preferably 3 times), centrifuging (preferably at 4deg.C and centrifuging at 8000rpm for 10 min), and collecting wet cells; (2) crude enzyme solution: the harvested wet cells were resuspended in pH 7.0, 100mM sodium phosphate buffer at 50g/L, the mixture sonicated for 5 minutes under ice-bath conditions, power 400W, working for 3 seconds,crushing for 7 seconds, collecting the crushed liquid after ultrasonic treatment, centrifuging for 10 minutes at the temperature of 4 ℃ at the speed of 8000rpm, and taking the supernatant to obtain ethanol dehydrogenase crude enzyme liquid; (3) pure enzyme solution: the ethanol dehydrogenase crude enzyme liquid and the chromatographic medium are mixed according to the volume ratio of 8:1 mixing (preferably in ice cubes, shaking slowly in a 40rpm shaker for 1 h), and then applying to Beyogold ^TM His-tag chromatographic column is washed by non-denatured washing liquid for 5 times to remove the impurity protein, and 1-2 column volumes are eluted each time; eluting with non-denaturing eluent for 6-10 times, eluting 1-2 column volumes each time, collecting eluent containing target protein, ultrafiltering, concentrating, and lyophilizing to obtain pure enzyme of R-omega-transaminase; the non-denaturing washing is pH8.0, 50mM sodium phosphate buffer containing 300mM NaCl and 2mM imidazole; the non-denaturing eluate is pH8.0, 50mM sodium phosphate buffer containing 300mM NaCl and 50mM imidazole.

The ultrafiltration concentration refers to the steps of loading crude enzyme liquid into an ultrafiltration tube (10000 MWCO) for ultrafiltration concentration treatment, and taking trapped liquid as concentrated liquid; the freeze-drying is to pre-freeze the concentrated solution in a refrigerator at-80 ℃ for 8 hours, and then put the concentrated solution into a freeze dryer at-65 ℃ for 12 hours in vacuum.

Further, the reaction system also comprises metal ions and reagents, and the reaction system comprises: r-omega-tranferase and alcohol dehydrogenase are used as double enzyme catalysts, 3,5-bis (trifluoromethyl) acetophenone (BPO) is used as an amino acceptor, isopropylamine hydrochloride (IPA) is used as an amine donor, pyridoxal phosphate (PLP) and NAD are used as an amino acceptor ⁺ Adding a cosolvent, metal ions and a reagent as auxiliary factors, and forming a reaction system by taking a buffer solution with pH of 6-10 as a reaction medium; the metal ion and the reagent are Ca ²⁺ 、Mg ²⁺ 、Cu ²⁺ 、Fe ²⁺ 、Zn ²⁺ 、Mn ²⁺ 、Na ⁺ 、K ⁺ Or EDTA, preferably EDTA; the final concentrations of the metal ions and the reagent added into the reaction system are respectively 10mM.

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

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

in practice, due to unfavorable thermodynamic equilibrium, it is often necessary to superstoichiometric isopropylamine and remove the corresponding acetone by-product in situ in order to shift the reaction equilibrium in the desired direction. In order to maximize the productivity of R- ω -transaminases, it is important to find an efficient method to accelerate bioconversion. According to the invention, the aminotransferase ATA117 and the yeast alcohol dehydrogenase ADH are used in combination, 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 a water phase system is enhanced. The first step of BPO amination reaction comprises the following steps: ATA117 catalyzes the amination of substrate BPO to R-BPA with isopropylamine as an amino donor and PLP as a coenzyme, with acetone as a byproduct. The second step of in-situ removal reaction process of byproduct acetone comprises the following steps: the regeneration of coenzyme NADH is realized by catalyzing the auxiliary substrate ethanol to generate acetaldehyde by ADH, and sufficient NADH promotes the ADH to catalyze acetone to generate propanol. The second step of reaction converts the acetone byproduct of the first step of reaction into ethanol, so that the amination reaction efficiency of the substrate BPO is improved. The ethanol not only plays a role of an organic substrate cosolvent, but also serves as a substrate for the cascade reaction of the second step for in-situ regeneration of the coenzyme NADH. According to the invention, the in-situ removal reaction cascade of the first-step substrate amination reaction catalyzed by ATA117 and the second-step byproduct acetone catalyzed by ADH is carried out, the catalytic yield 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, so that a process for preparing R-BPA by high-efficiency amination of the BPO is established, and the process control strategy provides a reference for later industrial application.

(IV) description of the drawings

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

FIG. 2 is a schematic diagram of the reaction mechanism of aminated BPO to R-BPA by the double enzyme constructed cascade reaction of the invention.

(fifth) detailed description of the invention

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

the R- ω -transaminase (ATA 117) 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 NCBI library (https:// www.ncbi.nlm.nih.gov/protein/NP-014555.1).

The Beyogold ^TM His-tag Purification Resin protein purification column is reduction-resistant chelate, purchased from Shanghai Biyun biotechnology Co., ltd.

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

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

Example 1: preparation of transaminase

1. Transaminase crude enzyme liquid

The R-omega-aminotransferase gene (gene accession number JA717225.1, the nucleotide sequence of which is shown as SEQ ID NO.1 and is marked as aminotransferase gene ATA 117) is inserted between BamHI and XhoI restriction sites of the pETdure-1 expression vector, transferred into E.coli DH5 alpha competence, extracted into recombinant plasmid pETdure-ATA 117 and transformed into E.coli BL21 (DE 3). The transformant obtained 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 E.coli. The seed solution was transferred to 0.15L of fresh LB liquid medium containing 50. Mu.g/mL ampicillin at a 3% by volume of the inoculum size, and cultured by fermentation at 37℃and 180rpm until an Optical Density (OD) at 600nm was reached ₆₀₀ ) Isopropyl- β -thiogalactoside (IPTG) was added to a final concentration of 1mM to induce gene expression, and then the culture was transferred to a shaker at 18 ℃ and 180rpm to induce expression for 15h. The culture broth was packed 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, 3 seconds of operation, 7 seconds of disruption). Collect the crushed liquid in 50mL centrifuge tubeAfter centrifugation for 10min at 8000rpm and 4 ℃, the supernatant is taken and put into an ultrafiltration tube (10000 MWCO) for ultrafiltration concentration treatment, and the trapped liquid is 8mL of concentrated transaminase crude enzyme liquid.

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

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

2. transaminase-purifying enzyme solution

(1) Chromatographic column pre-equilibrium

2ml of 50% Beyogold ^TM His-tag Purification Resin ℃,8000rpm centrifugation for 1min, storage liquid was discarded, and 1ml gel was obtained. 3mL of affinity column containing 1mL of gel was pre-equilibrated with non-denaturing lysate (pH 8.0, 50mM sodium phosphate buffer and 300mM NaCl), centrifuged at 8000rpm at 4℃for 1min, the liquid was discarded, and the supernatant was discarded, i.e., the chromatography after pre-equilibration.

(2) Loading and eluting

And (3) 8mL of transaminase crude enzyme solution prepared by the method in the step (1) (the volume ratio of the crude enzyme solution to the column-filling gel in the step (1) is 8:1), loading the transaminase crude enzyme solution into the chromatographic column after the pre-equilibrium in the step (1), shaking and mixing uniformly, putting the chromatographic column into ice cubes, slowly shaking the chromatographic column for 1h in a shaking table of 40rpm, transferring the chromatographic column into a new chromatographic column, eluting the chromatographic column by using non-denatured washing solution (pH 8.0, 50mM sodium phosphate buffer, 300mM NaCl and 2mM imidazole) depending on gravity for removing the foreign proteins, and eluting the chromatographic column for 5 times, wherein the using amount of the transaminase crude enzyme solution is 1 column volume each time, and the eluting speed is about 0.5mL/min. The protein for transaminase purification was then eluted with non-denaturing eluates (pH 8.0, 50mM sodium phosphate buffer, 300mM NaCl and 50mM imidazole) 8 times, 1 column volume each time, each column separately collected as Lane 1-Lane 8, and monitored by 12% SDS-PAGE, as shown in FIG. 1A. As can be seen from SDS-PAGE, lane 1-Lane 6 all contain target proteins, the molecular weight is about 35kDa, the content is reduced from small to large, lane7 and Lane8 do not elute the corresponding proteins, and therefore, the proteins are not shown. Finally, the eluates of Lane 1-Lane 6 are combined to obtain the eluent containing target protein, the eluent is pre-frozen for 8 hours in a refrigerator at the temperature of minus 80 ℃, and then the eluent is put into a freeze dryer for vacuum drying at the temperature of minus 65 ℃ for 12 hours, thus obtaining 15mg of transaminase pure enzyme.

3. Enzyme activity detection

Transaminase enzyme activity assay (2 mL) composition: 832mM isopropyl amine hydrochloride (IPA, amine donor), 1mM pyridoxal 5' -phosphate (PLP, cofactor), 0.3mL dimethyl sulfoxide (DMSO, co-solvent), 166.56 mM 3, 5-bis-trifluoromethyl acetophenone (BPO, substrate), 1mL 15mg/mL of the aqueous transaminase-purified enzyme solution prepared in step 2 were made up to 2mL with 100mM Tris-HCl (pH 9.0) buffer. The reaction was stopped by adding 1. Mu.L of trifluoroacetic acid ester (TFA, 1%) at 45℃and 180rpm for 2h. The reaction solution was subjected to Gas Chromatography (GC) to determine the peak area of (R) -1- [3,5-bis (trifluoromethyl) phenyl ] ethylamine ((R) -BPA), and the (R) -BPA content was obtained according to a standard curve, in the same manner 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 liquid of alcohol dehydrogenase

Ethanol dehydrogenase Gene (NP)014555.1, the amino acid sequence of which is shown in SEQ ID NO.2 and which is marked as ADH), is inserted between BamHI and XhoI restriction sites of pET-28a (+) expression vector, E.coli DH5 a competent cells are transformed, pET28a-ADH recombinant plasmid is extracted and introduced into E.coli BL21 (DE 3) competent cells, after activation for 1h in a shaker at 37℃and 180rpm, LB plates containing 50. Mu.g/mL Kan antibiotic are applied, and the plates are inverted and allowed to stand at 37℃for overnight. Single colonies were picked up in 50mL of LB liquid medium containing 50. Mu.g/mL Kan antibiotics, and placed in a shaking table at 37℃and 180rpm for overnight culture to obtain seed solution. Transferring into 0.15L LB liquid medium containing 50 μg/mL Kan according to inoculation amount of 3% volume concentration, and culturing at 37deg.C and 180rpm until OD ₆₀₀ 0.7 and IPTG was added at a final concentration of 1mM, and the reaction was induced at 23℃and 180rpm for 15 hours. After the induction is finished, the culture solution is split into centrifuge tubes and placed in a centrifugal machine at the temperature of 4 ℃ and the centrifugal machine at the speed of 8000rpm for 10min. The collected cell pellet was washed with 10mL of 0.9% (v/w) physiological saline each time, for a total of 3 times. Wet cells were collected by centrifugation at 8000rpm at 4℃for 10min. 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 under ice bath conditions for 5 minutes (power 400W, 3 seconds of operation, disruption 7 seconds). Collecting the ultrasonic crushed liquid, centrifuging at the speed of 8000rpm and the temperature of 4 ℃ for 10min, taking supernatant, loading into an ultrafiltration tube (10000 MWCO) for ultrafiltration concentration treatment (5000 Xg, 30 min), and obtaining the trapped liquid which is 8mL of concentrated ethanol dehydrogenase crude enzyme liquid.

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. alcohol dehydrogenase pure enzyme liquid

(1) Chromatographic column pre-equilibrium

The gel column was 1mL in volume as in example 1.

(2) And (3) loading 8mL of the ethanol dehydrogenase crude enzyme solution prepared in the step (1) (the volume ratio of the crude enzyme solution to the column-filling gel of the step (1) is 8:1), loading the crude enzyme solution into the chromatographic column subjected to pre-equilibrium in the step (1), shaking and mixing uniformly, putting the chromatographic column into ice cubes, slowly shaking the chromatographic column for 1h in a shaking table of 40rpm, transferring the chromatographic column into a new chromatographic column, eluting the chromatographic column by using non-denatured washing solution (pH 8.0, 50mM sodium phosphate buffer, 300mM NaCl and 2mM imidazole) depending on gravity for removing the impurity proteins, and washing the chromatographic column for 5 times, wherein the using amount of each time is 1 column volume, and the eluting speed is about 0.5mL/min. The ethanol dehydrogenase target protein was then purified by eluting with non-denaturing eluents (pH 8.0, 50mM sodium phosphate buffer, 300mM NaCl and 50mM imidazole) 8 times, 1 column volume each time, each column separately collected as Lane 1-Lane 8, and monitored by 12% SDS-PAGE, as shown in FIG. 1B. And 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 minus 80 ℃ for 8 hours, and then placing the eluent into a freeze dryer at the temperature of minus 65 ℃ for vacuum drying for 12 hours to obtain 18mg of pure enzyme of alcohol dehydrogenase.

3. Measurement of enzyme Activity

5mL of ethanol dehydrogenase enzyme activity assay system comprises: 2.5mL glycine-sodium hydroxide buffer (pH 8.6), final concentration 1X 10 ^-3 mol/L NAD ⁺ And final concentration 5X 10 ^-3 After warming in a 25℃water bath for 20 minutes, 1mL of 18mg/mL of the ethanol dehydrogenase-purified enzyme aqueous solution prepared in step 2 warmed under the same conditions was added. The time was then immediately counted, and the absorbance at 340nm was read every 1 minute for 5 consecutive minutes. The amount of enzyme required to increase at A340 by 0.001 per minute was one unit (U) at 25℃and pH 8.6. The enzyme activity was measured at 0.15U/mg.

Example 3: screening of transaminase substrate profiles

2mL systems were built in 13 vials each: 13 different substrates (added after dissolution with cosolvent ethanol) at a final concentration of 166mM, isopropyl amine hydrochloride (IPA) at a final concentration of 832mM, pyridoxal phosphate (PLP) at a final concentration of 1mM, 1mL of a 15mg/mL aqueous solution of transaminase-purified enzyme prepared in the same manner as in example 1 (total enzyme activity: 2.7U), and ethanol (cosolvent) at a final concentration of 15% by volume were made up to 2mL with 0.1M Tris-HCl buffer pH9.

The reaction system was simultaneously placed in a shaker at 45℃and 180rpm for reaction for 24 hours, the reaction solution was taken out, centrifuged at 8000rpm for 10 minutes at 4℃and the supernatant was collected and extracted with an equal volume of ethyl acetate, the extraction was repeated three times, the collected extract was dried over anhydrous sodium sulfate and evaporated at normal temperature to remove ethyl acetate, the peak areas of the bottoms 3, 5-Bistrifluoroacetophenone (BPO), (R) -1- [3,5-bis (trifluoromethyl) phenyl ] ethylamine ((R) -BPA), (S) -1- [3,5-bis (trifluoromethyl) phenyl ] ethylamine ((S) -BPA) were detected by gas chromatography, the content was calculated by an internal standard method (dodecane was added as an internal standard), the yield of (R) -BPA and the enantiomeric excess (eep) were calculated by the formula (1) and the formula (2), and the results are shown in Table 1.

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

The yield (X) and enantiomeric excess (eep) of product (R) -BPA are calculated by formulas (1) and (2):

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

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

TABLE 1 screening of transaminase substrate spectra

The aminotransferase can catalyze amination of various aldehydes and ketones, so that the substrate range is relatively wide, and engineering (R) -omega-TA (ATA 117) has successfully realized industrial application of producing sitagliptin (an oral hypoglycemic agent) by taking sitagliptin precursor ketone as a substrate. To examine the substrate specificity of transaminase ATA117 to expand the substrate spectrum of transaminase ATA117, the reactivity of 13 different substrates (amino-acceptors) was compared. From Table 1, it can be seen that the reaction using methyl acetoacetate, ethyl acetoacetate, and 3, 5-bistrifluoromethyl acetophenone as substrates all had good yields. However, the R-BPA enantiomer excess values corresponding to the three are slightly different. In the reaction with 3,5-bis (trifluoromethyl) acetophenone as substrate, the advantages of considerable yield and better enantioselectivity are taken into account, so that the method is selected as the next experimental study object.

Example 4: influence of pH on transaminase Single enzyme catalyzed amination

In a 2mL reaction system, 166mM substrate BPO (added after being dissolved in ethanol in advance), 832mM amino donor IPA, 1mM auxiliary factor pyridoxal phosphate (PLP) and 1mL of 15mg/mL transaminase pure enzyme aqueous solution (total enzyme activity 2.7U) prepared in the method of example 1 were added, and the buffer solutions with different pH values were made up to 2mL by using 15% ethanol as a cosolvent. The buffers were 0.1M sodium phosphate buffer pH6, sodium phosphate buffer pH7, tris-HCl pH8, tris-HCl pH9 and glycine-NaOH (pH 10), respectively. The reaction flask was left to react at 45℃and 180rpm for 24h. After that, centrifugation was carried out at 8000rpm for 10min at 4℃to collect the supernatant and extract it with an equal volume of ethyl acetate, and after repeating the extraction three times, the collected extract was dried over anhydrous sodium sulfate and ethyl acetate was volatilized at normal temperature, and the R-BPA yield and the enantiomeric excess were measured by the GC method in example 3, and the results are shown in Table 2.

TABLE 2 influence of pH on transaminase Single enzyme catalyzed amination reactions

In the pH range of 6-10, it was examined that the catalytic performance of the aminotransferase ATA117 was affected by the pH of the buffer, and in the pH range of 6-9, the conversion rate showed a tendency to gradually increase with increasing pH, and the conversion rate slightly decreased to pH 10, but still maintained at a higher level. It can be seen from Table 2 that the pH of the reaction medium can affect the activity of the enzyme, probably due to the change in the steric conformation of the enzyme active site caused by the change in the pH of the reaction medium. As can be seen from the trend of the yield, the enzyme is alkalophilic, the yield is controlled to be more than 80% when the pH of the buffer solution is more than 8, and all products at the moment are target products R-BPA. Therefore, 0.1M pH 9Tris-HCl was determined as the optimal buffer for the system.

Example 5: effect of temperature on double enzyme coupling reactions

Double enzyme-coupled group: in a 2mL reaction system, 166mM of substrate BPO (dissolved in advance in the cosolvent ethanol), 832mM of amino donor IPA, 1mM of cofactor pyridoxal phosphate (PLP) and 1mM of cofactor NAD were added ⁺ 1mL of the ethanol dehydrogenase-purified enzyme aqueous solution prepared by the method of example 2 (total enzyme activity: 2.7U) and 1mL of the transaminase-purified enzyme aqueous solution prepared by the method of example 1 (total enzyme activity: 2.7U) were mixed, and the mixture was supplemented to 2mL with 0.1M pH 9Tris-HCl buffer using 15% ethanol as a cosolvent.

Control group: the ethanol dehydrogenase in the coupled group was removed and the effect of temperature on the double enzyme catalyzed cascade was investigated in the same way.

The double enzyme-coupled group and the control group reaction flask were simultaneously placed at different temperatures of 30℃to 55℃and reacted at 180rpm for 24 hours. After that, centrifugation was carried out at 8000rpm for 10 minutes at 4℃to collect the supernatant and extract it with an equal volume of ethyl acetate, and after repeating the extraction three times, the collected extract was dried over anhydrous sodium sulfate and then volatilized off ethyl acetate at normal temperature, and the yield of R-BPA and the enantiomeric excess were measured by the GC method as in example 3, and the results are shown in Table 3.

TABLE 3 influence of temperature on the cascade of the double enzyme systems

The temperature significantly affects the catalytic activity of the enzyme, which activity changes with temperature. Due to the denaturation of proteins, enzymatic activity is lost or inhibited at high temperatures. As can be seen from the table, the yield of the transaminase-catalyzed reaction (control) tended to rise first and then fall over a range of temperatures (30-55 ℃) with a maximum value at 50 ℃. Whereas 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 alcohol dehydrogenase ADH significantly promoted the reaction to proceed in the forward direction in the range of 30℃to 40 ℃. Superstoichiometric added isopropylamine is subjected to ammonia conversion reaction to form corresponding acetone byproducts, and the added ADH catalyzes a reaction taking ethanol as an auxiliary substrate to remove the corresponding acetone byproducts in situ so as to shift the reaction balance forward and promote the catalytic efficiency. The reaction mechanism is shown in FIG. 2. When the temperature was further increased to 50 ℃, the cascade was no longer apparent, the yield was slightly increased compared to the control, at which time ADH was hardly affected. Thus, the optimal temperature for the reaction is 40℃for a two-enzyme catalyzed cascade.

Example 6: influence of Metal ions and reagents on double enzyme coupling reactions

To investigate the effect of metal ions and reagents on the double enzyme coupling reaction, ca was added at a final concentration of 10mM each ² ⁺ 、Mg ²⁺ 、Cu ²⁺ 、Fe ²⁺ 、Zn ²⁺ 、Mn ²⁺ 、Na ⁺ 、K ⁺ And EDTA into the reaction system.

2mL of reaction system: 166mM of substrate BPO (previously dissolved in the cosolvent ethanol), 832mM of amino donor IPA, 1mM of cofactor pyridoxal phosphate (PLP), 1mM of cofactor NAD ⁺ 1mL of the aqueous solution of pure enzyme of alcohol dehydrogenase (total enzyme activity 2.7U) prepared by the method of example 2 and 1mL of the aqueous solution of pure enzyme of transaminase (total enzyme activity 2.7U) prepared by the method of example 1 at 18mg/mL, the final concentrations of metal ion and reagent (Ca) were 10mM, respectively ²⁺ 、Mg ²⁺ 、Cu ²⁺ 、Fe ²⁺ 、Zn ²⁺ 、Mn ²⁺ 、 Na ⁺ 、K ⁺ And EDTA), ethanol with a final volume concentration of 15% was used as a cosolvent, and 2mL was made up with 0.1M pH 9Tris-HCl buffer. After 24h of reaction at 40℃and 180rpm, centrifugation is carried out for 10min at 4℃and 8000rpm, the supernatant is collected and extracted with an equal volume of ethyl acetate, the extraction is repeated three times, the collected extract is dried over anhydrous sodium sulfate and the ethyl acetate is volatilized off at normal temperature, and the yield and the enantiomeric excess of R-BPA are detected by the GC method as in example 3. The relative yields were calculated using the reaction without any metal ions and reagents added as a control (100%).

TABLE 4 influence of Metal ions and reagents on the cascade of double enzyme systems

This example examined the effect of high concentrations of metal ions and EDTA on a cascade of transaminase and alcohol dehydrogenase, and the results are shown in Table 4. As can be seen from this, the yields of the vast majority of the reaction groups were improved to some extent (1.2-1.6 fold) after addition of different types of metal ions and EDTA, with the effect of EDTA addition with 10mM metal ion chelating agent (1.533 fold) being most pronounced. While 10mM Mn is added ²⁺ And Fe (Fe) ³⁺ Then the reaction efficiency is inhibited to different degrees, wherein Fe is used as ³⁺ The inhibition effect of (2) is most obvious. The cascade reaction system is slightly dependent on metal ions.

Example 7: effect of co-solvent on double enzyme system cascade reaction

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

2mL of reaction system: 166mM substrate BPO (dissolved in different co-solvents), 832mM amino donor IPA, 1mM cofactor pyridoxal phosphate (PLP), 1mM cofactor NAD ⁺ 1mL of 18mg/mL ethanol prepared as in example 2Aqueous solutions of pure dehydrogenase (total enzyme activity 2.7U) and 1mL of the aqueous solution of pure transaminase (total enzyme activity 2.7U) prepared in example 1 at a final concentration of 15% by volume of cosolvent were made up to 2mL using 0.1M pH 9Tris-HCl buffer.

After 24h of reaction at 40℃and 180rpm, centrifugation is carried out at 4℃and 8000rpm for 10min, the supernatant is collected and extracted with an equal volume of ethyl acetate, the extraction is repeated three times, the collected extract is dried over anhydrous sodium sulfate and the ethyl acetate is volatilized at normal temperature, and the yield and enantiomeric excess of R-BPA are detected by the GC method as in example 3. The relative yields were calculated using the reaction without any metal ions and reagents added as a control (100%) and the results are shown in table 5.

TABLE 5 Effect of cosolvents on the Cascade reaction of double enzyme systems

As can be seen from Table 5, the species of the cosolvents have a major effect on the yield to some extent, while having little effect on the enantiomeric excess of R-BPA, ee _p (%) were controlled at a high level (. Gtoreq.96%). The two groups of data of Tween 20 and Tween 80 show that the dissolution assisting effect of the Tween solvent on the BPO is not great, the effect is generally slightly better than Yu Sipan, and the yield of the Tween solvent is slightly lower than that of methanol. Effects in yield and ee with DMSO, ethanol and glycerol as co-solvents _p (%) are suitable as co-solvents for the asymmetric amination of BPO to R-BPA. Among them, ethanol is the most effective. On the other hand, the ethanol can also be used as an auxiliary substrate in the cascade reaction to promote the in-situ removal of the acetone byproduct and promote the reaction. Ethanol is therefore chosen as the cosolvent for the cascade.

Example 8 double enzyme coupling reaction

The reaction flask was charged with 166mM final concentration of substrate BPO (first dissolved in cosolvent ethanol), 832mM final concentration of amino donor IPA, 1mM final concentration of cofactor pyridoxal phosphate (PLP), 1mM final concentration of cofactor NAD ⁺ 1mL of the ethanol dehydrogenase-purified enzyme aqueous solution prepared in example 2 (total enzyme activity: 2.7U), 1mL of the transaminase-purified enzyme aqueous solution prepared in example 1 (total enzyme activity: 2.7U) and 10mM EDTA were mixed, and the reaction system was supplemented with 2mL of the final volume concentration of 15% ethanol as a cosolvent using 0.1M Tris-HCl buffer at pH 9. After the system had reacted at 40℃and 180rpm for 24 hours, it was centrifuged at 8000rpm for 10 minutes at 4℃and the supernatant was collected and extracted with an equal volume of ethyl acetate, and after three repeated extractions, the collected extract was dried over anhydrous sodium sulfate and evaporated to ethyl acetate at room temperature, the yield of R-BPA was 99% and the enantiomeric excess was 99.9% as determined by the GC method in example 3.

Sequence listing

<110> Zhejiang university of industry

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

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 990

<212> DNA

<213> Unknown (Unknown)

<400> 1

atggcgttct cagcggacac ccctgaaatc gtttacaccc acgacaccgg tctggactat 60

atcacctact ctgactacga actggacccg gctaacccgc tggctggtgg tgctgcttgg 120

atcgaaggtg ctttcgttcc gccgtctgaa gctcgtatcc ctatcttcga ccagggtttt 180

tatacttctg acgctaccta caccaccttc cacgtttgga acggtaacgc tttccgtctg 240

ggggaccaca tcgaacgtct gttctctaat gcggaatcta ttcgtttgat cccgccgctg 300

acccaggacg aagttaaaga gatcgctctg gaactggttg ctaaaaccga actgcgtgaa 360

gcgatggtta ccgttacgat cacccgtggt tactcttcta ccccattcga gcgtgacatc 420

accaaacatc gtccgcaggt ttacatgagc gctagcccgt accagtggat cgtaccgttt 480

gaccgcatcc gtgacggtgt tcacctgatg gttgctcagt cagttcgtcg tacaccgcgt 540

agctctatcg acccgcaggt taaaaacttc cagtggggtg acctgatccg tgcaattcag 600

gaaacccacg ctcgtggttt cgagttgccg ctgctgctgg actgcgacaa cctgctggct 660

gaaggtccgg gcttcaacgt tgttgttatc aaagacggtg ttgttcgttc tccgggtcgt 720

gctgctctgc cgggtatcac ccgtaaaacc gttctggaaa tcgctgaatc tctgggtcac 780

gaagctatcc tggctgacat caccccggct gaactgtacg acgctgacga agttctgggt 840

tgctcaaccg gtggtggtgt ttggccgttc gtttctgttg acggtaactc tatctctgac 900

ggtgttccgg gtccggttac ccagtctatc atccgtcgtt actgggaact gaacgttgaa 960

ccttcttctc tgctgacccc ggtacagtac 990

<210> 2

<211> 334

<212> PRT

<213> Unknown (Unknown)

<400> 2

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. Synthesis of (R) -1- [3,5-bis (trifluoromethyl) phenyl by coupling R-omega-aminotransferase with alcohol dehydrogenase]A method for ethylamine, characterized in that the method is carried out according to the following steps: r-omega-transaminase and alcohol dehydrogenase are used as double enzyme catalysts, 3, 5-bistrifluoromethyl acetophenone is used as an amino acceptor, isopropylamine hydrochloride is used as an amine donor, pyridoxal phosphate and NAD are used ⁺ Adding cosolvent as auxiliary factor, forming reaction system with pH7-10 buffer solution as reaction medium, mixing and stirring at 30-50deg.C in 150-200rpm table for reaction 12-36-h, separating and purifying the reaction solution to obtain (R) -1- [3,5-bis (trifluoromethyl) phenyl]Ethylamine; the cosolvent is ethanol, glycerol or dimethyl sulfoxide; the addition amount of the 3,5-bis (trifluoromethyl) acetophenone is 25-170mmol/L based on the volume of the reaction system; the addition amount of the isopropylamine hydrochloride is 125-850mmol/L based on the volume of the reaction system.

2. The method according to claim 1, wherein the amount of the R- ω -transaminase added to the reaction system is 1 to 2U/mL by volume of the reaction system; the addition amount of the alcohol dehydrogenase is 1-2U/mL based on the volume of the reaction system; the adding amount of pyridoxal phosphate is 0.5-1.5mmol/L based on the volume of the reaction system; the NAD ⁺ Adding inThe amount is 0.5-1.5mmol/L based on the volume of the reaction system; the addition amount of the cosolvent is 10% -30% by volume of the reaction system.

3. The method of claim 1, wherein the R-omega-aminotransferase is pure enzyme obtained by ultrasonic disruption and extraction of wet bacterial cells which are fermented and cultured by engineering bacteria containing the R-omega-aminotransferase gene, and the nucleotide sequence of the R-omega-aminotransferase gene is shown as SEQ ID NO. 1; the pure enzyme is added in the form of an aqueous solution.

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

5. The method of claim 3, wherein the purified enzyme of R- ω -transaminase is prepared by:

(1) Induction culture: inoculating engineering bacteria containing R-omega-aminotransferase genes into LB liquid culture medium containing 30-80 mug/mL ampicillin, and culturing at 37 ℃ and 180rpm for overnight to obtain seed liquid; transferring the seed solution into fresh LB liquid culture medium containing 30-80 mug/mL ampicillin according to the inoculum size of 1% -5% of volume concentration, culturing at 37 ℃ and 180rpm until OD ₆₀₀ Adding isopropyl-beta-thiogalactoside with final concentration of 0.5-1.5mM into the mixture of 0.6-0.9, transferring to 16-26 ℃ to induce expression of 10-20h, centrifuging, collecting cell precipitate, washing with 0.9% physiological saline, and collecting wet bacterial cells; (2) crude enzyme solution: resuspending the wet bacterial cells obtained in the step (1) in a sodium phosphate buffer solution with the pH of 7.0 and 100mM, carrying out ultrasonic crushing for 5 minutes, carrying out power 400W, working for 3 seconds, crushing for 7 seconds, collecting crushed liquid, centrifuging, and taking supernatant to obtain a transaminase crude enzyme solution; (3) pure enzyme: mixing transaminase crude enzyme solution with chromatographic medium, loading onto Beyogold ™ His-tag chromatographic column, washing the column with non-denaturing washing liquid for 5 times to remove impurity protein, eluting 1-2 column volumes each time; eluting with non-denaturing eluent for 6-10 times, eluting 1-2 column volumes each time, collecting target proteinConcentrating the eluent by ultrafiltration, and freeze-drying to obtain pure enzyme of R-omega-aminotransferase; the non-denaturing wash is a pH8.0, 50mM sodium phosphate buffer containing 300mM NaCl and 2mM imidazole; the non-denaturing eluate is pH8.0, 50mM sodium phosphate buffer containing 300mM NaCl and 50mM imidazole.

6. The method of claim 1, wherein the alcohol dehydrogenase is a pure enzyme obtained by ultrasonic disruption and extraction of wet thalli obtained by fermenting and culturing engineering bacteria 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 engineering 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 method of claim 6, wherein the pure enzyme of alcohol dehydrogenase is prepared by: (1) induction culture: inoculating ethanol dehydrogenase genetic engineering bacteria to an LB liquid culture medium containing 30-80 mug/mL Kan antibiotics, and placing the LB liquid culture medium in a shaking table at 37 ℃ and 180rpm for overnight culture to obtain seed liquid; transferring the inoculated strain with the volume concentration of 1-5% into LB liquid culture medium containing 30-80 mug/mL Kan, and continuously culturing at 37 ℃ and 180rpm until OD ₆₀₀ Adding 0.6-0.9 mM IPTG with final concentration of 0.5-1.5mM, inducing expression at 23deg.C and 180rpm for 10-20 hr, centrifuging, collecting cell precipitate, washing with 0.9% physiological saline, centrifuging, and collecting wet cells; (2) crude enzyme solution: resuspending the obtained wet cells in a sodium phosphate buffer solution with pH of 7.0 and 100mM, carrying out ultrasonic treatment on the mixture for 5 minutes under ice bath conditions, carrying out power 400W, working for 3 seconds and crushing for 7 seconds, collecting the crushed liquid after ultrasonic treatment, centrifuging, and taking the supernatant to obtain ethanol dehydrogenase crude enzyme liquid; (3) pure enzyme solution: mixing the ethanol dehydrogenase crude enzyme solution and a chromatographic medium uniformly according to a volume ratio of 8:1, then loading the mixture to a Beyogold ™ His-tag chromatographic column, washing the column with a non-denaturing washing liquid for 5 times to remove the impurity protein, and eluting 1-2 column volumes each time; thenEluting for 6-10 times by using non-denaturing eluent, eluting for 1-2 column volumes each time, collecting eluent containing target protein, ultrafiltering, concentrating, and freeze-drying to obtain pure enzyme of R-omega-transaminase; the non-denaturing wash is a pH8.0, 50mM sodium phosphate buffer containing 300mM NaCl and 2mM imidazole; the non-denaturing eluate is pH8.0, 50mM sodium phosphate buffer containing 300mM NaCl and 50mM imidazole.

9. The method of claim 1, wherein the reaction system further comprises metal ions and a reagent, the metal ions and the reagent being Ca ²⁺ 、Mg ²⁺ 、Cu ²⁺ 、Fe ²⁺ 、Zn ²⁺ 、Mn ²⁺ 、Na ⁺ 、K ⁺ Or EDTA.

10. The method of claim 9, wherein the final concentrations of the metal ions and the reagent added to the reaction system are each 10mM.