Olefin reductase SeER mutant and application thereof in synthesis of chiral drugs

Abstract

The invention discloses an olefin reductase SeER mutant and application thereof in synthesis of chiral drugs, and provides a series of novel olefin reductase SeER mutants capable of efficiently and asymmetrically reducing beta-alkyl / aryl-beta-cyanoacrylate compounds, and the olefin reductase SeER mutants have high conversion rate (gt; 99% conversion rate), good enantioselectivity (gt; 99% ee), the substrate range is wide (gt; according to the present invention, the screened olefin reductase SeER mutant can be successfully synthesized into the chiral GABA derivative drugs such as pregabalin and brivaracetam through the chemical-enzyme coupling method, such that the overall reaction yield is improved, and the route is simple;

Description

(I) Technical Field

[0002] This invention belongs to the field of drug synthesis, and specifically relates to an olefin reductase. Se ER mutants and their application in the synthesis of chiral drugs from reduced olefin compounds. (II) Background Technology

[0004] olefin reductases (ERs) are oxidoreductases belonging to the FMN-dependent oxidoreductase superfamily. They catalyze the asymmetric reduction of alkenes to produce chiral molecules with up to two stereocenters. Olefin reductases offer advantages such as mild reaction conditions and high selectivity, thus gradually replacing traditional high-pressure hydrogenation methods catalyzed by chemical metals and becoming important tools in the construction of chiral drugs. However, olefin reductases typically exhibit low catalytic activity and poor enantioselectivity with non-natural substrates, limiting their application in the synthesis of key intermediates for chiral drugs. Therefore, enzyme molecular modification is needed to enhance their activity and enantioselectivity with non-natural substrates.

[0005] Chiral γ-amino acid butyric acid (GABA) and its derivatives are a class of molecules with diverse biological activities. β -alkyl / aryl cyano ester molecular skeletons are important precursors for the synthesis of chiral GABA derivatives. Asymmetric reduction via olefin reductases... β -alkyl / aryl- β The cyanoacrylate route provides a simple and atom-economical method for synthesizing chiral GABA derivatives. However, the olefin reductases used in this route have been reported to have problems such as low catalytic activity and poor enantioselectivity, making it difficult to meet the requirements for the synthesis of highly optically pure chiral molecules. (III) Summary of the Invention

[0007] The purpose of this invention is to provide an olefin reductase. Se This invention relates to ER mutants, encoding genes, vectors, engineered bacteria, and their application in the synthesis of chiral drugs from highly active, highly enantioselective reduction of olefin compounds. S. E. eubayanus olefin reductase Se ER was used for molecular modification to screen for molecules with high activity (>99% conversion rate) and high enantioselectivity (>99%). ee Mutants that catalyze the reduction of olefin compounds, such as ( Z Methyl 3-cyano-3-isobutyl acrylate and its series of non-natural substrates can be further converted into chiral GABA derivatives, such as pregabalin and buvacerostat, through coupling chemical ester hydrolysis and cyano reduction. This solves the problems of low activity and poor stereoselectivity of existing olefin reductases in the synthesis of chiral cyano ester compounds.

[0008] The technical solution adopted in this invention is:

[0009] In a first aspect, the present invention provides an olefin reductase. Se ER mutant, the olefin reductase Se The ER mutant is derived from *Saccharomyces cerevisiae* as shown in SEQ ID NO. 2. S. eubayanus olefin reductase Se The olefin reductase described in this invention is obtained by single- or multi-site site-directed mutation or site-directed saturation mutation at positions 38, 83, 87, and 117 of the ER amino acid sequence. Se The nucleotide sequence of the ER-encoding gene is shown in SEQ ID NO.1.

[0010] Furthermore, the olefin reductase is preferred. SeThe ER mutant is formed by mutating the amino acid sequence shown in SEQ ID NO.2 to one of the following: (1) deletion of the 117th tryptophan residue (Del_W117, denoted as M1); (2) site deletion of the 117th tryptophan and mutation of the 38th threonine to asparagine or serine (M1 / T38N, M1 / T38S); (3) site deletion of the 117th tryptophan and mutation of the 83rd tyrosine to arginine or proline (M1 / Y83R, M1 / Y38P); (4) site deletion of the 117th tryptophan and mutation of the 87th proline to cysteine ​​(M1 / P87C); (5) site deletion of the 117th tryptophan and mutation of the 38th threonine to asparagine or serine (M1 / T38N, M1 / T38S); (6) Delete tryptophan at position 117 and mutate threonine at position 38 to asparagine and tyrosine at position 83 to proline (M1 / T38N / Y83P); (7) Delete tryptophan at position 117 and mutate threonine at position 38 to serine and tyrosine at position 83 to arginine (M1 / T38S / Y83R); (8) Delete tryptophan at position 117 and mutate threonine at position 38 to serine and tyrosine at position 83 to arginine (M1 / T38S / Y83P); (9) Delete tryptophan at position 117 and mutate threonine at position 38 to serine and tyrosine at position 83 to proline (M1 / T38S / Y83P); (10) Delete tryptophan at position 117 and mutate threonine at position 38 to asparagine and proline at position 87 to cysteine ​​(M1 / T38N / P87C); (11) Delete tryptophan at position 117 and mutate threonine at position 38 to serine and proline at position 87 to cysteine ​​(M1 / T38S / P87C); (12) Delete tryptophan at position 117 and mutate threonine at position 38 to serine, tyrosine at position 83 to proline and proline at position 87 to cysteine ​​(M1 / T38S / Y83P / P87C); (13) The threonine at position 8 is mutated to serine, the tyrosine at position 83 is mutated to arginine, and the proline at position 87 is mutated to cysteine ​​(M1 / T38S / Y83R / P87C); (14) The tryptophan at position 117 is deleted and the threonine at position 38 is mutated to asparagine, the tyrosine at position 83 is mutated to proline, and the proline at position 87 is mutated to cysteine ​​(M1 / T38N / Y83P / P87C); (15) The tryptophan at position 117 is deleted and the threonine at position 38 is mutated to asparagine, the tyrosine at position 83 is mutated to arginine, and the proline at position 87 is mutated to cysteine ​​(M1 / T38N / Y83R / P87C).

[0011] This invention also relates to the olefin reductase. SeThe ER mutant encoding gene, recombinant expression vector, enzyme mutant, and recombinant genetically engineered bacteria are included. The recombinant expression vector is preferably pET21a(+), and the recombinant genetically engineered bacteria are preferably host bacteria. E. coli BL21(DE3).

[0012] Secondly, this invention also relates to the application of the olefin reductase mutant in the synthesis of chiral compounds from olefin compounds with high enantioselectivity. The method of application is as follows: using wet cells, lyophilized powder, or purified enzyme obtained by inducing culture of recombinant genetically engineered bacteria of the olefin reductase mutant as a catalyst, wet cells or lyophilized powder obtained by inducing culture of glucose dehydrogenase genetically engineered bacteria as a coenzyme, a prechiral olefin compound as a substrate, glucose as a co-substrate, dimethyl sulfoxide as a cosolvent, and NADP... + As a cofactor, a buffer solution with pH 5-9 (preferably pH 7.4, 100 mM potassium phosphate buffer) was used as the reaction medium to form the reaction system. The reaction was carried out at 25-40℃ (preferably 30℃) and 1000 rpm. After the reaction was completed, the reaction solution was separated and purified to obtain the chiral product.

[0013] Furthermore, the structure of the prochiral olefin compound is shown in Figure 1:

[0014]

[0015] In Equation 1, R 1 It is one of the following: methyl, ethyl, n-propyl, n-butyl, isopropyl, isobutyl, cyclobutyl, cyclopentyl, or phenyl; R 2 and R 4 Each group can be either a cyano group or an ester group; R 3 It can be methyl or hydrogen.

[0016] Further, the olefin compound is one of the following: (Z)-3-cyano-3-methyl methacrylate (1b), (Z)-3-cyano-3-ethyl methacrylate (1c), (Z)-3-cyano-3-n-propyl methacrylate (1d), (Z)-3-cyano-3-n-butyl methacrylate (1f), (Z)-3-cyano-3-isopropyl methacrylate (1e), (Z)-3-cyano-3-isobutyl methacrylate (1g), (Z)-3-cyano-3-isobutyl methacrylate (1a), (Z)-3-cyano-3- Ethyl cyclobutyl acrylate (1h), (Z)-3-cyano-3-cyclopentyl acrylate (1i), (Z)-3-cyano-3-(4-chloro)phenyl acrylate methyl ester (1j), (Z)-3-cyano-3-methacrylate ethyl ester (1k), (Z)-3-cyano-3-methacrylate isopropyl ester (1i), (Z)-3-cyano-3-methacrylate tert-butyl ester (1m), (Z)-2-methyl-3-cyano-3-methacrylate methyl ester (1n), 2-methyl maleate dimethyl ester (1o), 2-phenyl maleate dinitrile (1p).

[0017] Furthermore, in the reaction system, the substrate is added to a final concentration of 10–200 mM; the glucose is added to a final concentration of 20–300 mM; the catalyst dosage is 20–80 g / L (preferably 50 g / L) based on the wet cell weight, or 0.1–4 g / L based on the protein content in the purified enzyme; the coenzyme… Es GDH concentration was 1–2 mg / mL based on the weight of the purified lyophilized enzyme powder; dimethyl sulfoxide concentration was added at 5–10% by volume; NADP... + The concentration added was 0.01-20 mM. The glucose dehydrogenase gene (GenBank No. KM817194.1) was derived from a Siberian microbacterium (…). Exiguobacterium sibirium DSM 17290), the nucleotide sequence is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4.

[0018] Furthermore, the wet bacterial cells are prepared by the following method: [The method involves] preparing a solution containing olefin reductase. Se Engineered bacteria containing the ER mutant gene were inoculated into LB liquid medium containing a final concentration of 100 μg / mL ampicillin and incubated at 37°C. o C. Incubate at 200 rpm for 10 h to obtain seed culture; then, incubate the seed culture at a volume concentration of 1.0% (… v / v The inoculum was inoculated into sterilized LB liquid medium containing a final concentration of 100 μg / mL ampicillin, and inoculated at 37°C. o C. Incubate at 200 rpm until OD 600= 0.6~0.8, add isopropyl thiogalactoside (IPTG) to the culture medium to a final concentration of 0.15 mM, 16 o After culturing in C for 16 hours, 4 o C. Centrifuge at 8000 rpm for 10 min to obtain olefin reductase. Se The wet cells of the ER mutant; the method for preparing the wet cells of the engineered bacteria containing the glucose dehydrogenase gene through induction culture is the same as that for the olefin reductase-containing bacteria. Se Wet bacterial cells of the ER mutant gene.

[0019] Furthermore, the aforementioned Se The ER mutant purification enzyme was prepared as follows: [The enzyme contained in the olefin reductase was then prepared.] Se The engineered bacteria containing the ER mutant gene were resuspended at 100 g / L in 100 mM potassium phosphate buffer (pH 7.4) after fermentation. The mixture was then sonicated on an ice-water mixture for 25 min under the following conditions: 400 W power, 2 s sonication followed by a 3 s pause. The resulting mixture was collected to obtain a crude enzyme solution. 0.1 mg / mL of riboflavin sodium phosphate (FMN) was added to the crude enzyme solution, and the mixture was incubated on ice for 30 min, followed by sonication at 8000 rpm for 4 minutes. o Centrifuge at C for 20 min, collect the supernatant, filter through a 0.45 μm microfiltration membrane, and purify the mutant protein using a nickel affinity column (1.6 × 10 cm, Bio-Rad, USA). The purification procedure is as follows: ① Elute with gravity in a pH 7.4, 20 mM potassium phosphate buffer containing 0.5 M NaCl and 20 mM imidazole to remove unbound impurities until the conductivity stabilizes; ② Elute with gravity for one column volume in a pH 7.4, 20 mM potassium phosphate buffer containing 0.5 M NaCl and 500 mM imidazole; collect all eluent and transfer to an ultrafiltration tube with a molecular weight cutoff of 30 kDa. Use 20 mM, pH 7.4 potassium phosphate as a replacement buffer to remove imidazole, collect the cutoff protein, and obtain the desired protein. Se ER mutant purification enzyme. Es The preparation method of GDH purification enzyme is the same as that of GDH purification enzyme. Se The ER mutant was used to purify the enzyme, but no riboflavin sodium phosphate was added to the crude enzyme solution and the ultrafiltration tube used had a molecular weight cutoff of 10 kDa.

[0020] Thirdly, the present invention also provides an olefin reductase. Se The application of ER mutants in the catalytic asymmetric synthesis of pregabalin, wherein the application is as follows: using olefin reductase-containing enzymes... SeThe engineered bacteria encoding the ER mutant gene were fermented to obtain wet bacterial cells, purified enzyme, or lyophilized purified enzyme powder as catalysts. The purified enzyme lyophilized powder obtained from the fermentation of engineered bacteria containing the glucose dehydrogenase encoding gene was used as a coenzyme. Ethyl (Z)-3-cyano-3-isobutyl acrylate was used as the substrate, glucose as a co-substrate, dimethyl sulfoxide (DMSO) as a cosolvent, and NADP... + Using a cofactor, a reaction system was constructed using a pH 5-9 buffer solution (preferably pH 7.4) as the reaction medium. The reaction was carried out at 25-40 °C (preferably 30 °C) and 1000 rpm. After the reaction was completed, the reaction solution was separated and purified to obtain ( S ethyl 3-cyano-3-isobutylpropionate; S Ethyl 3-cyano-3-isobutylpropionate was hydrolyzed with potassium hydroxide and then hydrogenated with Raney nickel to synthesize pregabalin; the olefin reductase... Se ER mutant preferred Se ER-DelW117 / T38N / Y83P / P87C Se ER-DelW117 / T38N / Y83R / P87C.

[0021] Furthermore, the method for synthesizing pregabalin: at 0°C, ( S Ethyl 3-cyano-3-isobutylpropionate was dissolved in methanol, and potassium hydroxide aqueous solution was slowly added dropwise. After the addition was complete, the mixture was brought back to room temperature and stirred for 3 hours. The reaction was monitored by TLC until the reactants were fully reacted. Raney nickel was then added to the solution, and the mixture was stirred for 24 hours under a hydrogen atmosphere. After the reaction was complete, the insoluble solids were removed by filtration. The filtrate was concentrated and recrystallized using an isopropanol / water system. The resulting white solid was pregabalin. S The mass ratio of ethyl 3-cyano-3-isobutylpropionate to potassium hydroxide is 1.5:1, and the volume of methanol used is ( S The mass of ethyl 3-cyano-3-isobutylpropionate is 13-15 mL / g; S The mass ratio of ethyl 3-cyano-3-isobutylpropionate to Raney nickel was 20:1. w / w ).

[0022] Furthermore, the substrate was added to a final concentration of 200 mM, and the cosolvent was added to a final concentration of 10% by volume, with the substrate first dissolved in the cosolvent before addition; the glucose was added to a final concentration of 300 mM; the coenzyme was added to a final concentration of 1 mg / mL; the catalyst was added to a final concentration of 50 g / L based on the wet cell weight; NADP... + Add to a final concentration of 20 mM; the buffer solution is 100 mM, pH 7.4 potassium phosphate buffer.

[0023] Fourthly, the present invention provides an olefin reductase. Se Application of ER mutants in the asymmetric synthesis of brivaceran, wherein the application is based on olefin reductase. Se The engineered bacteria encoding the ER mutant gene were fermented to obtain wet bacterial cells, purified enzyme, or lyophilized purified enzyme powder as catalysts. The purified enzyme lyophilized powder obtained from the fermentation of engineered bacteria containing the glucose dehydrogenase encoding gene was used as a coenzyme. Methyl (Z)-3-cyano-3-n-propyl acrylate was used as the substrate, glucose as a co-substrate, dimethyl sulfoxide (DMSO) as a solubilizer, and NADP... + Using a cofactor, a reaction system was constructed using a pH 5-9 buffer solution (preferably pH 7.4) as the reaction medium. The reaction was carried out at 25-40 °C (preferably 30 °C) and 1000 rpm. After the reaction was completed, the reaction solution was separated and purified to obtain ( R ethyl 3-cyano-3-n-propylpropionate; R Ethyl 3-cyano-3-n-propylpropionate is generated by reduction with Raney nickel followed by spontaneous cyclization to form (4... R )-4-propylpyrrolidone-2-one, (4 R )-4-propylpyrrolidone-2-one and ( S 2-Bromobutyramide undergoes a substitution reaction to generate brivacertan, the olefin reductase. Se ER mutant preferred Se ER-DelW117 / T38N / Y83P / P87C Se ER-DelW117 / T38N / Y83R / P87C.

[0024] Furthermore, the substrate was added to a final concentration of 200 mM, and the cosolvent was added to a final concentration of 10% by volume, with the substrate first dissolved in the cosolvent before addition; the glucose was added to a final concentration of 300 mM; the coenzyme was added to a final concentration of 1 mg / mL; the catalyst was added to a final concentration of 50 g / L based on the wet cell weight; NADP... + Add to a final concentration of 20 mM; the buffer solution is 100 mM, pH 7.4 potassium phosphate buffer.

[0025] Furthermore, the method for synthesizing buvasidan: (…) R Methyl 3-cyano-3-n-propylpropionate was dissolved in methanol, Raney nickel was added, and the mixture was reacted under a hydrogen atmosphere for 24 h. After the reaction was complete, the mixture was filtered, and the filtrate was concentrated to dryness to obtain (4-) R )-4-propylpyrrolidone-2-one, the resulting (4 R )-4-propylpyrrolidone-2-one was slowly added dropwise to a tetrahydrofuran solution containing sodium hydride. After stirring at 0 °C for 20 min, ( S)-2-bromobutyramide, continue stirring at 0 °C until (4 R 4-propylpyrrolidone-2-one was completely converted. After the reaction was complete, a saturated ammonium chloride solution was added to quench the reaction, the organic phase was extracted, concentrated and dried, and the resulting mud-like substance was recrystallized from isopropanol / water to obtain a white solid, which was buvacerostatin. R The mass ratio of methyl 3-cyano-3-n-propylpropionate to Raney nickel was 20:1. w / w ), methanol volume usage ( R The mass of methyl 3-cyano-3-n-propylpropionate is 13-15 mL / g; R The mass ratio of methyl 3-cyano-3-n-propylpropionate to sodium hydride was 5.8:1. w / w ); ( R methyl 3-cyano-3-n-propylpropionate and ( S The feed ratio of 2-bromobutyramide is 1:1.5. w / w ).

[0026] The olefin reductase of the present invention Se Both the ER and olefin reductase mutants have a full-length base sequence of 1218 bp, from the first base to the 1194th base, with the start codon being ATG and the stop codon being TAA, encoding 405 amino acids.

[0027] The olefin reductase of the present invention Se The ER mutants were obtained through protein engineering, ultimately yielding six beneficial mutants: DelW117, T38S, T38N, Y83P, Y83R, and P87C. Further iterative combination mutations were used to obtain olefin reductase. Se ER quadratic mutant library. The obtained mutant recombinant plasmid was transformed into the library by heat shock. E. coli BL21(DE3) competent cells were used to inoculate, transfer, induce culture, and recover the obtained strain. For the inoculation, transfer, induction culture, and cell recovery of the olefin reductase mutant of this invention, the culture medium can be any medium in the art that can grow the strain of this invention, preferably LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, dissolved in water. There are no special limitations on the culture method and conditions; the culture method and conditions can be appropriately selected according to different host types and culture methods, based on common knowledge in the art.

[0028] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0029] (1) Olefin reductase Se The catalytic performance of the ER mutant is significantly improved.

[0030] This invention provides a series of new methods capable of efficient asymmetric reduction. β -alkyl / aryl- β olefin reductase of cyanoacrylate compounds Se ER mutants exhibit high transformation efficiency (>99%) and good enantioselectivity (>99%). ee It has a broad substrate range (>10 substrate types) and is tolerant to high substrate concentrations (200 mM).

[0031] (2) The synthetic route for chiral GABA drugs has high yield and good atom economy.

[0032] The olefin reductase screened in this invention Se ER mutants were successfully used to synthesize chiral GABA derivative drugs, such as pregabalin and buvacertan, via a chemical-enzyme coupling method, with improved overall reaction yield and a simpler route. (iv) Description of the attached drawings

[0034] Figure 1 For different pH values ​​in Example 5 Se Relative activity of ER-M1 / T38N / Y83P / P87C.

[0035] Figure 2 For different temperatures in Example 5 Se Relative activity of ER-M1 / T38N / Y83P / P87C.

[0036] Figure 3 For different NADPs in Example 5 + concentration Se Relative activity of ER-M1 / T38N / Y83P / P87C.

[0037] Figure 4 for Se Substrate profiles of ER and its mutants.

[0038] Figure 5 for Se Schematic diagram of pregabalin synthesis using ER mutant.

[0039] Figure 6 This is the NMR spectrum of pregabalin.

[0040] Figure 7 for Se Schematic diagram of ER mutant synthesis of Brucine. (V) Detailed Implementation Methods

[0042] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0043] The LB liquid medium used in this invention consists of 10 g / L peptone, 5 g / L yeast extract, and 10 g / L sodium chloride dissolved in water. The LB solid medium is the LB liquid medium with 18 g / L agar added.

[0044] Example 1: Se ER purification enzyme preparation and enzyme activity assay

[0045] 1. Se ER purification enzyme

[0046] (1) Wild-type olefin reductase genetically engineered strain: artificially synthesized from GenBank by *Saccharomyces cerevisiae* ( Saccharomyces eubayanus The olefin reductase gene sequence (GenBank No. XP_018218866.1), i.e., olefin reductase. Se The ER gene (nucleotide sequence as shown in SEQ ID NO.1, amino acid sequence as shown in SEQ ID NO.2) is ligated into the vector pET21a(+). Nde I and Xho A recombinant expression vector was constructed between the I restriction sites, and this recombinant expression vector was transformed into the host bacteria. E. coli BL21(DE3) was used to obtain a wild-type olefin reductase genetically engineered bacterium. E. coli BL21(DE3) / pET21a(+)-seer.

[0047] SEQ ID NO.2:

[0048] MPFVKDFEPPVSLENTNLFKPIKVGNTQLAHRVVMPPLTRMRASHPGNVPNKQWAAEYYRQRAQRPGTMIITEGTFISPQAGGYDNAPGIWSDEQVAQWKDI LLAIHDKKSFAWVQLWALGRASFPDAMARDGLRYDSASDAVYMEDTANAAKVPAHTLTKDDIKQYVKEYVHAAKNAVFLAGADGVEIHSANGYLLNQFLDPR SNTRNDEYGGSIENRARFTLEVVDAVSDAIGHDRVGLRLSPYGTFNSMSGGAEPHIVAQYAYVLGELERRAKAGKRLAFVHLVEPRVTDLTDEGEGDYDE GSNDFAYSIWKGPIVKAGNFALHPDAVKEEVKRDRTLIAYGRFFIANPDLVDRLEKGLPLNKYDRATFYTMSAEGYIDYPTYGEAVELGWDKNLEHHHHHH.

[0049] (2) Wet bacteria

[0050] The construction in step (1) E. coli BL21(DE3) / pET21a(+)-seer was inoculated into LB liquid medium containing a final concentration of 100 μg / mL ampicillin and incubated at 37°C. o C. Incubate at 200 rpm for 10 h to obtain seed culture; dilute the seed culture with a volume concentration of 1% ( v / v The inoculum was inoculated into fresh LB liquid medium containing a final concentration of 100 μg / mL ampicillin, and inoculated at 37°C. o C. Incubate at 200 rpm until OD 600 = 0.6~0.8, then add IPTG to the culture medium to a final concentration of 0.15 mM, 16 o After culturing in C for 16 hours, 4 o C. Centrifuge at 8000 rpm for 10 min and collect the wet cells of the engineered bacteria.

[0051] (3) Enzyme purification

[0052] The wet bacterial cells from step (2) were dispersed in a 100 g wet bacterial cell / L buffer solution and resuspended in a pH 7.4, 100 mM potassium phosphate buffer solution. The mixture was then sonicated on an ice-water mixture for 25 min. The sonication conditions were: 400 W power, 2 s of sonication followed by a 3 s pause. The resulting sonicated mixture was the crude enzyme solution.

[0053] Riboflavin sodium phosphate (FMN) was added to the obtained crude enzyme solution at a rate of 1 mg / g wet bacterial cells, and the solution was incubated on ice for 30 min, followed by incubation at 8000 rpm for 4 hours. o Centrifuge at C for 20 min, collect the supernatant, filter through a 0.45 μm filter membrane, and purify the permeate using a nickel affinity column (1.6 × 10 cm, Bio-Rad, USA).

[0054] The enzyme purification procedure is as follows: ① Load the sample at a flow rate of 1.0 mL / min and elute by gravity with a pH 7.4 buffer containing 0.5 M NaCl, 20 mM imidazole, and 20 mM potassium phosphate to remove unbound impurities until the conductivity stabilizes; ② Then elute by gravity for one column volume with a pH 7.4 buffer containing 0.5 M NaCl, 500 mM imidazole, and 20 mM potassium phosphate; pour all the collected eluent (one column volume, 10 mL) into a 30 kDa ultrafiltration tube, use 20 mM pH 6.0 potassium phosphate as the replacement buffer to remove imidazole, and collect the truncated protein, which is the final product. Se ER purified enzyme, denoted as wt- SeER. The purified enzyme was freeze-dried at -80℃ to prepare a purified enzyme lyophilized powder for subsequent experiments.

[0055] (4) Enzyme activity assay

[0056] An enzyme activity unit (U) is defined as the amount of enzyme required to consume 1 μmol of NADPH or produce 1 μmol of product per minute under optimal conditions. Specific enzyme activity is defined as the number of international units of enzyme activity per milligram of enzyme protein, U / mg.

[0057] Enzyme activity assay conditions: Add 10 mM (Z)-3-cyano-3-isobutylacrylate and 1 mM NAD to 500 μL phosphate buffer (PB, 100 mM, pH 7.4). Final concentration 25 mM glucose, final concentration 2 mg / mL, prepared in step 2. Es GDH freeze-dried powder and appropriate amount Se ER purification of enzyme was performed at 30℃ for 10–60 minutes. After extraction with ethyl acetate, the ethyl acetate layer was analyzed by gas chromatography to determine the substrate and product content, and the substrate conversion rate and product yield were calculated. ee value.

[0058] Gas chromatography was performed using an Agilent 7890 gas chromatograph with a chiral column (Astec). ® CHIRALDEX™ G-TA (30 m × 0.25 mm × 0.12 μm), carrier gas N2, flow rate 1 mL / min, 100 °C, 5 °C·min -1 Heat to 180 °C and hold for 5 min. Substrate elution time (13.41 min), product elution time ( R Configuration product 12.172 min, S Configuration product 12.690 min).

[0059] 2. Es GDH purification enzyme

[0060] Using the method in step 1, a strain of Siberian microbacterium (from GenBank) was artificially synthesized. Exiguobacterium sibirium The glucose dehydrogenase gene (GenBank No. KM817194.1) of DSM 17290, with the nucleotide sequence shown in SEQ ID NO.3, was analyzed under the same conditions (the difference being...). Es The GDH crude enzyme solution was prepared without the addition of riboflavin sodium phosphate and the ultrafiltration tube had a molecular weight cutoff of 10 kDa. Es GDH wet bacteria and Es GDH purified enzyme.Es GDH purified enzyme was freeze-dried at -80℃ to prepare purified enzyme lyophilized powder for subsequent experiments.

[0061] Enzyme activity assay conditions: 1 mM NADP + 0.01 mg protein content Es GDH purification enzyme, 1 mM glucose, pH 6.0, and 100 mM potassium phosphate buffer were added to bring the final volume to 200 μL. The solution was then incubated at 30°C. o The reaction was carried out at C for 2 min, and the amount of NADPH generated was detected using a Biotek Epoch microplate reader.

[0062] Enzyme activity is defined as the amount of enzyme required to generate 1 micromole of NADPH or consume 1 micromole of glucose per minute under optimal conditions.

[0063] Example 2: Se Construction of ER mutants

[0064] 1. WT- Se Selection of ER mutant sites

[0065] Using the docking software YASARA, the substrate (Z)-3-cyano-3-phenylmethyl acrylate was docked into the active pocket of the WT-SeER enzyme. Amino acid residues within a 5 Å range of the substrate were selected, as these amino acids are most likely to affect the selectivity and activity of the enzymatic reaction. After excluding the conserved catalytic triplet amino acids (H189, N192, Y194), T38, W117, Y83, and P87 were selected as mutation sites. These amino acids constitute the pocket on the ester side of the substrate and may interact with the ester functional group, thereby affecting the enantioselectivity of the reaction activity. At the same time, since W117 has a large side chain group, it is most likely to affect the reaction between the enzyme and the substrate, so it was preferentially saturated and mutated.

[0066] 2. Construction and screening of W117 site saturation mutants

[0067] (1) Saturation mutation

[0068] Extraction Example 1 E. coli The plasmid pET21a(+)-seer of BL21(DE3) / pET21a(+)-seer was amplified by PCR using the primers in Table 1. The tryptophan at position 117 was mutated. After digesting the PCR product with enzymes to remove the original template and clean up, it was transformed into [the desired product]. E. coli BL21(DE3) competent cells were seeded with clones into LB solid medium containing 100 μg / mL ampicillin and incubated at 37°C. oCultured at C for 12 h. Then, clones were picked and transferred to 10 mL of LB liquid medium containing 100 μg / mL ampicillin, and incubated at 37°C. o C. Incubate at 200 rpm for 10 h, then centrifuge the culture at 8000 rpm for 10 min and collect the wet cells. Store the selected strains at -80 ℃.

[0069] Table 1. Primer design for saturation mutant at the W117 site of olefin reductase

[0070]

[0071] Note: a W117TY represents the upstream universal primer for the 117 site mutation.

[0072] PCR reaction system (25 μL): 0.5 μL forward primer (100 μM), 0.5 μL reverse primer (100 μM), 12.5 μL 2×Phanta buffer, 0.5 μL dNTP mixture (10 mM each), 0.5 μL plasmid template, 0.5 μL DNA polymerase, and 10 μL ultrapure water. The PCR program was set up according to the Phanta Super-Fidelity DNA polymerase manual as follows: 95 o C pre-denaturation for 5 min, followed by 30 cycles (95) o C denaturation 15 s, 55-65 o Annealing at C for 15 seconds, 72°C o C extended 10 s), 72 o C extends for 10 minutes, 16 o C. Insulation.

[0073] (2) Screening of mutants

[0074] Mutant screening reaction system: 50 g / L of each wet bacterial cell prepared in step (1), 10 mM (Z)-3-cyano-3-isobutylacrylate (added in the form of DMSO solution), DMSO (5%) v / v ), 25 mM glucose, 2 g / L, prepared by the method in Example 1 Es GDH lyophilized powder, 1 mM NADP + The reaction medium was a pH 7.4, 100 mM potassium phosphate buffer solution, comprising 1 mL of the reaction system, and the reaction was carried out at 30°C. oAfter reacting at 1000 rpm for 24 h, the reaction solution was extracted with ethyl acetate. The substrate concentration was determined using the gas chromatography method described in Example 1, and the substrate conversion, specific enzyme activity, and stereoselectivity were calculated. The results are shown in Table 2. Each mutant was characterized in triplicate. The dominant mutant strains were selected as follows: E. coli BL21(DE3) / pET21a(+)-seer-Del_W117, denoted as E. coli BL21(DE3) / pET21a(+)-seer-M1.

[0075] Table 2 Results of saturation mutation at the W117 site

[0076]

[0077] 3. Mutations and screening at the T38 site

[0078] Extraction step 2 engineered bacteria E. coli Using the plasmid BL21(DE3) / pET21a(+)-seer-M1 as a template, PCR amplification was performed using the degenerate primers in Table 3, following the methods and conditions in step 2. The relative activity and stereoselectivity of each mutant wet cell to M1 were tested, and the results are shown in Table 4. The dominant strains obtained were... Se ER-M1 / T38N and Se ER-M1 / T38S should be stored at -80 ℃.

[0079] Table 3 Primers for T38 site saturation mutation

[0080]

[0081] N represents bases A, T, C, and G; K represents bases G and T; M represents bases A and C, and so on.

[0082] Table 4. Relative activity and stereoselectivity of dominant mutants at the T38 site

[0083]

[0084] 4. Mutation and screening at the Y83 site

[0085] Extraction step 2 engineered bacteria E. coli Using the plasmid BL21(DE3) / pET21a(+)-seer-M1 as a template, PCR amplification was performed using the primers in Table 5 and the methods and conditions in step 2. The relative activity and stereoselectivity of each mutant wet cell to M1 were tested, and the results are shown in Table 6. The dominant strains were screened out as follows: Se ER-M1 / Y83P and Se ER-M1 / Y83R should be stored at -80 ℃.

[0086] Table 5 Primers for Y83 site saturation mutation

[0087]

[0088] Table 6. Relative activity and stereoselectivity of dominant mutants at the Y83 site.

[0089]

[0090] 4. Mutation and screening at the P87 site

[0091] Extraction step 2 engineered bacteria E. coli Using the plasmid BL21(DE3) / pET21a(+)-seer-M1 as a template, PCR amplification was performed using the primers in Table 7 and the methods and conditions in step 2. The relative activity and stereoselectivity of each mutant wet cell to M1 were tested, and the results are shown in Table 8. The dominant strains were screened out as follows: Se ER-M1 / P87C, and store at -80 ℃.

[0092] Table 7 Primers for P87 site saturation mutation

[0093]

[0094] Table 8. Relative activity and stereoselectivity of dominant mutants at the P87 site.

[0095]

[0096] Example 3: Construction of the Triple Mutant

[0097] Extraction of engineered bacteria in Example 2 E. coli BL21(DE3) / pET21a(+)-seer-M1 / T38S、 E. coli BL21(DE3) / pET21a(+)-seer-M1 / T38N、 E. coli BL21(DE3) / pET21a(+)-seer-M1 / Y83R、 E. coli BL21(DE3) / pET21a(+)-seer-M1 / Y83P and E. coli Using the plasmid BL21(DE3) / pET21a(+)-seer-M1 / P87C as a template, and employing the primers in Table 9, PCR amplification was performed according to the method and conditions in step 2 of Example 2 to construct engineered bacteria. E. coli BL21(DE3) / pET21a(+)-seer-M1 / T38S / Y83R、 E. coliBL21(DE3) / pET21a(+)-seer-M1 / T38S / Y83P、 E. coli BL21(DE3) / pET21a(+)-seer-M1 / T38S / P87C, E. coli BL21(DE3) / pET21a(+)-seer-M1 / T38N / Y83P、 E. coli BL21(DE3) / pET21a(+)-seer-M1 / T38N / Y83R、 E. coli BL21(DE3) / pET21a(+)-seer-M1 / T38N / P87C was stored at -80 ℃. The relative activity and stereoselectivity of each engineered strain are shown in Table 10.

[0098] Table 9 Primers for Tri-mutants

[0099]

[0100] Note: a TY represents the upstream universal primer for the site mutation.

[0101] Table 10 Relative activity and stereoselectivity of the three mutations

[0102]

[0103] Example 4: Construction of a quad mutant

[0104] Extraction of engineered bacteria in Example 3 E. coli BL21(DE3) / pET21a(+)-seer-M1 / T38S / P87C, E. coli Using the plasmid BL21(DE3) / pET21a(+)-seer-M1 / T38N / P87C as a template, and with the primers in Table 9, a saturation mutation was performed at site 83. PCR amplification was then performed using the method and conditions in step 2 of Example 2 to construct engineered bacteria. E. coli BL21(DE3) / pET21a(+)-seer-M1 / T38S / Y83R / P87C、 E. coli BL21(DE3) / pET21a(+)-seer-M1 / T38S / Y83P / P87C、 E. coli BL21(DE3) / pET21a(+)-seer-M1 / T38N / Y83R / P87C、 E. coli BL21(DE3) / pET21a(+)-seer-M1 / T38N / Y83P / P87C were cultured and stored at -80 ℃. The relative activity and stereoselectivity of each engineered bacterium are shown in Table 11.

[0105] Table 11 Relative activity and stereoselectivity of the four mutants

[0106]

[0107] Example 5: Reaction temperature, pH, and NADP of mutant M1 / T38N / Y83P / P87C + Concentration optimization

[0108] 1. Optimal reaction pH

[0109] The mutant M1 / T38N / Y83P / P87C was prepared and purified using the method in Example 1, with a protein concentration of 0.1 mg / mL. The optimal reaction pH for the mutant was screened using acetate buffer (pH 5.0–5.5), phosphate buffer (pH 5.5–7.5), and Tris-hydrochloric acid buffer (pH 7.5–9.0).

[0110] Reaction system: In 1 mL of 100 mM buffer at different pH values ​​(pH 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 9.0), add protein at a concentration of 0.1 mg / mL. Se Purified enzyme of ER-M1 / T38N / Y83P / P87C, 10 mM substrate (Z)-3-cyano-3-isobutylacrylate (substrate dissolved in DMSO before addition), final concentration 5% DMSO, 30 mM glucose, 1 mM NADP + 1 mg / mL Es GDH purified enzyme lyophilized powder. (At 30...) o C. React at 1000 rpm for 10 min, and calculate the enzyme activity at different pH values ​​according to the method in Example 1. The results are as follows: Figure 1 As shown, Se The optimal reaction pH for ER-M1 / T38N / Y83P / P87C is 7.4.

[0111] 2. Optimal reaction temperature

[0112] Add the protein to 1 mL of pH 7.4, 100 mM potassium phosphate buffer at a concentration of 0.1 mg / mL. Se ER-M1 / T38N / Y83P / P87C purified enzyme, 10 mM substrate (Z)-3-cyano-3-isobutylacrylate (substrate dissolved in DMSO before addition), final volume concentration 5% DMSO, 30 mM glucose, 1 mM NADP + 1 mg / mL EsGDH purified enzyme lyophilized powder was reacted at 20–45℃ (20℃, 25℃, 30℃, 35℃, 40℃, 45℃) and 1000 rpm for 10 min. Enzyme activity at different temperatures was detected according to the method in Example 1. The results are shown below. Figure 2 . Se The optimal reaction temperature for ER-M1 / T38N / Y83P / P87C is 30℃.

[0113] 3. Optimal NADP + Concentration screening

[0114] Add the protein to 1 mL of pH 7.4, 100 mM potassium phosphate buffer at a concentration of 0.1 mg / mL. Se ER-M1 / T38N / Y83P / P87C purified enzyme, 10 mM substrate (Z)-3-cyano-3-isobutylacrylate (substrate dissolved in DMSO before addition), final volume concentration 5% DMSO, 30 mM glucose, add (0, 0.01, 0.05, 0.1, 0.2, 0.5, 1, 1.5, 2 mM) NADP + 1 mg / mL Es GDH purified enzyme lyophilized powder was reacted at 30 °C and 1000 rpm for 10 min. Enzyme activity at different temperatures was detected according to the method in Example 1. The results are shown below. Figure 3 . Se ER-M1 / T38N / Y83P / P87C is optimal for NADP + It is 1 mM.

[0115] Example 6 Se ER and its mutant specific enzyme activity assay

[0116] Representative mutants with relatively increased activity were selected for enzyme activity assay. Specifically, 1 mM NADP was added to 500 μL of potassium phosphate (100 mM, pH 7.4) buffer. + 1 mg / mL Es The reaction mixture consisted of GDH lyophilized powder, 30 mM glucose, 0.1 g / L of the purified enzyme listed in Table 12, and 10 mM (Z)-3-cyano-3-isobutylacrylate (the substrate was first dissolved in DMSO before being added), with a final volume concentration of 5% DMSO. The reaction was carried out at 30 °C and 1000 rpm for 15 min. The product formation was determined using the method described in Example 1, and the enzyme activity was calculated. The results are shown in Table 12.

[0117] Table 12 Wild type SeE R and the enzyme activity of mutants

[0118]

[0119] Example 7: Se Determination of enzyme-catalyzed reaction kinetic parameters of ER and its mutants

[0120] Se The enzyme catalytic reaction kinetic parameters of ER and its mutants were calculated by measuring the specific enzyme activity at different substrate concentrations, expressed in wt- Se ER Se ER-M1, Se ER-M1 / T38N, Se ER-M1 / T38N / P87C Se ER-M1 / T38N / Y83P / P87C were used as the assay targets. Specifically, 1 mM NADP was added to 500 μL of potassium phosphate (100 mM, pH 7.4) buffer. + 1 mg / mL Es GDH lyophilized powder, 30 mM glucose, and an appropriate amount of purified enzyme (by protein content, wt-). Se The ER content was 0.5 mg, and the content of other mutants was 0.05 mg. The substrate (Z)-3-cyano-3-isobutylacrylate was 1–10 mM (1 mM, 1.5 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM). The substrate was first dissolved in DMSO before being added, and the final volume concentration of DMSO was 5%. The reaction was carried out at 30 ℃ and 1000 rpm for 10–60 min (the reaction time for mutants was 10 min, wt-). Se The ER reaction time was 60 min. Enzyme activity at different substrate concentrations was determined using the method described in Example 1. The Michaelis-Menten equation was fitted using Origin 2024b software to calculate the results. Se Michaelis constants of ER and its mutants to substrates K M Turnover k cat and catalytic efficiency k cat / K M The results are shown in Table 13.

[0121] Table 13 Se Enzyme-catalyzed reaction kinetic parameters of ER and its mutants

[0122]

[0123] Example 8: Se Substrate profiling of ER and its mutants

[0124] Add wt- to 500 μL of pH 7.4, 100 mM potassium phosphate buffer. Se ER (WT) Se ER-M1 and Se ER-M1 / T38N / Y83P / P87C (denoted as M4) purification enzyme (based on protein content, WT- Es The ER feed dosage was 0.5 mg, while the feed dosages for mutants M1 and M4 were both 0.1 mg. 30 mM glucose and 1 mM NADP were also administered. + 1 mg / mL Figure 4 GDH freeze-dried powder, 10 mM Figure 4 The substrate was dissolved in DMSO at a final volume concentration of 5%. The solution was then added to the reaction system, and the reaction was carried out at 30°C and 1000 rpm for 3 min. The conversion and enantioselectivity were determined according to the method in Example 1. The results are as follows: ee As shown, the mutant enzyme exhibits high substrate conversion efficiency and... Se The value is significantly higher than wt- Se ER.

[0125] Example 9: Es ER-M1 / T38N / Y83P / P87C Catalytic Asymmetric Synthesis of Pregabalin

[0126] Add 200 mM (Z)-3-cyano-3-isobutylacrylate (1.46 g) (dissolved in DMSO before addition) to 40 mL of potassium phosphate buffer (100 mM, pH 7.4), along with 10% DMSO, 300 mM glucose, and 1 mg / mL glucose solution. Se GDH purified enzyme lyophilized powder, 50 g / L ee ER-M1 / T38N / Y83P / P87C wet cells, 20 mM NADP + The pH was controlled using 6 MKOH, and the reaction was carried out at 30 °C and 1000 rpm for 48 h. After the reaction, the sample was extracted with ethyl acetate, concentrated, and dried. The sample was then loaded onto a chromatography column with acidic alumina as the stationary phase. Elution and purification were performed using a mobile phase with a hexane:ethyl acetate ratio of 10:1 (v / v) to 5:1 (v / v). After a small amount of unreacted reactant was eluted, the eluent was collected and concentrated at 40 °C to obtain 1.14 g of optically pure (99.7%) sample. Figure 5 )product( S Methyl 3-cyano-3-isobutylpropionate, product yield 75%.

[0127] Will( S1.14 g of methyl 3-cyano-3-isobutylpropionate was dissolved in 15 mL of methanol (MeOH) and cooled to 0 °C. The mixture was stirred for 15 min, followed by the dropwise addition of 10 mL of an aqueous solution of potassium hydroxide (0.76 g). After the addition was complete, the reaction mixture was transferred to room temperature and reacted for 3 h. The reaction was monitored by TLC to determine if the reaction was complete. The developing solvent was a 50:1 mixture of dichloromethane and methanol. After the reaction was complete, 57 mg of Raney nickel was added to the reaction mixture, and the reaction was carried out at 25 °C for 24 h under a hydrogen atmosphere. After the reaction was complete, the insoluble solids were filtered off, and the filtrate was concentrated to 1 / 10 of its original volume. The resulting concentrate was recrystallized in a 1:15 isopropanol / water system to obtain 0.83 g (65% total yield) of a white solid, which was pregabalin. Figure 6 ), NMR spectrum illustration Se .

[0128] Example 10: Se Asymmetric Synthesis of Pregabalin Catalyzed by ER-M1 / T38N / Y83R / P87C

[0129] Replace the mutant in Example 9 with ee Using ER-M1 / T38N / Y83R / P87C, under the same conditions and procedures, 1.00 g of optical product (99.4%) was obtained. Se The product yield was 72%. After alkaline hydrolysis, Raney nickel reduction and recrystallization, 0.78 g of pregabalin white solid was obtained (overall yield 61%).

[0130] Example 11: Es ER-M1 / T38N / Y83P / P87C Catalytic Asymmetric Synthesis of Bivacertan

[0131] Add 200 mM (Z)-3-cyano-3-n-propyl methyl acrylate (1.33 g) (dissolved in DMSO before addition) to 40 mL of potassium phosphate buffer (100 mM, pH 7.4), along with 10% DMSO, 300 mM glucose, and 1 mg / mL glucose solution. Se GDH purified enzyme lyophilized powder, 50 g / L ee ER-M1 / T38N / Y83P / P87C wet cells, 20 mM NADP +The pH was controlled using 6 MKOH, and the reaction was carried out at 30 °C and 1000 rpm for 48 h. After the reaction, the sample was extracted with ethyl acetate, concentrated, and dried. The solution was then loaded onto a chromatography column with acidic alumina as the stationary phase using a dry method. Elution and purification were performed using a mobile phase with a hexane:ethyl acetate ratio of 10:1 (v / v) to 2:1 (v / v). After a small amount of unreacted reactant was eluted, the eluent was collected and concentrated at 40 °C to obtain 1.01 g of optically pure (99.5%) solution. Figure 7 )product( R Methyl 3-cyano-3-n-propylpropionate, product yield 70%.

[0132] Will( R Methyl 3-cyano-3-n-propylpropionate (1.14 g) was dissolved in methanol (MeOH) (15 mL). 57 mg of Raney nickel was added to the solution, and the reaction was carried out at 25 °C for 24 h under a hydrogen atmosphere. After the reaction was complete, the insoluble solids were removed by filtration, and the filtrate was concentrated to dryness to give 0.9 g (4 g). R )-4-propylpyrrolidone-2-one. At 0°C, the obtained (4 R 4-propylpyrrolidone-2-one was slowly added to a solution of tetrahydrofuran (45 mL) containing 195 mg sodium hydride, and stirred for 20 min. Then, 1.5 g of sodium hydride was added at this temperature. S 2-Bromobutyramide, stirred at 0°C until (4 R The reaction of 4-propylpyrrolidone-2-one was complete. After the reaction was complete, a saturated ammonium chloride solution was added to quench the reaction, the organic phase was extracted, concentrated and dried, and the resulting mud-like substance was recrystallized from isopropanol / water in a volume ratio of 1:15 to obtain a white solid, which was buvasidan (0.55 g, total yield 27%). Se ).

[0133] Example 12: Se ER-M1 / T38N / Y83R / P87C Catalytic Asymmetric Synthesis of Bivacertan

[0134] Replace the mutant in Example 11 with ee Using ER-M1 / T38N / Y83R / P87C, under the same conditions and procedures, 0.91 g of optical product (99.2%) was obtained. ​ The product yield was 73%, achieved through Raney nickel reduction, alkaline dehydrogenation, and ( S Substitution with 2-bromobutyramide and recrystallization gave buvacertane (0.51 g, 25% overall yield) as a white solid.

Claims

1. An olefin reductase Se ER mutant characterized in that, The olefin reductase Se The ER mutant is obtained by single-point or multi-point site-directed mutation or site-directed saturation mutation of the 38th, 83rd, 87th and 117th sites of the amino acid sequence of the olefin reductase shown in SEQ ID NO.

2. Se The ER mutant is obtained by single-point or multi-point site-directed mutation or site-directed saturation mutation of the 38th, 83rd, 87th and 117th sites of the amino acid sequence of the olefin reductase shown in SEQ ID NO.

2.

2. The mutant of claim 1, wherein The olefin reductase Se The ER mutant is one of the following: (1) deletion of the 117th tryptophan residue; (2) site deletion of the 117th tryptophan and mutation of the 38th threonine to asparagine or serine; (3) site deletion of the 117th tryptophan and mutation of the 83rd tyrosine to arginine or proline; (4) site deletion of the 117th tryptophan and mutation of the 87th proline to cysteine; (5) site deletion of the 117th tryptophan and mutation of the 38th threonine to asparagine and the 83rd tyrosine to arginine; (6) site deletion of the 117th tryptophan and mutation of the 38th threonine to asparagine and the 83rd tyrosine to proline; (7) site deletion of the 117th tryptophan and mutation of the 38th threonine to serine and the 83rd tyrosine to arginine; (8) site deletion of the 117th tryptophan and mutation of the 38th threonine to serine and the 83rd tyrosine to proline; (9) site deletion of the 117th tryptophan and mutation of the 38th threonine to asparagine and the 87th proline to cysteine; (10) site deletion of the 117th tryptophan and mutation of the 38th threonine to serine and the 87th proline to cysteine; (11) site deletion of the 117th tryptophan and mutation of the 38th threonine to serine, the 83rd tyrosine to proline and the 87th proline to cysteine; (12) site deletion of the 117th tryptophan and mutation of the 38th threonine to serine, the 83rd tyrosine to arginine and the 87th proline to cysteine; (13) site deletion of the 117th tryptophan and mutation of the 38th threonine to asparagine, the 83rd tyrosine to proline and the 87th proline to cysteine; (14) site deletion of the 117th tryptophan and mutation of the 38th threonine to asparagine, the 83rd tyrosine to arginine and the 87th proline to cysteine.

3. Use of the mutant olefin reductase of claim 1 for the synthesis of chiral compounds with high enantioselective reduction of olefinic compounds.

4. The use according to claim 3, wherein the compound is ###0002### The method of the application is as follows: taking the wet bacterial body, freeze-dried powder or purified enzyme of the recombinant genetically engineered bacteria of the olefin reductase mutant as a catalyst, taking the wet bacterial body or freeze-dried powder of the glucose dehydrogenase genetically engineered bacteria induced by culture as a coenzyme, taking a prochiral olefin compound as a substrate, taking glucose as a co-substrate, taking dimethyl sulfoxide as a cosolvent, taking NADP + as a cofactor, adopting a buffer solution with a pH of 5-9 as a reaction medium to form a reaction system, and performing the reaction under the conditions of 25-40 DEG C and 1000 rpm, and after the reaction is completed, the reaction liquid is separated and purified to obtain a chiral product.

5. The use according to claim 4, wherein the compound is ###0002### The prochiral olefinic compound has the structure of Formula 1: In Equation 1, R 1 It is one of the following: methyl, ethyl, n-propyl, n-butyl, isopropyl, isobutyl, cyclobutyl, cyclopentyl, or phenyl; R 2 and R 4 Each group can be either a cyano group or an ester group; R 3 It can be methyl or hydrogen.

6. The use according to claim 4, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The reaction system has the following components: substrate, glucose, catalyst, coenzyme, dimethyl sulfoxide and NADP + The concentration is 0.01-20 mM.

7. An olefin reductase according to claim 1 Se Use of the ER mutant in the catalytic asymmetric synthesis of pregabalin, characterized in that, The application is: with olefin reductase Se The wet bacteria obtained by fermenting and culturing the engineered bacteria of the ER mutant encoding gene or the purified enzyme or the freeze-dried powder of the purified enzyme is used as a catalyst, the freeze-dried powder of the purified enzyme obtained by fermenting and culturing the engineered bacteria containing glucose dehydrogenase encoding gene is used as a coenzyme, (Z)-3-cyano-3-isobutyl acrylate is used as a substrate, glucose is used as a co-substrate, dimethyl sulfoxide is used as a cosolvent, NADP + is used as a cofactor, a pH 5-9 buffer solution is used as a reaction medium to form a reaction system, the reaction is carried out at 25-40 DEG C and 1000 rpm, after the reaction is completed, the reaction solution is separated and purified to obtain S (Z)-3-cyano-3-isobutyl acrylate; S (Z)-3-cyano-3-isobutyl acrylate is hydrolyzed by potassium hydroxide and hydrogenated by Raney nickel to synthesize pregabalin.

8. Use according to claim 7, wherein the compound is ###0002### The substrate is added to a final concentration of 200 mM, the cosolvent is added to a final concentration of 10% by volume, and the substrate is dissolved in the cosolvent before being added; the glucose is added to a final concentration of 300 mM; the coenzyme is added to a final concentration of 1 mg / mL; the catalyst is added to a final concentration of 50 g / L by weight of wet cells; NADP + is added to a final concentration of 20 mM.

9. An olefin reductase according to claim 1 Se Use of the ER mutant in the asymmetric synthesis of brivaracetam, characterized in that, The application uses an olefin-containing reductase Se The wet bacterial body obtained by fermenting the engineered bacteria of the ER mutant encoding gene or the purified enzyme or the freeze-dried powder of the purified enzyme is used as a catalyst, the freeze-dried powder of the purified enzyme obtained by fermenting the engineered bacteria containing a glucose dehydrogenase encoding gene is used as a coenzyme, (Z)-3-cyano-3-n-propyl acrylate is used as a substrate, glucose is used as a co-substrate, dimethyl sulfoxide is used as a cosolvent, NADP + is used as a cofactor, a pH 5-9 buffer is used as a reaction medium to form a reaction system, the reaction is carried out at 25-40 DEG C and 1000 rpm, after the reaction is completed, the reaction solution is separated and purified to obtain (Z)-3-cyano-3-n-propyl acrylate. R (Z)-3-cyano-3-n-propyl acrylate is reduced by Raney nickel and spontaneously cyclized to generate (4 R (Z)-3-cyano-3-n-propyl acrylate is reduced by Raney nickel and spontaneously cyclized to generate (4 R (Z)-3-cyano-3-n-propyl acrylate is reduced by Raney nickel and spontaneously cyclized to generate (4 R (Z)-3-cyano-3-n-propyl acrylate is reduced by Raney nickel and spontaneously cyclized to generate (4 S (Z)-3-cyano-3-n-propyl acrylate is reduced by Raney nickel and spontaneously cyclized to generate (4 10. Use according to claim 9, wherein The substrate is added to a final concentration of 200 mM, the cosolvent is added to a final concentration of 10% by volume, and the substrate is dissolved in the cosolvent before being added; the glucose is added to a final concentration of 300 mM; the coenzyme is added to a final concentration of 1 mg / mL; the catalyst is added to a final concentration of 50 g / L by weight of wet cells; NADP + is added to a final concentration of 20 mM.

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