An olefin reductase mutant, encoding gene, engineering bacteria and application

Abstract

The invention discloses an ene reductase mutant, a coding gene, an engineering bacterium and an application for asymmetric synthesis of optically pure (2R, 5R)-dihydrocarvone. The ene reductase mutant is obtained by site-directed mutation of the 103rd, 351st or 237th position of the amino acid sequence shown in SEQ ID NO.2. Compared with the ene reductase CtOYE, the ene reductase mutant CtOYE-Mut of the invention has higher stereoselectivity in the asymmetric synthesis of (2R, 5R)-dihydrocarvone by bioenzymatic method. The enantiomeric selectivity of asymmetric synthesis of optically pure (2R, 5R)-dihydrocarvone catalyzed by the mutant CtOYE-Mut as a biocatalyst is increased to 99% (2R, 5R).

Description

(I) Technical field

[0001] The present invention relates to an ene reductase mutant, in particular to an ene reductase mutant CtOYE-Mut and a coding gene thereof, a plasmid and a recombinant bacterium containing the coding gene of the mutant, and application of the ene reductase mutant CtOYE-Mut in asymmetric synthesis of a chiral compound (2R, 5R)-dihydrocarvone. (II) Background technology

[0002] (2R,5R)-Dihydrocarvone is an interesting molecule that can be used to synthesize different compounds, such as (+)-decipienin A, (-)-thujopsene, or as a derivative of the insect antifeedant ketone decalin, as a precursor of the antimalarial compound 1,2,4,5-tetraoxane, and as a precursor for the monoterpene lactone derivative (+)-dihydrocarbamide ((+)-DHCD) that is polymerized to form shape memory polymers.

[0003] (2R,5R) dihydrocarvone can be obtained by reducing (R)-carvone. In chemical synthesis, sodium borohydride, epoxylimonene, trifluoroethylene and metal zinc are often used for asymmetric reduction. Zhong Lin et al. reduced (2R,5R)-dihydrocarvone by metal zinc and potassium hydroxide system reduction method, but this method requires excessive zinc powder as a reducing agent, and the subsequent treatment is cumbersome and will produce a large amount of solid and liquid and waste liquid. The asymmetric reduction of (2R,5R)-dihydrocarvone by ene reductase is simple and pollution-free. For example, Powell et al. synthesized dihydrocarvone with dep values ​​of 97% (2R,5R), 75% (2R,5R) and 93% (2R,5R) respectively by OYE1, OYE3 and PeTnR. The asymmetric reduction of (R)-carvone and (S)-carvone produces diastereomers, so these two configurations of carvone may have different utilization rates in the asymmetric reduction of different ene reductases.

[0004] Compared with chemical synthesis methods that use high temperatures and expensive metal catalysts, enzyme catalysis is simple to operate, has high enantiomeric selectivity, high yield, low cost, safety and environmental protection, and solves the problems of high energy consumption, high pollution, and cumbersome operation of chemical methods. However, wild-type ene reductase still has problems of poor enantiomer selectivity and low enzyme efficiency in industrial applications. Therefore, it is necessary to improve the existing ene reductase to improve its catalytic activity and / or stability, thereby improving the high production cost and other problems in the existing technology. (III) Summary of the invention

[0005] The present invention aims to provide a highly stereoselective enolate reductase mutant CtOYE-Mut, a coding gene, an engineered bacterium and an application thereof in catalyzing the asymmetric reduction of (5R)-carvone to synthesize high optical purity (2R, 5R)-dihydrocarvone. The present invention successfully obtains a high conversion rate and high enantiomeric selectivity enolate reductase mutant CtOYE-Mut by molecular modification of the enolate reductase CtOYE derived from cyanobacterium Chroococcidiopsis thermalis, and applies the enolate reductase mutant CtOYE-Mut to synthesize (2R, 5R)-dihydrocarvone by asymmetric reduction of (5R)-carvone, and the product ee value and conversion rate both reach more than 99%, which is significantly improved compared with the enantiomeric selectivity of 86.4% of the unmodified wild-type enolate reductase CtOYE.

[0006] The technical solution adopted by the present invention is:

[0007] The present invention provides an olefin reductase mutant (CtOYE-Mut), wherein the olefin reductase mutant is obtained by performing site-directed mutagenesis on the 103rd, 351st or 237th position of the amino acid sequence shown in SEQ ID NO.2, and preferably, the olefin reductase mutant is obtained by mutating the tryptophan at the 103rd position of the amino acid sequence shown in SEQ ID NO.2 to phenylalanine, which is recorded as CtOYE-Mut (the amino acid sequence is shown in SEQ ID NO.4, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.5).

[0008] The present invention also provides a gene encoding the ene reductase mutant, a recombinant vector containing the gene encoding the ene reductase mutant, and a recombinant genetic engineering bacterium constructed by the recombinant vector. The recombinant vector of the present invention is preferably a pET-28a+ vector as a basic vector, and the recombinant genetic engineering bacterium is preferably E. coli BL21 (DE3) as a host bacterium; the recombinant vector is obtained by inserting the ene reductase mutant encoding gene between NdeI and BamHI of the pET-28a+ vector; the recombinant genetic engineering bacterium uses the recombinant plasmid pET-28a+-CtOYE as a template, and uses primers with mutant bases to amplify the whole plasmid through reverse PCR, and the obtained PCR product is digested with DpnⅠ enzyme to methylate the template, and the enzyme digestion product is transformed into Escherichia coli E. coli BL21 (DE3), so as to obtain the recombinant genetic engineering bacterium E. coli BL21 (DE3) / pET-28a-CtOYE-Mut containing the ene reductase mutant CtOYE-Mut gene.

[0009] The present invention also provides an application of the ene reductase mutant in catalyzing (5R)-carvone to prepare a chiral compound (2R, 5R)-dihydrocarvone, and can also be used to catalyze α, β-unsaturated aldehyde and ketone compounds to prepare chiral compounds.

[0010] Preferably, the application is: the wet bacterial cells obtained by fermentation culture of the recombinant genetically engineered bacteria containing the olefin reductase mutant encoding gene are ultrasonically broken, the supernatant (i.e., crude enzyme solution) after centrifugation of the broken mixed liquid or the pure enzyme solution extracted from the supernatant is used as a catalyst, (5R)-carvone is used as a substrate, and NAD(P) + The reaction system is formed by using a cofactor, glucose dehydrogenase as a coenzyme, glucose as a cosubstrate, and a buffer solution of pH 6-9 (preferably pH 8.0) as a reaction medium. The reaction is carried out in a shaking table at 30-40° C. and 180-250 rpm for 12-48 h (preferably 30° C., 200 rpm, 24 h). After the reaction is complete, the reaction solution is extracted with ethyl acetate, and then dehydrated with anhydrous magnesium sulfate to obtain a chiral product (2R, 5R)-dihydrocarvone.

[0011] In the reaction system, the amount of catalyst used is 0.1-5 mg / mL (preferably 1 mg / mL) based on protein content, the substrate is added to a final concentration of 2-10 mM (preferably 5 mM), the NAD(P) + The final concentration is 0.01-1.0 mM (preferably 0.1 mM), the coenzyme is added to a final concentration of 0.2-2 U / mL (preferably 0.5 U / mL), and the auxiliary substrate is added to a final concentration of 30-100 mM (preferably 40 mM).

[0012] The reaction system is preferably: using pure enzyme liquid extracted by ultrasonic crushing of wet bacteria obtained by fermentation and culture of recombinant genetically engineered bacteria containing an olefin reductase mutant encoding gene as a catalyst, (5R)-carvone as a substrate, and NAD(P) + As a cofactor, glucose dehydrogenase as a coenzyme, glucose as a cosubstrate, and a 50 mM PBS buffer solution at pH 8.0.

[0013] Preferably, the wet bacterial cells are prepared as follows: a single colony of a recombinant genetically engineered bacterial plate containing an olefin reductase mutant encoding gene is inoculated into an LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C, 180 rpm, and shaken for 15-20 h; then the bacterial liquid is inoculated into a fresh LB liquid medium containing 50 μg / mL kanamycin at an inoculum concentration of 1% by volume, and cultured at 37°C, 180 rpm, and shaken until the OD 600nmReach 0.6-0.8; add IPTG to a final concentration of 0.2mM to the culture medium, induce culture at 25°C, 180rpm shaking for 12h, centrifuge at 1,0000rpm for 10min, suspend and wash the precipitate with sterile water, and collect the wet bacteria.

[0014] Preferably, the crude enzyme solution is prepared as follows: wet bacteria are suspended in 50 mM, pH 8.0 PBS buffer, and then ultrasonically disrupted in a 4°C low-temperature ice water bath, the ultrasonic disruption conditions are: power 400 W, 3s on, 4s off, temperature 4°C, and effective disruption time 8min; the ultrasonically disrupted mixed solution is centrifuged at 12,000 rpm for 10 min, and then the supernatant is centrifuged at 12,000 rpm for 20 min to obtain a supernatant, the supernatant is aspirated, and a crude enzyme solution containing the target ene reductase mutant is obtained.

[0015] Further, the pure enzyme solution is prepared as follows: using Ni-NTA metal chelate affinity chromatography, the crude enzyme solution obtained above is transferred to the Ni-NTA elution column through a chromatograph. 2+ In the column, after the sample is loaded, a large amount of impurities are first eluted with an eluent containing 5mM imidazole, and then the residual impurities and part of the target protein are eluted with an eluent containing 50mM imidazole, and then the eluent containing 100mM imidazole is used for elution. At this time, a large amount of target protein is eluted, and the effluent containing the target protein is collected. Finally, all the proteins remaining in the column are eluted with an eluent containing 250mM imidazole, and the effluent containing the target protein is desalted and concentrated by centrifugation at 4°C and 7000rpm for 30min using an ultrafiltration tube with a molecular weight cutoff of 10kDa, and the retentate is the pure enzyme solution; the eluent composition: 300mM sodium chloride, the solvent is 50mM PBS buffer, pH 8.0.

[0016] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in that the stereoselective enolate reductase mutant CtOYE-Mut of the present invention has higher stereoselectivity in the bioenzymatic asymmetric synthesis of (2R, 5R)-dihydrocarvone compared with the enolate reductase CtOYE. + As a coenzyme, glucose dehydrogenase and D-glucose were used to drive the coenzyme cycle, and a dual-enzyme cascade catalytic asymmetric synthesis of optically pure (2R, 5R)-dihydrocarvone was successfully constructed. Its enantiomeric selectivity was increased to 99% (2R, 5R), overcoming the low asymmetric selectivity of olefin reductase CtOYE for (5R)-carvone. (IV) Description of the drawings

[0017] Figure 1 Schematic diagram of the structure of the recombinant plasmid pET28a-CtOYE-Mut.

[0018] Figure 2 Schematic diagram of the asymmetric hydrogenation of (5R)-carvone (Ⅰ) catalyzed by ene reductase to produce (2R,5R)-dihydrocarvone (Ⅱ).

[0019] Figure 3 It is the agarose gel electrophoresis diagram of the recombinant plasmid pET28a-CtOYE, M is the standard protein marker, and L1 is the recombinant plasmid pET28a-CtOYE.

[0020] Figure 4 The GC analysis charts of the standard substrate (5R)-carvone (Ⅰ), the product (2R,5R)-dihydrocarvone (Ⅱ) and the by-product (2S,5R)-dihydrocarvone.

[0021] Figure 5 The SDS-PAGE gel electrophoresis diagram of the crude enzyme solution and the purified pET28a-CtOYE-W103F; the target protein size is 42.0 kDa, L1 is the electrophoresis band of the mutant CtOYE-W103F crude enzyme solution, L2 is the standard protein marker, and L3 is the electrophoresis band of the mutant CtOYE-W103F purified enzyme.

[0022] Figure 6 This is the GC analysis spectrum of the olefin reductase mutant CtOYE-Mut catalyzing the substrate (5R)-carvone (Ⅰ) to produce the product (2R,5R)-dihydrocarvone (Ⅱ). (V) Specific implementation methods

[0023] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0024] The experimental materials and reagents used in the examples of the present invention are:

[0025] 1. Enzymes and other biochemical reagents: Plasmid miniprep kit and BCA protein concentration assay kit were purchased from Bio-Tech Biotechnology Co., Ltd.; PrimeSTAR Max Premix (2X) and Dpn I methylation digestion enzyme were purchased from Takara; ClonExpress II One Step Cloning Kit was purchased from Nanjing Novozyme Biotechnology Co., Ltd.; AxyPrep DNA agarose gel extraciton kit was purchased from Axygen; ultrafiltration tubes were purchased from Millipore. Other reagents were analytically pure and purchased from Sinopharm Chemical Reagent Co., Ltd.

[0026] 2. Culture medium:

[0027] LB liquid medium: 5g / L yeast powder, 10g / L peptone, 10g / L sodium chloride, solvent is deionized water, pH 6.8-7.0. LB solid medium is based on LB liquid medium plus 30g / L agar.

[0028] The molecular biology experimental methods not specifically described in the following examples are all carried out with reference to the specific methods listed in the book "Biochemical Technology Experimental Guide" (Chemical Industry Press) edited by Sun Peilong and Wu Shijin, or according to the kits and product instructions.

[0029] Example 1: Construction of genetically engineered bacteria E. coli BL21 (DE3) / pET28a-CtOYE

[0030] The gene fragment (Chro_0590, nucleotide sequence as shown in SEQ ID NO.3) from cyanobacterium Chroococcidiopsis thermalis in Genbank was codon optimized by Hangzhou Guannan Biotechnology Co., Ltd. The gene fragment after codon optimization was recorded as CtOYE gene, and the CtOYE gene was artificially synthesized. The nucleotide sequence is shown in SEQ ID NO.1 and the amino acid sequence is shown in SEQ ID NO.2. The artificially synthesized CtOYE gene was inserted between NdeI and BamHI of pET28a to obtain the recombinant expression plasmid pET28a-CtOYE. The electrophoresis diagram is shown in Figure 3 shown.

[0031] Take out 6 μL of the synthesized recombinant expression plasmid pET28a-CtOYE and add it to 30 μL of E.coli BL21 (DE3) competent medium, flick the tube wall to mix, and place on ice for 30 minutes. Heat shock in a 42℃ water bath for 1 minute, and immediately place on ice for 2 minutes. Add 1mL of LB liquid culture medium to the tube and culture on a 37℃ shaker for 1.5 hours. Centrifuge the culture solution at 8000rpm for 4 minutes and discard the supernatant. Use the remaining culture medium to suspend the bacteria, take out 100 μL and apply it on LB solid culture medium containing 50 μg / mL kanamycin. Culture overnight at 37℃ incubator for 12-14 hours to obtain genetically engineered bacteria E.coli BL21 (DE3) / pET28a-CtOYE, and extract plasmid pET28a-CtOYE.

[0032] SEQ ID NO.1

[0033]

[0034] SEQ ID NO.2

[0035] MNTNIDLFSPVRLGRYELPNRMVMAPLTRNRAGEGNVPRELNAEYYAQRVSAGLIITEQVSPQGLGYPFTPGIHSQEQVEGWRLVTKAVHDRGGKIFLQLWHVGRISHPDLQVDGALPVAPSAIAPSEGMAATYEGEKPYVTPRALETAEPGIVEQYRQGAKNALAAGFDGVEIHSANGY LLDQFLHDGSNHRTDEYGGSIENRARLLMEVVSVWGADRVGVRLSPSGTFGSVYDSDLKALFTYVVDALNQFELAYLHLVEPRVAGNETVENPTSELSSKYFRPIYKGTLISAGGYDRESGNAVLASGDADLVAYGRLFISNPDLPQRFALNAQLNPYDRSSFYGGDKRGYTDYPSLELQ AAG*.

[0036] Example 2: Construction of recombinant expression plasmid pET28a-CtOYE-Mut and genetically engineered bacteria E. coli BL21 (DE3) / pET28a-CtOYE-Mut

[0037] 1. Design of mutation primers

[0038] According to the nucleotide sequence of olefin reductase CtOYE (SEQ ID NO. 1), site-directed mutagenesis primers pET28a-CtOYE-AXf and pET28a-CtOYE-AXr were designed, A represents several selected mutated amino acids and their specific positions, and X represents the remaining 19 amino acids except the original amino acids, as shown in Table 1.

[0039] Table 1: Upstream and downstream primers for site-directed mutagenesis of pET28a-CtOYE-AX

[0040]

[0041]

[0042] 2. Site-directed mutagenesis PCR

[0043] According to the instructions of Takara's Prime STAR Max Premix (2X) kit, the recombinant expression plasmid pET28a-CtOYE obtained in Example 1 was used as a template, the primers in Table 1 were used, the reaction system in Table 2 was used, and the site-directed mutagenesis PCR of a single site was performed under the reaction conditions in Table 3.

[0044] Table 2: Site-directed mutagenesis PCR reaction system

[0045]

[0046] Table 3: Site-directed mutagenesis PCR reaction conditions

[0047]

[0048]

[0049] After the PCR product was detected by 1% agarose gel electrophoresis, the following steps were performed.

[0050] 3. Site-directed mutagenesis PCR product processing

[0051] According to the instruction manual of Takara's Dpn I methylase digestion enzyme, the mutant PCR product was treated and the methylated template DNA was digested at 37°C / 2h according to the reaction system in Table 4. After the reaction, the PCR product was purified using AxyPrep DNA agarose gel extraciton kit.

[0052] Table 4: DpnⅠ digestion template reaction system

[0053]

[0054] According to Novozymes II One Step Cloning Kit instructions, at 37°C / 30min reaction conditions, use the recombinase Exnase II according to the reaction system in Table 5 to circularize the purified linear PCR product in vitro.

[0055] Table 5: In vitro circularization reaction system for linearized PCR products

[0056]

[0057] The exo-circularized PCR product was transformed into E. coli BL21 (DE3), inoculated into LB plate medium containing 50 μg / mL kanamycin, and cultured overnight at 37°C. A single colony was picked and inoculated into LB culture medium containing 50 μg / mL kanamycin. After shaking culture at 37°C and 180 rpm for 16-20 hours, the bacterial solution was a recombinant E. coli containing the olefin reductase mutant CtOYE-X, recorded as E. coli BL21 (DE3) -pET28a-CtOYE-X, and transferred to a glycerol aqueous solution with a final concentration of 15% (v / v), mixed and stored at -80°C.

[0058] Example 3: Induced expression and purification of olefin reductase CtOYE and olefin reductase mutant CtOYE-X

[0059] 1. Bacterial induction culture

[0060] The genetically engineered bacteria E. coli BL21 (DE3) -pET28a -CtOYE constructed in Example 1 and the mutants E. coli BL21 (DE3) -pET28a -CtOYE-X obtained by site-directed mutagenesis in Example 2 were inoculated into LB liquid medium containing 50 μg / mL kanamycin, respectively, and cultured at 37 ° C, 180 rpm for 16-20 h. Then, the bacterial solution was inoculated into a fresh LB liquid medium containing 50 μg / mL kanamycin at an inoculum concentration of 1% by volume, and cultured at 37 ° C, 180 rpm until the OD 600nm Reach 0.6-0.8. Add IPTG (isopropylthiogalactoside) with a final concentration of 0.2mM to each culture solution, and induce culture at 25℃, 180rpm shaking for 12h. Then centrifuge at 1,0000rpm for 10min at 4℃, collect wet cells, wash with sterile water, and then add PBS buffer at a ratio of 1g wet cells: 15mL PBS buffer (pH8.0, 50mM) to suspend the cells.

[0061] 2. Preparation of crude enzyme solution

[0062] The bacterial suspension in step 1 was ultrasonically disrupted in a 4°C low-temperature ice water bath. The ultrasonic disruption conditions were: power 400W, 3s on, 4s off, temperature 4°C, and effective disruption time 8min. The ultrasonically disrupted mixed solution was centrifuged at 12,000rpm for 30min, and the supernatant was aspirated to obtain a crude enzyme solution containing the target ene reductase.

[0063] 3. Enzyme Purification

[0064] According to the instructions for Ni-NTA metal chelate affinity chromatography, take the crude enzyme solution from step 2 and load it onto the pre-equilibrated Ni 2+In the column, a large amount of impurity proteins were first eluted with an eluent containing 5 mM imidazole, and then the residual impurity proteins and part of the target protein were eluted with an eluent containing 50 mM imidazole, and then eluted with an eluent containing 100 mM imidazole. At this time, a large amount of target protein was eluted, and the effluent containing the target protein was collected. Finally, all the proteins remaining in the column were eluted with an eluent containing 250 mM imidazole. The effluent containing the target protein was desalted and concentrated by centrifugation at 4°C and 7000 rpm for 30 min using an ultrafiltration tube with a molecular weight cutoff of 10 kDa. The retentate was the pure enzyme solution, and the pure enzyme solutions of ene reductase CtOYE and ene reductase mutant GsOYE-X were obtained, respectively, and stored at -20°C for use.

[0065] The eluent composition: 300 mM sodium chloride, solvent is 50 mM PBS buffer, pH 8.0.

[0066] The pure enzyme solution obtained above was subjected to SDS-PAGE protein electrophoresis, and the protein electrophoresis results of the olefin reductase mutant CtOYE-W103F were shown in Figure 5 .

[0067] Example 4: Analysis of the chemoselectivity and stereoselectivity of olefin reductase CtOYE and olefin reductase mutant CtOYE-X

[0068] Reaction system 0.5 mL: crude enzyme solution (olefin reductase CtOYE and olefin reductase mutant GsOYE-X) prepared by the method of Example 3 was used as catalyst, the added amount was 1 mg / mL based on protein content, and the final concentration of 0.1 μM NAD(P) + As a cofactor, glucose dehydrogenase (purchased from Aladdin) with a final concentration of 0.5U / mL was used as a coenzyme, glucose with a final concentration of 40mM was used as a cosubstrate, (5R)-carvone with a final concentration of 5mM was used as a substrate, and 50mM PBS buffer with a pH of 8.0 was used as a reaction medium to form a reaction system of 0.5mL; the reaction was carried out in a shaker at 30°C and 200rpm for 24h. After the reaction was completed, an equal volume of ethyl acetate was added to the reaction solution for extraction, and the upper organic phase was dehydrated by anhydrous MgSO4, allowed to stand for 10min, and centrifuged at 12,000rpm for 10min. The supernatant was taken and the contents of the substrate (5R)-carvone and the products (2R,5R)-dihydrocarvone and (2S,5R)-dihydrocarvone were detected by gas chromatography, and the de value and product yield were calculated. The results are shown in Table 6. The gas chromatograms of the standard substrate (5R)-carvone and the product (2R,5R)-dihydrocarvone and the by-product (2S,5R)-dihydrocarvone are shown in Figure 4 shown.

[0069] Gas chromatography detection conditions: Determined by 2010-GC equipped with FID detector and chiral capillary column; FID detection temperature 250℃, injector 250℃; column inlet pressure 60kPa; the flow rate and split ratio of N2 as carrier gas were set to 1mL / min and 1:100 respectively, and the injection volume was 1μL. The sample was separated by Lipodex-E column: Macherey-Nagel LipodexE, 25m×250μm, and the detection conditions were constant temperature at 110℃ for 9.5min, then increased to 160℃ at 30℃ / min and maintained for 2min. The retention times of product (2R,5R)-dihydrocarvone, (2S,5R)-dihydrocarvone and substrate (5R)-carvone were 6.3min, 7.3min, and 8.8min respectively.

[0070] Table 6: Asymmetric reduction of (5R)-carvone by GsOYE and mutant GsOYE-X [a]

[0071]

[0072] [a] Data present mean ± SD from three independent experiments.

[0073] The results showed that the enolate reductase mutant CtOYE-W103F exhibited excellent stereoselectivity and chemoselectivity, and diastereoselectivity was p The conversion rate was 100%, and the mutant was marked as CtOYE-Mut. The amino acid sequence was shown in SEQ ID NO.4, and the nucleotide sequence was shown in SEQ ID NO.5.

[0074] Example 5: Activity analysis of olefin reductase CtOYE and olefin reductase mutant CtOYE-Mut

[0075] Reaction system 2 mL: At 30°C, 10 mM substrate (5R)-carvone and 0.1 mM NADPH were added to 2 mL of PBS (50 mM, pH 8.0) buffer, and finally 0.06 mg of pure enzyme solution of ene reductase CtOYE and ene reductase mutant CtOYE-Mut (based on protein content, the minimum enzyme amount that makes the curve of light absorption value changing with time remain linear) prepared by the method of Example 3 were added, and the change of absorbance at 340 nm within 2 min was immediately measured, and the blank control was taken as the blank control without adding pure enzyme.

[0076] One unit of enzyme activity is defined as the oxidation of 1 μmol NADPH (or NADP + Reducing) enzyme amount.

[0077] The specific enzyme activity was calculated by the formula: specific activity (U / mg) = ΔA × V / ε·m;

[0078] ΔA: change in absorbance at 340 nm within 1 min;

[0079] V: reaction solution volume, 0.2 mL in this experiment;

[0080] ε: NADPH molar absorption coefficient at 340nm, 6.22mM in this experiment -1 cm -1 ;

[0081] m: the amount of enzyme added to the reaction solution, 0.06 mg in this experiment;

[0082] Enzyme activity analysis was performed on ene reductase CtOYE and ene reductase mutant CtOYE-Mut. The amount of enzyme solution added was 0.06 mg, and the specific activity of GsOYE was 0.680 U / mg, and the specific activity of ene reductase mutant CtOYE-Mut was 0.8 U / mg.

[0083] Example 6: Stereoselectivity of the preparation of (2R,5R)-dihydrocarvone from (5R)-carvone catalyzed by pure enzymes of olefin reductase CtOYE and olefin reductase mutant CtOYE-Mut

[0084] Reaction system 0.5 mL: pure enzyme solution (olefin reductase CtOYE and olefin reductase mutant CtOYE-Mut) prepared by the method of Example 3 was used as a catalyst, the added amount was 1 mg / mL in terms of protein content, NAD(P)+ with a final concentration of 0.1 μM was used as a cofactor, glucose dehydrogenase with a final concentration of 0.5 U / mL (purchased from Aladdin) was used as a coenzyme, glucose with a final concentration of 40 mM was used as a cosubstrate, (5R)-carvone with a final concentration of 5 mM was used as a substrate, and 50 mM, pH 8.0 PBS buffer was used as a reaction medium to form a reaction system 0.5 mL; the reaction was carried out in a shaker at 30° C. and 200 rpm for 24 h. After the reaction was completed, an equal volume of ethyl acetate was added to the reaction solution for extraction. The upper organic phase was dehydrated with anhydrous MgSO4, allowed to stand for 10 minutes, and centrifuged at 12,000 rpm for 10 minutes. The supernatant was taken and the contents of the substrate (5R)-carvone and the products (2R, 5R)-dihydrocarvone and (2S, 5R)-dihydrocarvone were detected by gas chromatography, and the de value and product yield were calculated. The results showed that the de value and conversion rate of the ene reductase CtOYE were 86.4% (2R, 5R) and 100.0%, respectively, and the de value and conversion rate of the ene reductase mutant CtOYE-Mut were 100% (2R, 5R) and 100%, respectively. The GC analysis spectrum of the ene reductase mutant CtOYE-Mut catalyzing the substrate (5R)-carvone to prepare the product (2R, 5R)-dihydrocarvone is shown in Figure 6 Show.

[0085] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that numerous changes, modifications, substitutions and variations may be made to the examples without departing from the principles and spirit of the invention, the scope of the invention being defined by the appended claims and their equivalents.

Claims

1. An olefin reductase mutant, characterized in that The amino acid sequence of the olefin reductase mutant is shown in SEQ ID NO.

4.

2. A gene encoding the olefin reductase mutant according to claim 1.

3. A recombinant vector containing the gene encoding the olefin reductase mutant according to claim 1.

4. A recombinant genetically engineered bacterium constructed with the recombinant vector according to claim 3.

5. Use of the ene reductase mutant according to claim 1 in catalyzing the preparation of a chiral compound (2R, 5R)-dihydrocarvone from (5R)-carvone.

6. The use according to claim 5, characterized in that The application is as follows: the wet bacterial cells obtained by fermentation and culture of the recombinant genetic engineering bacteria containing the olefin reductase mutant encoding gene are ultrasonically broken, the supernatant after centrifugation of the broken mixed liquid or the pure enzyme liquid extracted from the supernatant is taken as a catalyst, (5R)-carvone is taken as a substrate, and NAD(P) + The reaction system is composed of a cofactor, glucose dehydrogenase as a coenzyme, glucose as a cosubstrate, and a pH 6-9 buffer as a reaction medium. The reaction is carried out in a shaker at 30-40°C and 180-250 rpm for 12-48 h. After the reaction is complete, the reaction solution is extracted with ethyl acetate and then dehydrated with anhydrous magnesium sulfate to obtain a chiral product (2R, 5R)-dihydrocarvone.

7. The use according to claim 6, characterized in that In the reaction system, the amount of catalyst used is 0.1-5 mg / mL based on protein content, the substrate is added to a final concentration of 2-10 mM, and the NAD(P) + The final concentration is 0.01-1.0 mM, the coenzyme is added to a final concentration of 0.2-2 U / mL, and the auxiliary substrate is added to a final concentration of 30-100 mM.

8. The use according to claim 6, characterized in that The wet bacterial cells were prepared as follows: a single colony of a recombinant genetic engineering bacterial plate containing an olefin reductase mutant encoding gene was inoculated into a LB liquid culture medium containing 50 μg / mL kanamycin, and cultured at 37° C. and 180 rpm for 15-20 h; the bacterial liquid was then inoculated into a fresh LB liquid culture medium containing 50 μg / mL kanamycin at an inoculum concentration of 1% by volume, and cultured at 37° C. and 180 rpm until the OD 600nm Reach 0.6-0.8; add IPTG to a final concentration of 0.2mM to the culture medium, induce culture at 25°C, 180rpm shaking for 12h, centrifuge at 1,0000rpm for 10min, suspend and wash the precipitate with sterile water, and collect the wet bacteria.

9. The use according to claim 6, characterized in that The pure enzyme solution was prepared as follows: wet bacteria were diluted with 50 mM, pH After suspending in 8.0 PBS buffer, the bacteria were ultrasonically disrupted in a 4°C low-temperature ice water bath, the ultrasonic disruption conditions were: power 400 W, 3s on, 4s off, temperature 4°C, and effective disruption time 8min; the ultrasonic disruption mixed solution was centrifuged at 12,000 rpm for 10 min, and then the supernatant was centrifuged at 12,000 rpm for 20 min to obtain a supernatant, the supernatant was aspirated to obtain a crude enzyme solution containing the target ene reductase mutant; the obtained crude enzyme solution was loaded onto a Ni-NTA metal chelate affinity chromatography column, and eluted with eluents containing 5mM imidazole, 50mM imidazole, 100mM imidazole and 250mM imidazole in sequence, the effluent containing the target protein was collected, and desalted and concentrated by centrifugation at 4°C and 7000 rpm for 30 min using an ultrafiltration tube with a molecular weight cutoff of 10kDa, and the retentate was taken as the pure enzyme solution; the eluent composition: 300mM sodium chloride, the solvent is 50mM PBS buffer, pH 8.0.

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