Alcohol dehydrogenase mutant and application thereof

Abstract

The invention relates to the field of biochemical engineering, in particular to an alcohol dehydrogenase mutant and application thereof. The mutant is obtained by mutating alcohol dehydrogenase with an amino acid sequence encoding SEQ ID NO: 4 at one or more sites selected from 62nd, 108th, 130th, 132, 134, 182nd, 206th, 287th, 303th, 313th and 314, and the mutation is that the amino acid at the site is mutated into one of A, G, V, L, I, P, F, Y, W, S, T, C, M, N, Q, D, E, K, R and H. According to the alcohol dehydrogenase mutant and the application of the alcohol dehydrogenase mutant, the feeding amount of substrates is increased; the high conversion rate (gt; 99%) of the substrate is realized; according to the present invention, the new YqjM has the good product ee value (gt; 98%), and it is determined that the YqjM derived from the bacillus subtilis old yellow enzyme (belonging to ketene reductase) has the optimal catalytic activity, such that the good industrial application performance is provided.

Description

Technical Field

[0001] The invention relates to the field of biochemical engineering, and in particular to an alcohol dehydrogenase mutant and application thereof. Background Art

[0002] Alcohol dehydrogenase (ADH), also known as alcohol dehydrogenase, is widely present in the digestive system of the human body or other animals, especially in the liver. It also exists in plants and microbial cells. In animals, it is mainly distributed in the liver, and also exists in other tissues such as mesenteric Leuconostoc. In plants, the activity of alcohol dehydrogenase increases during germination. It is an enzyme compound widely present in organisms, which has the function of catalyzing the dehydrogenation reaction of alcohol substances. It plays a key role in the alcohol metabolism process of organisms and has important physiological and pathological significance.

[0003] Brivaracetam (chemical name: (S)-2-((R)-2-oxo-4-propylpyrrolidin-1-yl)butanamide, chemical structure shown below) was developed by UCB of Belgium and is a derivative of the levetiracetam LEV structure. Its mechanism of action is consistent with that of LEV, but its binding to the synaptic vesicle protein (SV2A) in the brain is ten times stronger than that of LEV, and its pharmacokinetic parameters, pharmacological properties and safety are also significantly better than those of LEV. In recent years, the market size of anti-epileptic drugs in my country has grown rapidly, reaching more than 7 billion yuan, and levetiracetam accounts for 19.6% of anti-epileptic drugs, ranking second. Combined with the good market performance of LEV and its better clinical effects than LEV, the development of brivaracetam synthesis technology has great economic value and social significance.

[0004] The reported synthesis methods of brivaracetam include chemical asymmetric synthesis, chemical chiral resolution, enzymatic chiral resolution and enzymatic asymmetric synthesis. First, the chiral column, such as the method disclosed in the patent AU5214401A of the original UCB company, obtains the diastereomers and then obtains brivaracetam by the method of preparing the chiral chromatographic column, and finally obtains an 80% yield, but the chiral column chromatographic resolution of the method requires expensive equipment and consumables. Although there is a patent CN106748950B that uses a chemical resolution method to obtain brivaracetam, the yield is only 26%.

[0005] In contrast, the bioenzymatic reaction is mild, the raw materials are cheap and readily available, and the environmental friendliness has driven researchers to study bioenzymatic chiral separation. In recent years, methods for synthesizing buvacethamine intermediates by enzymatic C=C asymmetric hydrogenation have also been reported, including CN107604018A, CN109852644A, CN111154735A, CN111286509A, and CN112143764A. Although CN107604018A discloses a C=C asymmetric hydrogenation method of enone reductase to substrate 4-propyl-2(5H)-furanone, the method for preparing buvacethamine intermediate (R)-4-propyl-dihydrofuran-2-one (Compound VII) lacks key enzyme information, and its actual application effect is unknown. Although CN113444702B discloses a mutant strain of enone reductase and improves the C=C asymmetric hydrogenation activity of the substrate 4-propyl-2(5H)-furanone, it still has the problem of low catalytic activity and certain limitations on industrial production. Summary of the invention

[0006] In order to overcome the problems of low substrate input amount and low substrate conversion rate, the present invention provides an alcohol dehydrogenase enzyme mutant and application thereof.

[0007] In order to achieve the above object, the present invention is implemented by the following technical solutions: In the first aspect, the mutant is obtained by mutating one or more sites selected from the group consisting of sites 62, 108, 130, 132, 134, 182, 206, 287, 303, 313, and 314 for an alcohol dehydrogenase having an amino acid sequence encoding SEQ ID NO: 4, and the mutation is to mutate the amino acid at the site to one of A, G, V, L, I, P, F, Y, W, S, T, C, M, N, Q, D, E, K, R, and H.

[0008] Preferably, mutations are performed at the 313th, 132nd and 182nd positions, and the mutations are tryptophan W or tyrosine Y mutations.

[0009] Preferably, the mutation at position 313 is selected from the group consisting of M313A, M313C, M313D, M313E, M313F, M313G, M313H, M313L, M313I, M313N, M313Q, M313R, M313S, M313P, M313T, M313V, M313W, M313K, and M313Y; the mutation at position 132 is selected from the group consisting of G132A, G132C, G132D, G132E, G132F, G132P, G132H, G132L, G132 32I, G132M, G132N, G132Q, G132R, G132S, G132T, G132V, G132W, G132K, and G132Y; the mutation at position 182 is selected from one of T182A, T182C, T182D, T182E, T182F, T182P, T182H, T182L, T182I, T182M, T182N, T182Q, T182R, T182S, T182G, T182V, T182W, T182K, and T182Y.

[0010] The mutation of the present invention is a combined mutation of mutations at positions 313, 132 and 182. The position 313 is mutated, and the point mutation at position 313 is selected from M313R, M313K, M313Y, M313E, M313Q, M313N, M313H, M313D, preferably M313R. The mutant M313R is further subjected to a point mutation. The point mutation using mutant M313R as a template is selected from M313R / G132N, M313R / G132H, M313R / G132D, M313R / G132S, M313R / G132C, M313R / G132T, M313R / T182N, M313R / T182A, M313R / T182D, M313R / T182H, M313R / M130E, M313R / M130Y, M313R / M130K, and M313R / M130Q, preferably double mutants M313R / G132T, M313R / T182A, M313R / T182N, and M313R / M130Y. The double mutant M313R / G132T is subjected to point mutation. The point mutation using the mutant M313R / G132T as a template is selected from M313R / G132T / T182A, M313R / G132T / T182N, M313R / G132T / M130Y, preferably the triple mutant M313R / G132T / T182A.

[0011] Preferably, the amino acid sequence of the alcohol dehydrogenase mutant is as shown in SEQ ID NO: 6 or has a greater than 70% identity with SEQ ID NO: 6.

[0012] The amino acid sequence of the alcohol dehydrogenase mutant of the present invention is shown in SEQ ID NO:6, or a variant having 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with SEQ ID NO:6, as well as an amino acid fragment or a sequence that hybridizes under stringent conditions.

[0013] In a second aspect, the present invention provides a gene of an alcohol dehydrogenase mutant.

[0014] Preferably, the nucleotide sequence of the present invention is as shown in SEQ ID NO:5 or has a nucleotide sequence with a homology of more than 70% with SEQ ID NO:5.

[0015] In a third aspect, the present invention provides a recombinant plasmid containing a coding gene.

[0016] In a fourth aspect, the present invention provides a recombinant bacterium containing a coding gene.

[0017] In a fifth aspect, the present invention provides the use of the alcohol dehydrogenase mutant or the encoding gene in the enzymatic reduction of 3-aldehyde hexanoic acid (compounds IV, V) to prepare the brivaracetam intermediate (R)-4-propyl-dihydrofuran-2-one, compound VII.

[0018] Preferably, the application comprises: constructing recombinant bacteria containing the genes encoding ketone reductase and alcohol dehydrogenase respectively, using wet cells obtained by fermentation culture of the recombinant bacteria or pure enzymes obtained by crushing and purifying the cells as catalysts, performing an asymmetric hydrogenation reaction on compound I to obtain compound VII.

[0019] The one-pot enzymatic catalytic system of the present invention is based on NAD(P) + / NAD(P)H coenzyme circulation system, wherein the coenzyme circulation system is selected from alcohol dehydrogenase / isopropanol, formate dehydrogenase / formate, and glucose dehydrogenase / glucose.

[0020] The one-pot reaction system of the present invention is characterized in that it contains ketone reductase, alcohol dehydrogenase mutant and coenzyme circulation system, wherein the protein concentration of ketone reductase is 0.1 mg / mL to 10 mg / mL, the dosage of coenzyme system protein concentration is 0.1 mg / mL to 10 mg / mL, the protein concentration of alcohol dehydrogenase mutant enzyme is 0.1 mg / mL to 10 mg / mL, the dosage of compound I is 0.1%-2.0%, NAD(P) + The dosage is 0.05-2 mmol / L, the dosage of glucose is 10-500 mmol / L, and the pH value of the hydrolysis process is controlled at 7.0-9.0 and the temperature at 20°C-40°C.

[0021] After comparing the catalytic activities of ketone reductases from various sources, the present invention determined that ketone reductase YqjM from Bacillus subtilis subsp. subtilis str.168 (NCBI accession number: CP022391.1) has the best catalytic activity, that is, it catalyzes the asymmetric hydrogenation of compound I to synthesize compound II. Subsequently, after comparing the catalytic activities of alcohol dehydrogenases from various sources, it was determined that alcohol dehydrogenase YahK from Bacillus subtilis (NCBI accession number: ACT42179) has the best catalytic activity, that is, it catalyzes the asymmetric hydrogenation of compound I to synthesize compound (IV, V) to synthesize compound VII. Semi-rational design and mutational modification of YahK were performed, and variants with mutations at positions 62, 108, 130, 132, 134, 182, 206, 287, 303, 313, and 314 were obtained based on the wild-type sequence, and the optimal mutation site, position 313, was obtained. Further mutation screening was performed at position 313, and the mutations were selected from M313R, M313K, M313Y, M313E, M313Q, M313N, M313H, and M313D. The optimal first-generation single-point mutant strain was M313R, and a YahK mutant YahK-M313R with significantly improved stereoselectivity was obtained. M313R was further used as a template and the 132, 182, and 130 mutations were selected from M313R / G132N, M313R / G132H, M313R / G132D, M313R / G132S, M313R / G132C, M313R / G132T, M313R / T182N, M313R / T182A, M313R / T182D, M313R / T182H, M313R / M130E, M313R / M130Y, M313R / M130K, and M313R / M130Q. The best second-generation double-point mutant strains were M313R / G132T, M313R / T182A, M313R / T182N, and M313R / M130Y, and the YahK mutant M313R / G132T with significantly improved stereoselectivity was obtained. Further, M313R / G132T was used as a template and combined with 182 and 130 for mutation, selected from M313R / G132T / T182A, M313R / G132T / T182N, and M313R / G132T / M130Y, and the best mutation YahK mutant M313R / G132T / T182A with significantly improved stereoselectivity was obtained.

[0022] The amino acid sequence of the alcohol dehydrogenase mutant M313R / G132T / T182A is SEQ ID NO: 6. The three-dimensional crystal structure of the enzyme (PDB ID: 1UUF) was used to perform molecular docking simulation analysis with the substrate, and combined with the enzyme catalytic mechanism (GVDhoke, et al, ACS Catalysis, 2015, 5, 3207-3215.), semi-rational design and mutation modification were performed, that is, tryptophan / tyrosine mutation analysis was performed on the potential key amino acid residues in the substrate structure pocket of alcohol dehydrogenase YahK, and then combined mutations were performed on specific sites, so as to screen and obtain the alcohol dehydrogenase mutant strain M313R / G132T / T182A.

[0023] The nucleotide sequence of the alcohol dehydrogenase mutant strain M313R / G132T / T182A can be artificially synthesized according to its amino acid sequence, and its preferred sequence is SEQ ID NO:6. The mutant strain M313R / G132T / T182A gene sequence and recombinant expression plasmid are constructed as follows: first, according to the amino acid sequence of alcohol dehydrogenase YahK (SEQ ID NO:4), codon optimization and artificial gene synthesis are performed, and its preferred nucleotide sequence is SEQ ID NO:3; the YahK gene is cloned into the Escherichia coli expression vector pET28a to construct a recombinant expression plasmid pET28a-YahK; by a site-directed mutagenesis method, mutations are introduced at positions 313, 132, and 182, that is, the methionine at position 313 is mutated to arginine, the glycine at position 132 is mutated to threonine, and the threonine at position 182 is mutated to alanine, to obtain the recombinant expression plasmid pET28a-YahK-M313R / G132T / T182A, thereby obtaining the amino acid sequence SEQ ID NO:6 of the alcohol dehydrogenase mutant strain YahK-M313R / G132T / T182A.

[0024] The application of the alcohol dehydrogenase mutant is to clone the gene containing the alcohol dehydrogenase mutant YahK-M313R / G132T / T182A into an expression vector and overexpress it through a host cell to prepare the alcohol dehydrogenase mutant YahK-M313R / G132T / T182A containing the amino acid sequence of SEQ ID NO: 6, and use it to catalyze the synthesis of compound VII from compound (IV, V).

[0025] The expression plasmid and host cell, the preferred expression vector is pET28a, the preferred host cell is E. coli BL21 (DE3). The recombinant expression plasmid pET28a-YahK-M313R / G132T / T182A is transformed into the E. coli BL21 (DE3) strain to obtain the YahK-M313R / G132T / T182A expression engineering strain E. coli BL21 (DE3) (pET28a-YahK-M313R / G132T / T182A), referred to as E. coli IEF-YahK-M313R / G132T / T182A.

[0026] The application method of the alcohol dehydrogenase mutant of the present invention is to obtain YahK-M313R / G132T / T182A fermentation liquid by fermenting the engineered strain E. coli YahK-M313R / G132T / T182A, purify and collect pure enzyme as a YahK-M313R / G132T / T182A enzyme preparation for catalytic reaction by cell crushing.

[0027] The one-pot catalytic system contains ketone reductase at a protein concentration of 0.1 mg / mL to 10 mg / mL, a coenzyme cell protein concentration of 0.1 mg / mL to 10 mg / mL, an alcohol dehydrogenase mutant enzyme protein concentration of 0.1 mg / mL to 10 mg / mL, a compound I dosage of 0.1% to 2.0% (preferably 1%), and NAD(P) + The dosage is 0.05-2 mmol / L (preferably 0.2 mmol / L), the dosage of glucose is 10-500 mmol / L (preferably 100 mmol / L), and the hydrolysis process is controlled at pH 7.0-9.0 (preferably pH 8.0) and temperature 20° C.-40° C. (preferably 35° C.).

[0028] The NAD(P) + The NAD(P)H coenzyme circulation system is a commonly used coenzyme circulation system, including alcohol dehydrogenase / isopropanol, formate dehydrogenase / formate, glucose dehydrogenase / glucose, etc., preferably glucose dehydrogenase and glucose derived from Bacillus subtilis.

[0029] The alcohol dehydrogenase mutant can be used for one-pot enzymatic method to efficiently catalyze 0.1%-2.0% of substrate compound I to prepare brivaracetam intermediate compound VII, the conversion rate of substrate I is greater than 99%, and the ee value of the product brivaracetam intermediate compound VII is greater than 98%.

[0030] The present invention has the following beneficial effects: (1) Compared with the existing enzymatic reduction technology for preparing the target product brivaracetam intermediate (such as CN107604018A, CN109852644A, CN111154735A, CN113444702A, etc.), the alcohol dehydrogenase mutant and its application of the present invention increase the substrate feed amount; achieve a high conversion rate of the substrate (>99%); and have a good product ee value (>98%). (2) The present invention studies the catalytic activity of ketone reductases from various sources, i.e., using compound I as a substrate, asymmetric hydrogenation is performed to prepare compound II, and it is determined that the old yellow enzyme (belonging to ketone reductase) YqjM from Bacillus subtilis has the best catalytic activity. (3) The present invention compares the catalytic activities of alcohol dehydrogenases from various sources, that is, using compounds IV and V as substrates to hydrogenate and prepare compound VII, and determines that the alcohol dehydrogenase YahK from Bacillus subtilis has the best catalytic activity. (4) The present invention screened mutants with improved catalytic performance through semi-rational design and mutation modification, and the stereoselectivity was significantly improved. The optimized mutants achieved a product ee value of >91%. (5) The present invention optimizes the one-pot enzymatic catalytic system, resulting in significantly improved stereoselectivity. The optimized system achieves a product ee value of >98%, showing good industrial application characteristics. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the reaction flow of the one-pot enzymatic synthesis of product VII (Brivaracetam intermediate).

[0032] Figure 2 Schematic diagram of the substrate binding pocket of alcohol dehydrogenase YahK.

[0033] Figure 3 This is the GC chromatogram of the one-pot catalytic synthesis of brivaracetam intermediates by wild-type YahK.

[0034] Figure 4 This is the GC chromatogram of the standard sample compound VII of the intermediate of brivaracetam. DETAILED DESCRIPTION

[0035] The present invention is further described below in conjunction with specific embodiments. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are generally only embodiments of a part of the present invention, rather than all embodiments. Therefore, based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work should belong to the scope of protection of the present invention. In the following embodiments, the methods used are all conventional methods unless otherwise specified, and the reagents used can be obtained from commercial sources.

[0036] LB medium: yeast powder 5.0 g / L, peptone 10.0 g / L, NaCl 10.0 g / L.

[0037] Fermentation medium: yeast powder 12.0 g / L, peptone 15.0 g / L, Na2HPO4·12H2O 8.9 g / L, KH2PO4 3.4 g / L, NH4Cl 2.67 g / L, Na2SO4 0.71 g / L, MgSO4·7H2O 0.49 g / L, kanamycin 50 μg / L, pH 7.0.

[0038] 100mmol / L phosphate buffer solution (pH 8.0): Na2HPO4·12H2O 32.94g / L, NaH2PO4·H2O 1.10g / L. Example 1: Construction of recombinant expression system of ketone reductase from different sources

[0039] 14 candidate enone reductases were screened from the NCBI database and analyzed for their catalytic activity for C=C asymmetric hydrogenation of compound I. Their NCBI accession numbers are shown in Table 1. Based on the amino acid sequences of these enzymes, they were sent to a gene synthesis company (BGI Qinglan Biotechnology Co., Ltd.), and after codon optimization, the genes were artificially synthesized and cloned into the NdeI / BamHI region of the expression vector pET28a to obtain recombinant plasmids, such as pET28a-OYE1.

[0040] The recombinant plasmids of each enzyme gene were transformed into the expression host E. coli BL21 (DE3). The specific operation was as follows: 50 ng of the recombinant plasmid was added to 100 μL of E. coli BL21 (DE3) competent cells, and the tube wall was flicked several times to mix, and placed in an ice water bath for 30 minutes. Heat shock at 42°C for 45 seconds, and incubated in ice water for 3 minutes. 900 μL of LB medium without antibiotics was added, and incubated at 37°C for 60 minutes to recover its resistance. 50 μL of bacterial solution was evenly spread on an LB plate containing 50 μg / mL kanamycin. The plate was inverted and cultured at 37°C overnight. The clones with positive colony PCR were picked, and after streaking purification and shaking culture, the plasmids were extracted, and enzyme cutting and sequencing verification were verified. Finally, the positive clones with correct verification were obtained, and the recombinant E. coli engineering bacteria of each enzyme were obtained for enzyme expression and enzyme catalytic activity analysis. Example 2: Construction of recombinant expression system of alcohol dehydrogenase from different sources

[0041] 17 candidate alcohol dehydrogenases from Escherichia coli and Bacillus subtilis were screened from the KEGG database to analyze the catalytic activity of hydrogenation of racemic compounds IV and V. Primers were designed based on the nucleotide sequences of these enzymes for PCR amplification, as shown in Tables 1 and 2, and the target gene fragments were cloned between NdeI / BamHI of the expression vector pET28a to obtain recombinant plasmids, such as pET28a-YahK.

[0042] The recombinant plasmids of each enzyme gene were transformed into the expression host E. coli BL21 (DE3). The specific operation was as follows: 50 ng of the recombinant plasmid was added to 100 μL of E. coli BL21 (DE3) competent cells, and the tube wall was flicked several times to mix, and placed in an ice water bath for 30 minutes. Heat shock at 42°C for 45 seconds, and incubated in ice water for 3 minutes. 900 μL of LB medium without antibiotics was added, and incubated at 37°C for 60 minutes to recover its resistance. 50 μL of bacterial solution was evenly spread on an LB plate containing 50 μg / mL kanamycin. The plate was inverted and cultured at 37°C overnight. The clones with positive colony PCR were picked, and after streaking purification and shaking culture, the plasmids were extracted, and enzyme cutting and sequencing verification were verified. Finally, the positive clones with correct verification were obtained, and the recombinant E. coli engineering bacteria of each enzyme were obtained for enzyme expression and enzyme catalytic activity analysis. Table 1 Primer design for PCR amplification of alcohol dehydrogenase from Escherichia coli Table 2 Primer design table for PCR amplification of alcohol dehydrogenase from Bacillus subtilis Example 3: Construction of recombinant Escherichia coli with coenzyme recycling system

[0043] Application of coenzyme NAD(P) + / NAD(P)H circulation system, avoiding the use of expensive NADPH raw materials and using relatively low-priced NADP instead + As raw material, the dosage is significantly reduced, reducing production costs. Relatively low NAD (P) +As raw material, and the dosage is significantly reduced, reducing production costs. A coenzyme circulation system based on alcohol dehydrogenase GDH and glucose from Bacillus subtilis was selected. The amino acid sequence of GDH (NCBI ID: WP_003246720.1) was sent to a gene synthesis company (BGI Qinglan Biotechnology Co., Ltd.), and the gene of GDH was artificially synthesized after codon optimization and cloned into the NdeⅠ / BamHI of the expression vector pET28a to obtain the recombinant plasmid pET28a-GDH of the coenzyme circulation. The above recombinant plasmid was transformed into the E. coli BL21 (DE3) strain to obtain a recombinant Escherichia coli E. coli BL21 (DE3) (pET28a-GDH) expressing the coenzyme circulation system, which were named E. coli IEF-GDH selection coenzyme circulation system, and the amino acid sequence of the coenzyme GDH was as described in SEQ ID NO:7. Example 4: Expression and purification of ketone reductase, alcohol dehydrogenase and glucose dehydrogenase

[0044] Recombinant Escherichia coli expressing ketone reductase, alcohol dehydrogenase and glucose dehydrogenase were inoculated into LB medium containing 50 μg / mL kanamycin, respectively, and cultured at 37° C. and 200 rpm to the mid-logarithmic growth phase to obtain freshly cultured seed solution.

[0045] The freshly cultured seed liquid was inoculated into the Escherichia coli fermentation medium containing 50 mg / L of kanamycin at an inoculum concentration of 5% by volume, cultured at 37°C for 3 h, added with a final concentration of 1.0 mmol / L IPTG, controlled the fermentation temperature at 25°C, and continued to ferment for 6 h to obtain fermentation broths expressing ketone reductase, alcohol dehydrogenase and glucose dehydrogenase, respectively.

[0046] The fermentation broth was centrifuged and resuspended with pH 7.5, 50 mmol / L Tris-HCl buffer. The cells were broken using a high-pressure cell homogenizer to obtain a crude enzyme solution, which needed to be purified as soon as possible.

[0047] Take 3.0-4.0mL of metal nickel chelated agarose gel filler in a chromatography column, let it stand and then wash it with water for 10 column volumes for use; use 10 column volumes of equilibrium buffer (50mmol / L sodium phosphate, 300mmol / LNaCl, 20mmol / L imidazole, pH7.4) to balance the adsorption column. After the equilibrium is completed, the crude enzyme solution obtained in the above operation is centrifuged at 12000rpm at 4°C for 10min, and an appropriate amount of supernatant is taken for sample loading. After the sample is loaded, both ends of the chromatography column are sealed and placed on ice for slow shaking for 40-60min. Re-fix the chromatography column vertically and discard the waste liquid; wash the non-specifically adsorbed impurities with 10 column volumes of washing buffer (50mmol / L sodium phosphate, 300mmol / LNaCl, 40mmol / L imidazole, pH7.4), and then elute the target protein with 10 column volumes of elution buffer (50mmol / L sodium phosphate, 300mmol / LNaCl, 300mmol / L imidazole, pH7.4), collect the eluate in separate tubes, and store it on ice. SDS-PAGE verifies the target protein in the collected solution, collects the collected solution with the target protein into a dialysis bag, and places it in a low temperature environment to desalt with 50mmol / L, pH 7.0 phosphate buffer solution as the dialysis solution. After the dialysis is completed, the dialysis buffer is replaced every 4h, and PEG20000 is added to the outside of the dialysis bag to concentrate the enzyme solution. After concentration, the protein concentration of the purified enzyme solution is determined according to the instruction manual of the Bradford kit. Example 5: Comparison of the catalytic activity of wild-type ketone reductase from various sources towards compound I

[0048] The purified enone reductase and glucose dehydrogenase GDH were used for the asymmetric hydrogenation reaction of compound I. The catalytic reaction system was as follows: 1.0 mg / mL purified enone reductase, 0.5 mg / mL pure GDH enzyme, 2 g / L substrate 5-hydroxy-4-propyl-2(5H)-furanone, 100 mmol / L glucose, 0.2 mmol / L NADPNa2 and 100 mmol / L phosphate buffer (pH 8.0). The above reaction solution was placed in a 50 mL round-bottom flask, stirred magnetically, and reacted at 30°C for 12 h. Under magnetic stirring, 100 μL of the reaction solution was sampled in 1.0 mL of ethyl acetate, centrifuged at 15000×g for 5 min, and the supernatant was taken for gas chromatography analysis.

[0049] Gas chromatography analysis method. Capillary column: SUPELCO column Beta Dex-225 (30m×0.25mmol / L×0.25μm; column temperature: 100℃, keep warm for 5min, increase the temperature to 160℃ at 10℃ / min, keep warm for 10min; then increase the temperature to 170℃ at 5℃ / min, keep for 7min. Finally, increase the temperature to 210℃ at 10℃ / min, keep for 8min. Inlet temperature: 230℃; detector temperature: 240℃; carrier gas (N2): 1ml / min; split ratio: 100:1; injection volume: 1μL; blank solution: ethyl acetate. Table 3 Comparison of catalytic activity of wild-type enone reductase towards compound Ⅰ As shown in Table 3, YqjM has the highest enzyme activity. Example 6: Comparison of catalytic activity of wild-type alcohol dehydrogenases from various sources on substrate compounds IV and V

[0050] The alcohol dehydrogenase purified in Example 4 was used for the asymmetric hydrogenation reaction of the self-racemic mixture (Ⅳ and Ⅴ). The catalytic reaction system was as follows: 1.0 mg / mL of purified alcohol dehydrogenase pure enzyme, 10 g / L of the self-racemic mixture (Ⅳ and Ⅴ), 50 mmol / L of NADPH and 100 mmol / L of phosphate buffer (pH 8.0). The above reaction solution was placed in a 2 mL EP tube, metal bath, and reacted at 35°C for 10 min. 500 μL of the reaction solution was sampled in 500 μL of ethyl acetate, centrifuged at 15000×g for 5 min, and the supernatant was taken for gas chromatography analysis. Table 4 Comparison of catalytic activity of wild-type alcohol dehydrogenase on compounds Ⅳ and Ⅴ The results of the catalytic reaction are shown in Table 4, where YahK has the highest specific enzyme activity, but prefers the S-type stereoselectivity. Example 7: Site-directed mutagenesis and screening of key amino acids in the active pocket of alcohol dehydrogenase YahK (1) Construction of site-directed mutagenesis of key amino acids in the active pocket of alcohol dehydrogenase YahK

[0051] In order to improve the stereoselectivity of alcohol dehydrogenase YahK for R-type, computer-aided design was used to select site-directed mutation sites of YahK. Based on the reported crystal structure of alcohol dehydrogenase (PDB ID: 1UUF), homology modeling of alcohol dehydrogenase YahK was performed. Protein three-dimensional structure analysis software and molecular docking software were used for simulation analysis. Comprehensive consideration was given to the docking results of alcohol dehydrogenase and substrate, the characteristics of the enzyme's substrate binding pocket, the structural characteristics of the enzyme's stereoselective recognition of substrates, and the enzyme's catalytic mechanism, such as Figure 2 As shown, finally, the 62nd serine, the 108th cysteine, and the 182nd threonine of the amino acid sequence SEQ ID NO:4 were subjected to point mutation modification, and were mutated into tyrosine respectively; the 130th methionine, the 132nd glycine, the 134th tyrosine, the 206th leucine, the 287th valine, the 303rd isoleucine, the 313th methionine, and the 314th isoleucine were subjected to point mutation modification, and were mutated into tryptophan respectively.

[0052] Site-directed mutagenesis primers for 11 sites were designed respectively, as shown in Table 5. Using vector pET28a-YahK as template, site-directed mutagenesis primers were used to PCR amplify the entire plasmid, and the amplified band of the correct size was obtained after 1% agarose gel electrophoresis. The PCR product was digested with restriction endonuclease DpnI for 1h to digest the methylated plasmid template. The digested PCR product was subjected to a one-step cloning method for ligation reaction, and then transformed into Escherichia coli BL21 (DE3) cells. After colony PCR verification and sequencing verification, the enone reductase mutant with mutation at the target site was obtained. Table 5 Site-directed mutagenesis primers for YahK (2) Comparison of catalytic activities of site-directed mutants of alcohol dehydrogenase YahK

[0053] Comparative analysis of the catalytic activities of 11 site-directed mutants was performed.

[0054] The recombinant protein purification method described in Example 4 was used to obtain pure enzymes of 11 mutants. The hydrogenation reaction system catalyzing the self-racemic mixture (Ⅳ and Ⅴ) was as follows: 10 g / L substrate compound (Ⅳ and Ⅴ), 100 mmol / L PBS buffer (pH 8.0), 50 mmol / L NADPH and 1 mg / mL purified YahK mutant. The above reaction solution was placed in a 2 mL EP tube and reacted at 35°C for 10 min on a metal bath. 1% trifluoroacetic acid was added to terminate the enzyme catalytic reaction and the cyclization reaction was carried out for 6 h. 500 μL of the reaction solution was sampled in 500 μL of ethyl acetate and centrifuged at 15000×g for 5 min. The supernatant was analyzed according to the gas chromatography analysis method of Example 5 to calculate its specific enzyme activity.

[0055] The specific enzyme activities and product ee values ​​of alcohol dehydrogenase YahK and 11 mutants are shown in the table below. The results show that mutant M313W achieves stereoselective inversion, i.e., R-selectivity, and the product ee reaches 23.1% (R). Table 6 Comparison of enzyme activities and substrate stereoselectivity of wild-type YahK and 11 YahK mutants Example 8: Further mutation modification of the M313 site of alcohol dehydrogenase YahK (1) Construction of a site-directed mutant of the M313 site of alcohol dehydrogenase YahK

[0056] The design of site-directed mutagenesis primers is shown in Table 7. Using the vector pET28a-YahK as a template, the site-directed mutagenesis primers were used to PCR amplify the entire plasmid, and the amplified band of the correct size was obtained after 1% agarose gel electrophoresis. The PCR product was digested with the restriction endonuclease DpnI for 1 hour to digest the methylated plasmid template. The digested PCR product was subjected to a one-step cloning method for ligation reaction, and then transformed into Escherichia coli BL21 (DE3) cells. After colony PCR verification and sequencing verification, the enone reductase mutant with a mutation at the target site was obtained. Table 7 Site-directed mutagenesis primers for the M313 site of YahK (2) Comparison of enzyme activities of site-directed mutants at M313

[0057] The catalytic activities of the eight mutants obtained were compared.

[0058] According to the recombinant protein purification method of Example 4, 8 mutant pure enzymes were obtained. The hydrogenation reaction system for catalyzing the self-racemic mixture (Ⅳ and Ⅴ) is as follows: 10g / L of the self-racemic mixture (Ⅳ and Ⅴ), 100mmol / L phosphate buffer (pH8.0), 50mmol / L NADPH and 1mg / mL purified YahK mutant. The above reaction solution was placed in a 2mL EP tube and reacted at 35°C for 10min on a metal bath. 1% trifluoroacetic acid was added to terminate the enzyme catalytic reaction and the cyclization reaction was carried out for 6h. 500μL of the reaction solution was sampled in 500μL of ethyl acetate, centrifuged at 15000×g for 5min, and the supernatant was analyzed according to the gas chromatography analysis method of Example 5 to calculate its specific enzyme activity.

[0059] The specific enzyme activity and product ee value of the site-directed mutant at the M313 site are shown in Table 8. The results show that the R-type stereoselectivity of the site-directed mutant M313R is significantly improved, and the product ee value reaches 80.5% (R). Table 8 Comparison of enzyme activities and substrate stereoselectivity of wild-type YahK and 8 YahK mutants Example 9: Further combined multi-site mutation modification of mutant YahK M313R (1) Construction of multi-site mutants using alcohol dehydrogenase YahKM313R as template

[0060] Eight double mutants with point mutations were designed and constructed, and the mutant primers are shown in Table 9. Using the vector pET28a-YahK-M313R as a template, PCR amplification of the entire plasmid was performed, and the amplified band of the correct size was obtained after 1% agarose gel electrophoresis. The PCR product was digested with the restriction endonuclease DpnI for 1 hour to digest the methylated plasmid template. The digested PCR product was subjected to a one-step cloning method for ligation reaction, and then transformed into Escherichia coli BL21 (DE3) cells. After colony PCR verification and sequencing verification, the enone reductase mutant with mutations at the target site was obtained. Table 9 Mutation primers for combined site mutation modification of YahK M313R (2) Comparison of the enzymatic activities and stereoselectivities of multiple mutants of alcohol dehydrogenase YahK

[0061] According to the method of Example 4, 8 kinds of pure enzymes of double mutants or triple mutants were obtained after induced expression and nickel column purification. The hydrogenation reaction system catalyzing the self-racemic compounds (Ⅳ and Ⅴ) is as follows: 10g / L substrate compounds (Ⅳ and Ⅴ), 100mmol / L PBS buffer (pH 8.0), 50mmol / L NADPH and 1mg / mL purified YahK multiple mutants. The above reaction solution was placed in a 2mL EP tube and reacted at 35°C for 10min on a metal bath. 1% trifluoroacetic acid was added to terminate the enzyme catalytic reaction and the cyclization reaction was carried out for 6h. 500μL of the reaction solution was sampled in 500μL of ethyl acetate and centrifuged at 15000×g for 5min. The supernatant was analyzed according to the gas chromatography analysis method of Example 5 to calculate its specific enzyme activity.

[0062] The specific enzyme activities and product ee values ​​of the wild-type alcohol dehydrogenase YahK and multiple mutants are shown in Table 10. The results show that the triple mutant M313R / G132T / T182A has the best stereoselectivity, with an ee of 91.62% (R). Table 10 Comparison of catalytic activity and stereoselectivity of multiple mutants Example 10: Comparison of different one-pot enzymatic catalysis methods

[0063] According to the method of Example 4, pure enzymes of M313R / G132T / T182A, GDH and ketone reductase YqjM were prepared respectively through induced expression and nickel column purification, and used for comparison of one-pot enzymatic reactions with different catalytic modes.

[0064] Catalytic method I, the three enzymes are added simultaneously for one-pot reaction. The one-pot enzymatic reaction system of catalytic method I is as follows: 10 g / L substrate compound (I), 100 mmol / L phosphate buffer (pH 8.0), 0.2 mmol / L NADP + , 1mg / mL triple mutant M313R / G132T / T182A of alcohol dehydrogenase YahK, 1mg / mL GDH, 1mg / mL enoketone reductase YqjM, 100mmol / L glucose. The above reaction solution was placed in a 2mL ep tube and reacted at 35°C for 1h on a metal bath. 1% trifluoroacetic acid was added to terminate the enzyme catalytic reaction and the cyclization reaction was carried out for 6h. 500μL of the reaction solution was sampled and placed in 500μL of ethyl acetate, centrifuged at 15000×g for 5min, and the supernatant was analyzed and calculated according to the gas chromatography analysis method of Example 5 for its conversion rate and ee value.

[0065] Catalytic method II, i.e., a two-step enzyme catalytic method, olefin reductase and alcohol dehydrogenase are added successively, the first step is C=C hydrogenation of olefin reductase, and the second step is aldehyde reduction of alcohol dehydrogenase. The one-pot method of catalytic method II has the following first step reaction system: 10 g / L substrate compound (I), 100 mmol / L phosphate buffer (pH 8.0), 0.2 mmol / L NADP + , 1mg / mL GDH, 1mg / mL ketone reductase YqjM, 100mmol / L glucose. The above reaction solution was placed in a 2mL EP tube and reacted at 35°C on a metal bath for 0.4h. Then 1mg / mL of the triple mutant enzyme M313R / G132T / T182A of alcohol dehydrogenase YahK was added to the reaction system, and the second step reaction was continued for 0.6h. 1% trifluoroacetic acid was added to terminate the enzyme catalytic reaction and the cyclization reaction was carried out for 6h. 500μL of the reaction solution was sampled in 500μL of ethyl acetate, centrifuged at 15000×g for 5min, and the supernatant was analyzed according to the gas chromatography analysis method of Example 5 to calculate its conversion rate and ee value.

[0066] The comparison results of the two one-pot enzymatic catalytic methods are shown in Table 11. The results show that the synchronous catalytic method, namely catalytic method I, is superior, not only the conversion rate is improved, but also the ee value is improved to >98%. Table 11 Comparison of different one-pot enzymatic catalysis methods Example 11: Application of one-pot enzymatic asymmetric synthesis to prepare brivaracetam intermediates

[0067] According to the method of Example 4, after induced expression and nickel column purification, pure enzymes of triple mutant enzymes M313R / G132T / T182A of alcohol dehydrogenase YahK, glucose dehydrogenase GDH, and ketone reductase YqjM were obtained for the preparation of brivaracetam intermediates.

[0068] Catalytic reaction with 1% substrate compound (I) feed amount. The reaction system is as follows: 10g / L substrate compound (I), 100mmol / L phosphate buffer (pH 8.0), 0.2mmol / L NADPNa2, 3mg / mL triple mutant enzyme M313R / G132T / T182A of alcohol dehydrogenase YahK, 1mg / mL glucose dehydrogenase GDH, 1mg / mL ketone reductase YqjM, 100mmol / L glucose. The above reaction solution is placed in a 2mL EP tube and reacted at 35°C for 5h on a metal bath. 1% trifluoroacetic acid is added to terminate the enzyme catalytic reaction and the cyclization reaction is carried out for 6h. Sample 500μL of the reaction solution in 500μL ethyl acetate, centrifuge at 15000×g for 5min, and take the supernatant to analyze and calculate its conversion rate and ee value according to the gas chromatography analysis method of Example 5. The results showed that the conversion rate of the substrate compound I was >99, and the ee value of the product compound VII was >98%.

[0069] Catalytic reaction with 2% substrate compound (I) added. The reaction system is as follows: 20g / L substrate compound (I), 100mmol / L phosphate buffer (pH 8.0), 0.2mmol / L NADPNa2, 5mg / mL alcohol dehydrogenase M313R / G132T / T182A, 1mg / mL GDH, 1mg / mL enoketone reductase YqjM, 200mmol / L glucose. The above reaction solution is placed in a 2mL EP tube and reacted at 35°C for 12h on a metal bath. 1% trifluoroacetic acid is added to terminate the enzyme catalytic reaction and the cyclization reaction is carried out for 6h. 500μL of the reaction solution is sampled and placed in 500μL ethyl acetate, centrifuged at 15000×g for 5min, and the supernatant is analyzed and calculated according to the gas chromatography analysis method of Example 5 for its conversion rate and ee value. The results show that the conversion rate of substrate compound I is >99, and the ee value of product compound VII is >98%.

Claims

1. An alcohol dehydrogenase mutant, characterized in that The mutant is obtained by mutating one or more sites selected from the group consisting of 62, 108, 130, 132, 134, 182, 206, 287, 303, 313, and 314 of an alcohol dehydrogenase having an amino acid sequence encoding SEQ ID NO: 4, wherein the mutation is to mutate the amino acid at the site to one of A, G, V, L, I, P, F, Y, W, S, T, C, M, N, Q, D, E, K, R, and H.

2. An alcohol dehydrogenase mutant as claimed in claim 1, characterized in that mutations are made at the 313th, 132nd and 182nd positions, and the mutations are tryptophan W or tyrosine Y mutations.

3. An alcohol dehydrogenase mutant according to claim 2, characterized in that: The mutation at position 313 is selected from one of M313A, M313C, M313D, M313E, M313F, M313G, M313H, M313L, M313I, M313N, M313Q, M313R, M313S, M313P, M313T, M313V, M313W, M313K, and M313Y; the mutation at position 132 is selected from one of G132A, G132C, G132D, G132E, G132F, G132P, G132H, G132L, G132 I, one of G132M, G132N, G132Q, G132R, G132S, G132T, G132V, G132W, G132K, and G132Y; the mutation at position 182 is selected from one of T182A, T182C, T182D, T182E, T182F, T182P, T182H, T182L, T182I, T182M, T182N, T182Q, T182R, T182S, T182G, T182V, T182W, T182K, and T182Y.

4. The alcohol dehydrogenase mutant according to claim 1, characterized in that The amino acid sequence of the alcohol dehydrogenase mutant is shown in SEQ ID NO: 6 or has a greater than 70% identity with SEQ ID NO:

6.

5. A gene encoding an alcohol dehydrogenase mutant according to any one of claims 1 to 4.

6. The gene encoding the alcohol dehydrogenase mutant according to claim 5, characterized in that Its nucleotide sequence is as shown in SEQ ID NO:5 or has a nucleotide sequence that is more than 70% identical to SEQ ID NO:

5.

7. A recombinant plasmid containing the coding gene as described in claim 5 or 6.

8. A recombinant bacterium containing the coding gene as described in claim 5 or 6.

9. An alcohol dehydrogenase mutant according to any one of claims 1 to 4 or a gene encoding the same according to any one of claims 5 to 6 for preparing a brivaracetam intermediate by enzymatic reduction of 3-aldehyde hexanoic acid ( R )-4-propyl-dihydrofuran-2-one, application in compound VII.

10. The use according to claim 9, characterized in that The application includes: constructing recombinant bacteria containing the genes encoding ketone reductase and alcohol dehydrogenase respectively, using wet bacteria obtained by fermentation culture of the recombinant bacteria or pure enzymes obtained by crushing and purifying the bacteria as catalysts, performing an asymmetric hydrogenation reaction on compound I to obtain compound VII.

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