Alcohol dehydrogenase enzyme mutants and uses thereof

Abstract

The present application relates to the field of biological chemical industry, in particular to an alcohol dehydrogenase mutant and application thereof, the mutant is obtained by mutating one or more sites selected from 62, 108, 130, 132, 134, 182, 206, 287, 303, 313, 314 of the alcohol dehydrogenase with the amino acid sequence of SEQ ID NO: 4, the mutation is to mutate the amino acid of the site into one of A, G, V, L, I, P, F, Y, W, S, T, C, M, N, Q, D, E, K, R, H, the alcohol dehydrogenase mutant and application of the present application increase the substrate feeding amount, realize high conversion rate (> 99%) of the substrate, have good product ee value (> 98%), and it is determined that the old yellow enzyme (belongs to enone reductase) YqjM from Bacillus subtilis has the best catalytic activity, and shows good industrial application performance.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological chemical industry, and in particular to an alcohol dehydrogenase mutant and application thereof. BACKGROUND

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

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

[0004]

[0005] The reported synthesis methods of brivaracetam include chemical asymmetric synthesis, chemical chiral resolution, enzymatic chiral resolution and enzymatic asymmetric synthesis. First, the method disclosed in the patent AU5214401A of the original research UCB Company is to obtain diastereoisomers by chiral column, and then obtain brivaracetam by the method of chiral column preparation, finally obtain 80% yield, but the chiral column resolution of this method needs expensive equipment and consumables. Although there is a patent CN106748950B which obtains brivaracetam by chemical resolution method, but the yield is only 26%.

[0006] In comparison, the biological enzyme method is mild, the raw materials are cheap and easy to obtain, and it is environmentally friendly. These factors promote researchers to study the chiral separation of biological enzymes. In recent years, the method of synthesizing the intermediate of brivudine by the enzyme C=C asymmetric hydrogenation has also been reported, including CN107604018A, CN109852644A, CN111154735A, CN111286509A and CN112143764A. Although CN107604018A discloses the method of asymmetric hydrogenation of C=C of enone reductase on the substrate 4-propyl-2(5H)-furanone, the method of preparing the intermediate of brivudine (R)-4-propyl-dihydrofuran-2-ketone (compound VII) lacks key enzyme information, and its actual application effect is unknown. Although CN113444702B discloses the mutant strain of enone reductase, and improves the C=C asymmetric hydrogenation activity of the substrate 4-propyl-2(5H)-furanone, but the catalytic activity is still not high, and the industrial production is limited to a certain extent. SUMMARY

[0007] In order to overcome the problems of low substrate feeding amount and low substrate conversion rate, the present application provides an alcohol dehydrogenase mutant and its application.

[0008] In order to achieve the above-mentioned purpose, the present application realizes the following technical scheme:

[0009] In the first aspect, the mutant is obtained by mutating the alcohol dehydrogenase having the amino acid sequence of SEQ ID NO: 4 at one or more sites selected from the group consisting of 62, 108, 130, 132, 134, 182, 206, 287, 303, 313, 314, 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, H.

[0010] As a preferred, the mutation is performed at the 313th, 132nd and 182nd sites, and the mutation is to mutate the tryptophan W or tyrosine Y.

[0011] As a preferred, the mutation at the 313th site is selected from one of M313A, M313C, M313D, M313E, M313F, M313G, M313H, M313L, M313I, M313N, M313Q, M313R, M313S, M313P, M313T, M313V, M313W, M313K, M313Y; the mutation at the 132nd site is selected from one of G132A, G132C, G132D, G132E, G132F, G132P, G132H, G132L, G132I, G132M, G132N, G132Q, G132R, G132S, G132T, G132V, G132W, G132K, G132Y; the mutation at the 182nd site is selected from one of T182A, T182C, T182D, T182E, T182F, T182P, T182H, T182L, T182I, T182M, T182N, T182Q, T182R, T182S, T182G, T182V, T182W, T182K, T182Y.

[0012] The mutation according to the present application is a combined mutation of the mutations at the 313th, 132nd and 182nd sites. The mutation at the 313th site is selected from M313R, M313K, M313Y, M313E, M313Q, M313N, M313H, M313D, preferably M313R. The mutant M313R is further mutated. The mutation of the mutant M313R 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, M313R / M130Q, preferably the double mutant M313R / G132T, M313R / T182A, M313R / T182N, M313R / M130Y. The double mutant M313R / G132T is further mutated. The mutation of the mutant M313R / G132T is selected from M313R / G132T / T182A, M313R / G132T / T182N, M313R / G132T / M130Y, preferably the triple mutant M313R / G132T / T182A.

[0013] As preferred, the amino acid sequence of the alcohol dehydrogenase mutant is shown in SEQ ID NO: 6 or is more than 70% identical to SEQ ID NO: 6.

[0014] The amino acid sequence of the alcohol dehydrogenase mutant of the present application is shown in SEQ ID NO: 6, or a variant more than 70%, more than 71%, more than 72%, more than 73%, more than 74%, more than 75%, more than 76%, more than 77%, more than 78%, more than 79%, more than 80%, more than 81%, more than 82%, more than 83%, more than 84%, more than 85%, more than 86%, more than 87%, more than 88%, more than 89%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% identical to SEQ ID NO: 6, and an amino acid fragment or a sequence hybridized under stringent conditions.

[0015] In the second aspect, the present application provides a gene of the alcohol dehydrogenase mutant.

[0016] As preferred, the nucleotide sequence of the present application is shown in SEQ ID NO: 5 or is more than 70% identical to SEQ ID NO: 5.

[0017] In the third aspect, the present application provides a recombinant plasmid containing the gene.

[0018] In the fourth aspect, the present application provides a recombinant bacterium containing the gene.

[0019] In the fifth aspect, the present application provides the use of the alcohol dehydrogenase mutant or the gene in the enzymatic catalytic reduction of 3-oxo hexanoic acid (compound IV, V) to prepare the intermediate (R)-4-propyl-dihydrofuran-2-one, compound VII, of brivudine.

[0020] As preferred, the use comprises: constructing a recombinant bacterium containing the gene of the alcohol dehydrogenase mutant, respectively, and using the wet bacterium or the purified enzyme obtained by crushing and purifying the wet bacterium obtained by fermentation culture of the recombinant bacterium as the catalyst to perform asymmetric hydrogenation reaction on compound I to obtain compound VII.

[0021] The one-pot enzyme catalytic system of the present application is carried out under the synergistic action of NAD(P) + / NAD(P)H coenzyme circulation system, and the coenzyme circulation system is selected from alcohol dehydrogenase / isopropanol, formate dehydrogenase / formate, glucose dehydrogenase / glucose.

[0022] The one-pot reaction system of the present application is characterized in containing an enone reductase, an alcohol dehydrogenase mutant and a coenzyme cycle system, wherein the protein concentration of the enone reductase is 0.1 mg / mL-10 mg / mL, the protein concentration of the coenzyme system is 0.1 mg / mL-10 mg / mL, the protein concentration of the alcohol dehydrogenase mutant is 0.1 mg / mL-10 mg / mL, the dosage of compound I is 0.1%-2.0%, the dosage of NAD(P) + The dosage of compound I is 0.1%-2.0%, the dosage of NAD(P)

[0023] After comparing the catalytic activities of enone reductases from various sources, the inventors determined that the enone reductase YqjM from Bacillus subtilis subsp. subtilis str. 168 (NCBI Accession No. CP022391.1) has the optimal catalytic activity, i.e., catalyzing the asymmetric hydrogenation of Compound I to synthesize Compound II. Subsequently, after comparing the catalytic activities of alcohol dehydrogenases from various sources, the inventors determined that the alcohol dehydrogenase YahK from Bacillus subtilis (NCBI Accession No. ACT42179) has the optimal catalytic activity, i.e., catalyzing the asymmetric hydrogenation of Compound I to synthesize Compound (IV, V) to synthesize Compound VII. The inventors performed semi-rational design and mutation modification on YahK, obtained variants in which the 62nd, 108th, 130th, 132nd, 134th, 182nd, 206th, 287th, 303rd, 313rd, and 314th positions were mutated based on the wild-type sequence, and obtained the optimal mutation site, i.e., the 313rd position, and further performed mutation screening at the 313rd position, selected from M313R, M313K, M313Y, M313E, M313Q, M313N, M313H, and M313D. The optimal one-generation single-point mutant strain is M313R, and the inventors obtained a YahK mutant, YahK-M313R, which has significantly improved stereoselectivity. Further, using M313R as a template and combining with 132nd, 182nd, and 130th mutations, the inventors selected 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 optimal two-generation double-point mutant strain is M313R / G132T, M313R / T182A, M313R / T182N, and M313R / M130Y, and the inventors obtained a YahK mutant, M313R / G132T, which has significantly improved stereoselectivity. Further, using M313R / G132T as a template and combining with 182nd and 130th mutations, the inventors selected M313R / G132T / T182A, M313R / G132T / T182N, and M313R / G132T / M130Y, and obtained the optimal mutant YahK mutant M313R / G132T / T182A, which has significantly improved stereoselectivity.

[0024] The amino acid sequence of the alcohol dehydrogenase mutant M313R / G132T / T182A is SEQ ID NO: 6. Based on the three-dimensional crystal structure of the enzyme (PDB ID: 1UUF), molecular docking simulation analysis with the substrate was performed, combined with the enzyme catalytic mechanism (G.V. Dhoke, et al, ACS Catalysis, 2015, 5, 3207-3215.), and semi-rational design and mutation were performed, i.e. tryptophan / tyrosine mutation analysis was performed on potential key amino acid residues in the substrate structure pocket of alcohol dehydrogenase YahK, and then combination mutation was performed on specific sites, thereby screening the alcohol dehydrogenase mutant M313R / G132T / T182A.

[0025] The nucleotide sequence of the alcohol dehydrogenase mutant M313R / G132T / T182A can be artificially synthesized according to its amino acid sequence, and its preferred sequence is SEQ ID NO: 6. The gene sequence of the mutant M313R / G132T / T182A and the construction of the recombinant expression plasmid are as follows: first, according to the amino acid sequence of alcohol dehydrogenase YahK (SEQ ID NO: 4), the codon is optimized and the gene is artificially synthesized, and its preferred nucleotide sequence is SEQ ID NO: 3; the YahK gene is cloned into the E. coli expression vector pET28a to construct the recombinant expression plasmid pET28a-YahK; by the method of site-directed mutagenesis, mutations are introduced at positions 313, 132 and 182, i.e. 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 YahK-M313R / G132T / T182A.

[0026] The application of the alcohol dehydrogenase mutant is to clone the alcohol dehydrogenase mutant YahK-M313R / G132T / T182A gene into an expression vector and overexpress it by a host cell to prepare the alcohol dehydrogenase mutant YahK-M313R / G132T / T182A containing the amino acid sequence SEQ ID NO: 6, and apply it to catalyze the synthesis of compound (IV, V) to compound VII.

[0027] The expression plasmid and the host cell, preferably, the expression vector is pET28a, and preferably, the 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 engineering strain E.coli BL21(DE3) (pET28a-YahK-M313R / G132T / T182A) for expressing YahK-M313R / G132T / T182A, which is abbreviated as E.coli IEF-YahK-M313R / G132T / T182A.

[0028] The application method of the alcohol dehydrogenase mutant of the application is as follows: after the engineering strain E.coli YahK-M313R / G132T / T182A is fermented, the YahK-M313R / G132T / T182A fermentation liquor is obtained, the cells are broken, and the purified enzyme is collected as a preparation of the YahK-M313R / G132T / T182A enzyme for catalyzing a reaction.

[0029] The protein concentration of the enone reductase in the one-pot catalytic system is 0.1 mg / mL to 10 mg / mL, the protein concentration of the coenzyme cell is 0.1 mg / mL to 10 mg / mL, the protein concentration of the alcohol dehydrogenase mutant enzyme is 0.1 mg / mL to 10 mg / mL, the dosage of the compound I is 0.1% to 2.0% (preferably 1%), and the dosage of NAD(P) + The dosage of the compound I is 0.1% to 2.0% (preferably 1%), and the dosage of NAD(P)

[0030] The NAD(P) + The NAD(P)

[0031] The alcohol dehydrogenase mutant can be used for one-pot enzyme catalysis to efficiently catalyze 0.1% to 2.0% of the substrate compound I to prepare the brivudine intermediate compound VII, the conversion rate of the substrate I is >99%, and the ee value of the product brivudine intermediate compound VII is >98%.

[0032] The application has the following beneficial effects:

[0033] (1) The alcohol dehydrogenase mutant and application of the present application compared with the existing enzyme method for reducing preparation of target product Boixitane intermediate technology (such as CN107604018A, CN109852644A, CN111154735A, CN113444702A, etc.), the substrate feeding amount is increased; high conversion rate (> 99%) is achieved; good product ee value (> 98%) is obtained.

[0034] (2) The catalytic activity of the enone reductase from various sources, i.e. asymmetric hydrogenation of compound I to prepare compound II, and the catalytic activity of YqjM from Bacillus subtilis old yellow enzyme (belonging to enone reductase) is determined.

[0035] (3) The catalytic activity of the alcohol dehydrogenase from various sources, i.e. hydrogenation of compound IV, V to prepare compound VII, and the catalytic activity of YqjM from Bacillus subtilis alcohol dehydrogenase YahK is determined.

[0036] (4) The mutant with improved catalytic performance is screened by semi-rational design and mutation, and the stereoselectivity is significantly improved, and the optimized mutant realizes the product ee value > 91%.

[0037] (5) The one-pot enzyme catalytic system is optimized, and the stereoselectivity is significantly improved, and the optimized system realizes the product ee value > 98%, which shows good industrial application characteristics. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 The reaction process schematic diagram for one-pot enzyme catalytic synthesis of product VII (Boixitane intermediate).

[0039] Figure 2 The substrate binding pocket schematic diagram of alcohol dehydrogenase YahK.

[0040] Figure 3 The GC chromatogram of one-pot method catalytic synthesis of Boixitane intermediate reaction process of wild type YahK.

[0041] Figure 4 The GC chromatogram of Boixitane intermediate standard compound VII. DETAILED DESCRIPTION

[0042] The application will be further described in conjunction with specific embodiments. Those skilled in the art will be able to implement the application based on these descriptions. In addition, the embodiments of the application described in the following description are generally only embodiments of a part of the application, not all embodiments. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments in the application without creative labor shall fall within the scope of protection of the application. In the following examples, the methods used are conventional methods unless otherwise specified, and the reagents used are commercially available.

[0043] LB medium: yeast powder 5.0 g / L, protein peptone 10.0 g / L, NaCl 10.0 g / L.

[0044] Fermentation medium: yeast powder 12.0 g / L, protein 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.

[0045] 100 mmol / L phosphate buffer solution (pH 8.0): Na2HPO4·12H2O 32.94 g / L, NaH2PO4·H2O 1.10 g / L.

[0046] Example 1: Construction of recombinant expression system of enone reductase from different sources

[0047] Fourteen candidate enone reductases were screened from the NCBI database, and their catalytic activities for asymmetric hydrogenation of the C=C of compound I were analyzed. The NCBI accession numbers are shown in Table 1. According to the amino acid sequences of these enzymes, the gene synthesis company (Huada Genetech Qinglan Biotechnology Co., Ltd.) was sent, and after codon optimization, the artificial synthesis gene was cloned into the NdeI / BamHI of the expression vector pET28a to obtain a recombinant plasmid, such as pET28a-OYE1.

[0048] The recombinant plasmid of each enzyme gene was transformed into the expression host E. coli BL21(DE3) as follows: 50 ng of the recombinant plasmid was added to 100 μL of E. coli BL21(DE3) competent cells, mixed by tapping the tube wall several times, and placed in an ice water bath for 30 min. 42°C heat shock for 45 s, ice water incubation for 3 min. Add 900 μL of LB medium without antibiotics, incubate at 37°C for 60 min to recover the resistance. Take 50 μL of bacterial solution, evenly spread on LB plate containing 50 μg / mL kanamycin. Invert the plate and incubate at 37°C overnight. Pick the colony PCR positive clones, purified by streaking and incubated on a shaker, extract the plasmid, and verify by enzyme digestion and sequencing. Finally, the correct positive clones were obtained, and the recombinant E. coli engineering bacteria of each enzyme were obtained for enzyme expression and enzyme catalytic activity analysis.

[0049] Example 2: Construction of recombinant expression system of alcohol dehydrogenase from different sources

[0050] Seventeen candidate alcohol dehydrogenases from E. coli and B. subtilis were screened from the KEGG database, and the catalytic activity of hydrogenation of the self-racemic compounds IV and V was analyzed. Primers were designed according to the nucleotide sequences of these enzymes for PCR amplification, as shown in Tables 1 and 2, and the target gene fragments were cloned into the NdeI / BamHI site of the expression vector pET28a to obtain recombinant plasmids, such as pET28a-YahK.

[0051] The recombinant plasmid of each enzyme gene was transformed into the expression host E. coli BL21(DE3) as follows: 50 ng of the recombinant plasmid was added to 100 μL of E. coli BL21(DE3) competent cells, mixed by tapping the tube wall several times, and placed in an ice water bath for 30 min. 42°C heat shock for 45 s, ice water incubation for 3 min. Add 900 μL of LB medium without antibiotics, incubate at 37°C for 60 min to recover the resistance. Take 50 μL of bacterial solution, evenly spread on LB plate containing 50 μg / mL kanamycin. Invert the plate and incubate at 37°C overnight. Pick the colony PCR positive clones, purified by streaking and incubated on a shaker, extract the plasmid, and verify by enzyme digestion and sequencing. Finally, the correct positive clones were obtained, and the recombinant E. coli engineering bacteria of each enzyme were obtained for enzyme expression and enzyme catalytic activity analysis.

[0052] Table 1 Design of PCR amplification primers for alcohol dehydrogenase from E. coli

[0053]

[0054]

[0055] Table 2 Design table of alcohol dehydrogenase PCR amplification primers from Bacillus subtilis

[0056]

[0057] Example 3: Construction of recombinant E. coli of coenzyme cycle system

[0058] Application of coenzyme NAD(P) + The cycle system of NAD(P) / NAD(P)H avoids the use of expensive NADPH raw materials and instead uses relatively low-cost NADP + as a raw material, and the amount is significantly reduced, thereby reducing production costs. The relatively low-cost NAD(P) + as a raw material, and the amount is significantly reduced, thereby reducing production costs. The coenzyme cycle system based on alcohol dehydrogenase GDH from Bacillus subtilis and glucose is selected. The amino acid sequence of GDH (NCBI ID: WP_003246720.1) is sent to a gene synthesis company (Huada Gene Qinglan Biotechnology Co., Ltd.), and the gene of GDH is artificially synthesized after codon optimization and cloned into the Nde I / BamH I between the expression vector pET28a, to obtain the recombinant plasmid pET28a-GDH of the coenzyme cycle. The above recombinant plasmid is transformed into the E. coli BL21 (DE3) strain to obtain the recombinant E. coli E. coli BL21 (DE3) (pET28a-GDH) expressing the coenzyme cycle system, which is named E. coli IEF-GDH, respectively. The amino acid sequence of the coenzyme GDH is described in SEQ ID NO: 7.

[0059] Example 4: Expression and purification of enone reductase, alcohol dehydrogenase and glucose dehydrogenase

[0060] The recombinant E. coli expressing enone reductase, alcohol dehydrogenase and glucose dehydrogenase is inoculated in LB medium containing 50 μg / mL kanamycin, 37°C, 200 rpm, and cultured to the mid-logarithmic growth phase to obtain fresh culture seed liquid.

[0061] The fresh culture seed liquid is inoculated in E. coli fermentation medium containing kanamycin 50 mg / L at a volume concentration of 5%, 37°C, and cultured for 3 h. The final concentration of IPTG is 1.0 mmol / L, and the fermentation temperature is controlled at 25°C. The fermentation is continued for 6 h to obtain the fermentation liquid expressing enone reductase, alcohol dehydrogenase and glucose dehydrogenase, respectively.

[0062] The fermentation liquid is centrifuged, resuspended with pH 7.5, 50 mmol / L Tris-HCl buffer, and the cells are broken by a high-pressure cell homogenizer to obtain the crude enzyme liquid, which needs to be purified as soon as possible.

[0063] Take 3.0-4.0 mL of metal nickel chelated sepharose gel filler in the chromatography column, after standing, wash with 10 column volumes of water, then use 10 column volumes of equilibration buffer (50 mmol / L sodium phosphate, 300 mmol / L NaCl, 20 mmol / L imidazole, pH 7.4) for adsorption column equilibration, after equilibration, centrifuge the crude enzyme solution obtained in the above operation at 12000 rpm for 10 min at 4°C, take an appropriate amount of supernatant for loading, after loading, seal the ends of the chromatography column, and place it on ice for slow shaking for 40-60 min. Revert the chromatography column to vertical position, discard the sample waste; use 10 times the column volume of washing buffer (50 mmol / L sodium phosphate, 300 mmol / L NaCl, 40 mmol / L imidazole, pH 7.4) to wash off the non-specifically adsorbed impurities, then use 10 times the column volume of elution buffer (50 mmol / L sodium phosphate, 300 mmol / L NaCl, 300 mmol / L imidazole, pH 7.4) for elution of the target protein, collect the eluate in separate tubes and store on ice. Verify the target protein in the collected liquid by SDS-PAGE, and pool the collected liquid containing the target protein into a dialysis bag, and place it in a low-temperature environment with 50 mmol / L, pH 7.0 phosphate buffer solution as the dialysis liquid for desalination. Replace the dialysis buffer every 4 h, and after dialysis is completed, concentrate the enzyme solution outside the dialysis bag with PEG20000. After concentration is completed, measure the protein concentration of the purified enzyme solution according to the Bradford reagent kit manual.

[0064] Example 5: Comparison of catalytic activity of wild-type enone reductases from various sources on compound I

[0065] The purified enone reductase and glucose dehydrogenase GDH described above are used for asymmetric hydrogenation of compound I. The catalytic reaction system is as follows: 1.0 mg / mL of purified enone reductase, 0.5 mg / mL of GDH pure enzyme, 2 g / L of substrate 5-hydroxy-4-propyl-2(5H)-furanone, 100 mmol / L of glucose, 0.2 mmol / L of NADPNa2, and 100 mmol / L of phosphate buffer (pH 8.0), and the above reaction solution is placed in a 50 mL round-bottom flask, magnetically stirred, and reacted at 30°C for 12 h. Under the condition of magnetic stirring, take 100 μL of the reaction solution in 1.0 mL of ethyl acetate, centrifuge at 15000 x g for 5 min, and take the supernatant for gas chromatography analysis.

[0066] Gas chromatography analysis method. Capillary chromatographic column: SUPELCO chromatographic column Beta Dex-225 (30 m x 0.25 mmol / L x 0.25 μm; column temperature: 100 °C, 5 min, 10 °C / min to 160 °C, 10 min, then 5 °C / min to 170 °C, 7 min, finally 10 °C / min to 210 °C, 8 min. Injection port temperature: 230 °C; detector temperature: 240 °C; carrier gas (N2): 1 ml / min; split ratio: 100:1; injection volume: 1 μL; blank solution: ethyl acetate.

[0067] Table 3 Comparison of catalytic activity of wild-type enone reductases on compound I

[0068]

[0069]

[0070] From Table 3, it can be seen that YqjM has the highest enzyme activity.

[0071] Example 6: Comparison of catalytic activity of wild-type alcohol dehydrogenases from various sources on substrates compound IV and V

[0072] The purified alcohol dehydrogenase in Example 4 was used for asymmetric hydrogenation reaction of the self-racemic mixture (IV and V). The catalytic reaction system was as follows: 1.0 mg / mL of the purified alcohol dehydrogenase, 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), and the above reaction solution was placed in a 2 mL EP tube, a metal bath, 35 °C for 10 min. 500 μL of the reaction solution was taken in 500 μL of ethyl acetate, centrifuged at 15000 x g for 5 min, and the supernatant was used for gas chromatography analysis.

[0073] Table 4 Comparison of catalytic activity of wild-type alcohol dehydrogenases on compound IV and V

[0074]

[0075]

[0076] The catalytic reaction results are shown in Table 4, in which the specific enzyme activity of YahK is the highest, but it prefers the stereoselectivity of S-type.

[0077] In order to improve the stereoselectivity of alcohol dehydrogenase YahK to R-type, the site-directed mutation sites of YahK were selected by computer-aided design. According to the reported crystal structure of alcohol dehydrogenase (PDB ID: 1UUF), homology modeling was performed on alcohol dehydrogenase YahK. The simulation analysis was performed by using protein three-dimensional structure analysis software and molecular docking software. Considering the docking results of alcohol dehydrogenase and substrate, the characteristics of enzyme substrate binding pocket, the structural characteristics of enzyme recognizing substrate stereoselectivity, and the catalytic mechanism of enzyme, such as Figure 2 As shown in FIG. 1, finally, the serine at position 62, the cysteine at position 108, and the threonine at position 182 of the amino acid sequence SEQ ID NO: 4 were mutated to tyrosine, respectively; the methionine at position 130, the glycine at position 132, the tyrosine at position 134, the leucine at position 206, the valine at position 287, the isoleucine at position 303, the methionine at position 313, and the isoleucine at position 314 were mutated to tryptophan, respectively.

[0078] The site-directed mutation primers of 11 sites were designed, as shown in Table 5. The entire plasmid was amplified by PCR using the vector pET28a-YahK as a template and the site-directed mutation primers. The correct size of the amplified band was detected by 1% agarose gel electrophoresis. The PCR product was treated with restriction endonuclease DpnI for 1 h to digest the methylated plasmid template. The digested PCR product was subjected to one-step cloning method for ligation reaction, and then transformed into E. coli BL21 (DE3) cells. The colony PCR and sequencing verification were performed to obtain the target site mutant of enone reductase.

[0079] Table 5 Site-directed mutation primers of YahK

[0080]

[0081]

[0082] (2) Comparison of catalytic activity of alcohol dehydrogenase YahK site-directed mutants

[0083] The catalytic activity of 11 site-directed mutants was compared and analyzed.

[0084] The purified enzymes of 11 mutants were obtained by using the recombinant protein purification method described in Example 4. The hydrogenation reaction system catalyzed by the self-racemic mixture (IV and V) was as follows: 10 g / L of substrate compound (IV and V), 100 mmol / L PBS buffer (pH 8.0), 50 mmol / L of NADPH and 1 mg / mL of purified YahK mutant. The above reaction solution was placed in a 2 mL EP tube, and reacted on a metal bath at 35°C for 10 min. 1% trifluoroacetic acid was added to terminate the enzyme catalysis and to perform the cyclization reaction for 6 h. 500 μL of the reaction solution was taken in 500 μL of ethyl acetate, centrifuged at 15000 x g for 5 min, and the supernatant was analyzed according to the gas chromatography analysis method of Example 5 to calculate the specific enzyme activity.

[0085] The specific enzyme activities and product ee values of the alcohol dehydrogenase YahK and 11 mutants are shown in the following table. The results show that the mutant M313W realizes the inversion of stereoselectivity, i.e. R-selectivity, and the product ee reaches 23.1% (R).

[0086] Table 6 Comparison of enzyme activity and substrate stereoselectivity of wild-type YahK and 11 YahK mutants

[0087]

[0088]

[0089] Example 8: Further mutation modification at M313 site of alcohol dehydrogenase YahK

[0090] (1) Construction of site-directed mutant at M313 site of alcohol dehydrogenase YahK

[0091] The design of the site-directed mutation primer is shown in Table 7. The entire plasmid was amplified by PCR using the site-directed mutation primer with the vector pET28a-YahK as the template, and the correct size of the amplified band was obtained by 1% agarose gel electrophoresis detection. The PCR product was treated with restriction endonuclease DpnI for 1 h to digest the methylated plasmid template. The digested PCR product was subjected to one-step cloning for ligation reaction, followed by transformation into Escherichia coli BL21 (DE3) cells, and the mutant of enone reductase with mutation at the target site was obtained by colony PCR verification and sequencing verification.

[0092] Table 7 Site-directed mutation primer at M313 site of YahK

[0093]

[0094]

[0095] (2) Comparison of enzyme activity of site-directed mutant at M313 site

[0096] The catalytic activity of the obtained eight mutants was compared.

[0097] The purified enzymes of the eight mutants were obtained according to the recombinant protein purification method of Example 4. The hydrogenation reaction system for catalyzing the self-racemic mixture (IV and V) was as follows: 10 g / L of the self-racemic mixture (IV and V), 100 mmol / L of a phosphate buffer (pH 8.0), 50 mmol / L of NADPH, and 1 mg / mL of the purified YahK mutant. The above reaction solution was placed in a 2 mL EP tube, and was reacted for 10 min at 35°C on a metal bath. 1% trifluoroacetic acid was added to terminate the enzyme catalysis and to perform the cyclization reaction for 6 h. 500 μL of the reaction solution was taken in 500 μL of ethyl acetate, centrifuged at 15000 x g for 5 min, and the supernatant was analyzed according to the gas chromatography analysis method of Example 5 to calculate the specific enzyme activity.

[0098] The specific enzyme activity and product ee value of the site-directed mutant at M313 are shown in Table 8, and 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).

[0099] Table 8 Comparison of enzyme activity and substrate stereoselectivity of wild-type YahK and eight YahK mutants

[0100]

[0101]

[0102] Example 9: Further combined multi-site mutation modification of mutant YahK M313R

[0103] Eight point mutant double mutants were designed and constructed, and the mutation primers are shown in Table 9. The entire plasmid was amplified by PCR using the vector pET28a-YahK-M313R as a template, and the correct size of the amplified band was obtained by 1% agarose gel electrophoresis detection. The PCR product was treated with restriction endonuclease DpnI for 1 h to digest the methylated plasmid template. The digested PCR product was subjected to one-step cloning for a ligation reaction, and then transformed into E. coli BL21 (DE3) cells. The mutant enone reductase mutant with mutation at the target site was obtained through colony PCR verification and sequencing verification.

[0104] Table 9 Mutation primers for combined site mutation modification of YahK M313R

[0105]

[0106]

[0107]

[0108] (2) Comparison of enzyme activity and stereoselectivity of multiple mutants of alcohol dehydrogenase YahK

[0109] According to the method of Example 4, the pure enzymes of the eight double mutants or triple mutants were obtained through induced expression and nickel column purification. The hydrogenation reaction system catalyzed by the self-racemization compounds (IV and V) was as follows: 10 g / L of substrate compound (IV and V), 100 mmol / L PBS buffer (pH 8.0), 50 mmol / L NADPH and 1 mg / mL of purified YahK multiple mutants. The above reaction solution was placed in a 2 mL EP tube, and the reaction was carried out at 35°C for 10 min on a metal bath. 1% trifluoroacetic acid was added to terminate the enzyme catalysis and the cyclization reaction was carried out for 6 h. 500 μL of the reaction solution was taken in 500 μL of ethyl acetate, centrifuged at 15000 x g for 5 min, and the supernatant was analyzed according to the gas chromatography analysis method of Example 5 to calculate the specific enzyme activity.

[0110] The specific enzyme activity and product ee value of the wild-type alcohol dehydrogenase YahK and the multiple mutants are shown in Table 10 below, and the results show that the triple mutant M313R / G132T / T182A has the best stereoselectivity, with an ee of 91.62% (R).

[0111] Table 10 Comparison of catalytic activity and stereoselectivity of multiple mutants

[0112]

[0113]

[0114] Example 10: Comparison of different one-pot enzyme catalysis modes

[0115] According to the method of Example 4, the pure enzymes of M313R / G132T / T182A, GDH and enone reductase YqjM were prepared respectively through induced expression and nickel column purification, and were used for one-pot enzyme reaction comparison of different catalytic modes.

[0116] Catalytic mode I, three enzymes were added synchronously for one-pot reaction. The one-pot enzyme reaction system of catalytic mode I was as follows: 10 g / L of substrate compound (I), 100 mmol / L phosphate buffer (pH 8.0), 0.2 mmol / L NADPH, 1 mg / mL of GDH, 1 mg / mL of enone reductase YqjM and 1 mg / mL of M313R / G132T / T182A. +, 1 mg / mL triple mutant of alcohol dehydrogenase YahK M313R / G132T / T182A, 1 mg / mL GDH, 1 mg / mL enoate reductase YqjM, 100 mmol / L glucose. The above reaction solution was placed in a 2 mL EP tube, and reacted for 1 h at 35°C on a metal bath. The enzyme catalysis reaction was terminated by adding 1% trifluoroacetic acid, and the cyclization reaction was performed for 6 h. 500 μL of the reaction solution was sampled in 500 μL of ethyl acetate, centrifuged at 15000 x g for 5 min, and the supernatant was analyzed according to the gas chromatography analysis method of Example 5 to calculate the conversion rate and ee value.

[0117] Catalytic mode II, i.e. two-step enzyme catalytic mode, the enoate reductase and the alcohol dehydrogenase were added in sequence, the first step was C=C hydrogenation of the enoate reductase, and the second step was aldehyde group reduction of the alcohol dehydrogenase. The one-pot method of catalytic mode II was as follows: 10 g / L of the substrate compound (I), 100 mmol / L phosphate buffer (pH 8.0), 0.2 mmol / L NADP + , 1 mg / mL GDH, 1 mg / mL enoate reductase YqjM, 100 mmol / L glucose. The above reaction solution was placed in a 2 mL EP tube, and reacted for 0.4 h at 35°C on a metal bath. Then 1 mg / 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 0.6 h. The enzyme catalysis reaction was terminated by adding 1% trifluoroacetic acid, and the cyclization reaction was performed for 6 h. 500 μL of the reaction solution was sampled in 500 μL of ethyl acetate, centrifuged at 15000 x g for 5 min, and the supernatant was analyzed according to the gas chromatography analysis method of Example 5 to calculate the conversion rate and ee value.

[0118] The comparison results of the two one-pot enzyme catalytic modes are shown in Table 11. The results show that the simultaneous catalytic mode, i.e. catalytic mode I, is more superior, not only the conversion rate is improved, but also the ee value is improved to >98%.

[0119] Table 11 Comparison of different one-pot enzyme catalytic modes

[0120]

[0121] Example 11: Application of one-pot enzyme asymmetric synthesis reaction in preparation of a Boixitant intermediate

[0122] According to the method of Example 4, after induction expression and nickel column purification, the pure enzymes of the triple mutant enzyme M313R / G132T / T182A of alcohol dehydrogenase YahK, glucose dehydrogenase GDH, and enoate reductase YqjM were obtained, which were used for the preparation of a Boixitant intermediate.

[0123] 1% substrate compound (I) feeding catalytic reaction. The reaction system is as follows: 10 g / L of substrate compound (I), 100 mmol / L phosphate buffer (pH 8.0), 0.2 mmol / L of NADPNa2, 3 mg / mL of alcohol dehydrogenase YahK triple mutant enzyme M313R / G132T / T182A, 1 mg / mL of glucose dehydrogenase GDH, 1 mg / mL of enone reductase YqjM, 100 mmol / L of glucose. The above reaction solution is placed in a 2 mL EP tube, and the reaction is carried out at 35°C for 5 h on a metal bath. 1% trifluoroacetic acid is added to terminate the enzyme catalytic reaction and carry out the cyclization reaction for 6 h. 500 μL of the reaction solution is taken in 500 μL of ethyl acetate, centrifuged at 15000 x g for 5 min, and the supernatant is analyzed according to the gas chromatography analysis method of Example 5 to calculate the 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%.

[0124] 2% substrate compound (I) feeding catalytic reaction. The reaction system is as follows: 20 g / L of substrate compound (I), 100 mmol / L phosphate buffer (pH 8.0), 0.2 mmol / L of NADPNa2, 5 mg / mL of alcohol dehydrogenase M313R / G132T / T182A, 1 mg / mL of GDH, 1 mg / mL of enone reductase YqjM, 200 mmol / L of glucose. The above reaction solution is placed in a 2 mL EP tube, and the reaction is carried out at 35°C for 12 h on a metal bath. 1% trifluoroacetic acid is added to terminate the enzyme catalytic reaction and carry out the cyclization reaction for 6 h. 500 μL of the reaction solution is taken in 500 μL of ethyl acetate, centrifuged at 15000 x g for 5 min, and the supernatant is analyzed according to the gas chromatography analysis method of Example 5 to calculate the 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 amino acid sequence is shown as SEQ ID NO:

6.

2. A gene encoding the alcohol dehydrogenase mutant according to claim 1.

3. The gene encoding the alcohol dehydrogenase mutant as described in claim 2, characterized in that, The nucleotide sequence thereof is shown as SEQ ID NO:

5.

4. A recombinant plasmid containing the gene according to claim 2.

5. A recombinant bacterium containing the gene according to claim 2.

6. Use of the alcohol dehydrogenase mutant of claim 1 or the gene of claim 2 in the enzymatic reduction of 3-oxo hexanoic acid to prepare a Bucillamine intermediate, -4-propyl-dihydrofuran-2-one. R -4-propyl-dihydrofuran-2-one.

7. Use according to claim 6, wherein The application comprises: constructing recombinant bacteria containing genes encoding enone reductase, glucose dehydrogenase and alcohol dehydrogenase, respectively, and using wet bacteria or pure enzyme obtained by crushing and purifying the bacteria obtained by fermentation culture of the recombinant bacteria as a catalyst to perform asymmetric hydrogenation reaction on 3-aldehyde hexanoic acid to obtain (R)-4-propyl-dihydrofuran-2-one. R )-4-propyl-dihydrofuran-2-one.

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