A method for biocatalytic synthesis of methyl epoxycinnamate compounds
Through the protein engineering transformation of P450BM3 enzyme, the mutation of the p450BM3 enzyme was solved, and the catalytic efficiency of catalyzing α,β-unsaturated ester epoxy compounds in the prior art was solved, and the efficient catalysis of various derivatives was achieved, and the ee value was achieved. 99%.
Patent Information
- Application Number
- CN202211119058.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-09-13
- Publication Date
- 2024-12-31
- Estimated Expiration
- 2042-09-13
AI Technical Summary
In the prior art, in the enzyme catalytic method of catalyzing epoxy compounds with α,β-unsaturated esters, there are problems of low catalytic efficiency and insufficient enantioselectivity, especially in the construction of antihypertensive drugs Diltiazem and beniserine paclitaxel. When chains and antibiotic chloramphenicol A are difficult to effectively improve the reaction efficiency.
By protein engineering of the P450BM3 enzyme from Bacillus mega-derived, especially the mutation of its 68th position aspartic acid (D68) to valine (V), to improve the reaction efficiency of methyl cinnamate olefin epoxy reaction and the required genes and expression vectors were amplified to construct recombinant E. coli to express the mutant P450BM3 enzyme.
The catalytic activity of the mutant D68V has been increased by 10 times, and has high olefin epoxy activity for a variety of derivatives such as methyl p-fluorocinnamate, methyl meta-fluorocinnamate and methyl o-fluorocinnamate, with an ee value of 99%. Significantly improves catalytic efficiency and selectivity.
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Abstract
Description
Technical Field
[0001] The invention relates to a method for synthesizing methyl epoxycinnamate compounds by biocatalysis, and belongs to the technical field of bioengineering. Background Art
[0002] Epoxides are three-membered ring building blocks that exist in a wide variety of natural product structures. Based on the fact that these compounds have epoxy structures, these structures have unique advantages in pharmacological, chemical and physical properties. There is considerable interest in preparing these scaffolds by synthetic methods or in combination with biocatalytic pathways. The simplest method is to produce enantioselective epoxides from olefin-rich raw materials. Catalysts used in chemical synthesis methods such as metal complexes, phase transfer catalysts (PTCs), chiral N-oxides and peptides have been applied to asymmetric epoxidation reactions of these compounds with varying degrees of success. These synthetic methods are generally efficient and stereoselective, but these methods often violate the principles of green chemistry, produce heavy metal waste, and have poor atom economy based on the synthesis of complex chiral ligand catalysts, and often cannot become sustainable methods. Enzyme catalysis has the advantages of high chemical selectivity, regioselectivity and stereoselectivity as well as non-toxicity, and has become an ideal tool for enantioselective and green synthesis. Among them, cytochrome P450 is particularly attractive because they can catalyze the oxidation of C=C bonds in substrates of specific sizes and shapes. These transformations utilize molecular oxygen and NADPH to form reactive ferroxo intermediates, often affording epoxides with high regio- and stereoselectivity.
[0003] The ring opening of α,β-unsaturated ester epoxy compounds can construct the antihypertensive drug diltiazem, the side chain of phenylisoserine paclitaxel, the antibiotic chloramphenicol A, etc. There are few studies on the enzymatic catalysis of such substrates. Due to the recognition of the substrate by the enzyme catalysis, the catalytic efficiency, regio- and stereo-selectivity of the enzyme are affected. Therefore, it is urgent to screen new P450BM3 enzymes with improved catalytic reaction efficiency and good enantioselectivity. Summary of the invention
[0004] The invention aims to improve the reaction efficiency of olefin epoxidation reaction of methyl cinnamate by transforming amino acid residues in a P450BM3 channel derived from Bacillus megaterium through protein engineering means.
[0005] The present invention provides a P450BM3 enzyme mutant, wherein the mutant contains an amino acid sequence as shown in SEQ ID NO.3.
[0006] The present invention also provides a gene encoding the mutant.
[0007] In one embodiment, the nucleotide sequence of the gene is shown as SEQ ID NO.4.
[0008] The invention also provides an expression vector containing the gene.
[0009] The invention also provides a genetically engineered bacterium for expressing the mutant.
[0010] In one embodiment, the host cell of the genetically engineered bacteria includes but is not limited to Escherichia coli.
[0011] In one embodiment, the genetically engineered bacteria are constructed using Escherichia coli as a host and a pRSF-Duet1 series plasmid as a vector.
[0012] The present invention also provides a recombinant Escherichia coli, which uses a pRSF-Duet1 series plasmid as an expression vector to express the P450BM3 enzyme mutant.
[0013] The present invention also provides a cell catalyst containing the P450BM3 enzyme mutant.
[0014] The present invention also provides a method for preparing the P450BM3 enzyme mutant, which comprises culturing the genetically engineered bacteria or the recombinant Escherichia coli in a culture medium and collecting the P450BM3 enzyme mutant.
[0015] In one embodiment, the culture medium is a TB medium containing, by final concentration, 1 mM vitamin B1 and 500 μM 5-aminolevulinic acid hydrochloride.
[0016] In one embodiment, the culture process is induced by IPTG, the final concentration of IPTG is 150-250 μM, the induction temperature is 22-28° C., the rotation speed is 160 rpm, and the induction time is 18-20 h.
[0017] In one embodiment, the culture process is induced by IPTG, the final concentration of IPTG is 200 μM, the induction temperature is 25° C., the rotation speed is 160 rpm, and the induction time is 18-20 h.
[0018] In one embodiment, the collection of P450BM3 enzyme mutants includes the following steps: collecting the cells of the genetically engineered bacteria or the recombinant Escherichia coli using a pre-cooled centrifuge, washing twice with a buffer solution, adding a small amount of lysozyme, quick freezing with liquid nitrogen, thawing in an ice water bath, ultrasonic disruption, high-speed centrifugation, and affinity purification using a His-nickel column and desalting with a desalting column.
[0019] The present invention also provides a method for improving the epoxide activity of the P450BM3 enzyme, which comprises mutating the 68th aspartic acid of the P450BM3 enzyme shown in SEQ ID NO.1 into valine.
[0020] The present invention also provides the use of the P450BM3 enzyme mutant or the cell catalyst in catalyzing methyl cinnamate or its derivatives.
[0021] In one embodiment, the methyl cinnamate derivatives include but are not limited to: methyl p-fluorocinnamate, methyl o-fluorocinnamate, and methyl m-fluorocinnamate.
[0022] In one embodiment, the method is to add enzyme to 100mM KH2PO4-K2HPO4 buffer system with OD=40-100, pH=7.0, at 30°C, with receptor substrate concentration of 10-20mM, reaction speed of 280-1200rpm and reaction time of 4-12h.
[0023] Beneficial effects:
[0024] (1) The present invention uses P450BM3 from Bacillus megaterium to perform protein engineering through cinnamic acid methyl olefin epoxidation reaction, and the obtained mutant D68V is used as a biocatalyst, and the transformation number is increased by 10 times compared with the wild type.
[0025] (2) After the enzyme was modified, its mutant D68V had high olefin epoxidation activity for methyl p-fluorocinnamate, methyl m-fluorocinnamate, and methyl o-fluorocinnamate, and the ee value reached 99%, which has important application value. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 The reaction substrate diagram of mutant D68V and wild type with methyl cinnamate as substrate.
[0027] Figure 2 This is a comparison chart of the product after the reaction of P450BM3 mutant D68V with methyl cinnamate as substrate and the standard.
[0028] Figure 3 This is the gas phase diagram of the product after the P450BM3 mutant D68V reacts with methyl p-fluorocinnamate as substrate.
[0029] Figure 4 This is a comparison chart of the product after the reaction of P450BM3 mutant D68V with methyl p-fluorocinnamate as substrate and the standard.
[0030] Figure 5 This is the gas phase diagram of the product after the P450BM3 mutant D68V reacts with methyl m-fluorocinnamate as substrate.
[0031] Figure 6 This is a comparison chart of the product after the reaction of P450BM3 mutant D68V with methyl m-fluorocinnamate as substrate and the standard.
[0032] Figure 7 This is the gas phase diagram of the product after the P450BM3 mutant D68V reacts with methyl o-fluorocinnamate as substrate.
[0033] Figure 8 This is a comparison chart of the product after the reaction of P450BM3 mutant D68V with methyl o-fluorocinnamate as substrate and the standard.
[0034] Fig. 9 Hydrogen spectrum of the product of (2S,3R)-3-phenoxyhexane-2-carboxylic acid methyl ester prepared by the mutant.
[0035] Fig.10 Carbon spectrum of the product of (2S,3R)-3-phenoxyhexane-2-carboxylic acid methyl ester prepared by the mutant.
[0036] Fig.11 Hydrogen spectrum of the product of (2S,3R)-3-(4-fluorophenyl)oxirane-2-carboxylic acid methyl ester prepared by the mutant.
[0037] Fig.12 Carbon spectrum of the product of (2S,3R)-3-(4-fluorophenyl)ethylene oxide-2-carboxylic acid methyl ester prepared by the mutant.
[0038] Fig.13 Fluorine spectrum of the product of (2S,3R)-3-(4-fluorophenyl)oxirane-2-carboxylic acid methyl ester prepared by the mutant.
[0039] Fig.14 Hydrogen spectrum of the product of (2S,3R)-3-(3-fluorophenyl)oxirane-2-carboxylic acid methyl ester prepared by the mutant.
[0040] Fig.15 Carbon spectrum of the product of (2S,3R)-3-(3-fluorophenyl)oxirane-2-carboxylic acid methyl ester prepared by the mutant.
[0041] Fig.16 Fluorine spectrum of the product of (2S,3R)-3-(3-fluorophenyl)oxirane-2-carboxylic acid methyl ester prepared by the mutant.
[0042] Fig.17 Hydrogen spectrum of the product of (2S,3R)-3-(2-fluorophenyl)oxirane-2-carboxylic acid methyl ester prepared by the mutant.
[0043] Fig.18 Carbon spectrum of the product of (2S,3R)-3-(2-fluorophenyl)oxirane-2-carboxylic acid methyl ester prepared by the mutant.
[0044] Fig.19Fluorine spectrum of the product of (2S,3R)-3-(2-fluorophenyl)oxirane-2-carboxylic acid methyl ester prepared by the mutant. DETAILED DESCRIPTION
[0045] The chemicals used in the present invention were all purchased from commercial sources.
[0046] The culture medium formula used in the embodiment is as follows:
[0047] Solid culture medium formula (1L): 5g yeast extract, 10g peptone, 10g sodium chloride, 15g agar. Make up to volume with deionized water and sterilize with high pressure steam.
[0048] LB medium formula (1L): 5g yeast extract powder, 10g peptone, 10g sodium chloride, dilute to volume with deionized water, and sterilize with high-pressure steam.
[0049] TB medium formula (1L): 24g yeast extract, 12g peptone, 4mL glycerol. Add 900mL deionized water, dissolve and sterilize with high pressure steam, then add 100mL of 0.17M KH2PO4 / 0.72M K2HPO4 solution that has been sterilized in the same way. (Note: Sodium chloride was purchased from Aladdin, and the rest were purchased from Sangon Biotech Co., Ltd.)
[0050] Specific activity assay of P450BM3 enzyme: The activity assay of P450BM3 was performed in 300 μL reaction buffer, which included 1 mM methyl cinnamate, 50 mM KH2PO4-K2HPO4 (pH 7.0), glucose 100 mM, sodium chloride 2 M and 25 μg of mutant purified enzyme, incubated at 30°C for 10 min, and then terminated by adding the same volume of methanol and analyzed by HPLC. One unit of enzyme activity is defined as the amount of enzyme that consumes 1 μmol of receptor substrate or generates 1 μmol of product in 1 min.
[0051] Product analysis method: gas chromatography and nuclear magnetic resonance hydrogen spectrum carbon spectrum fluorine spectrum. Conventional gas phase monitoring method: DM-5MS column, heating program: 250℃ injection port, 280℃ detector, 100℃ for 0 minutes, then 10℃ / min gradient to 300℃. The gas phase detection method for calculating the ee value of the standard product is column, and the temperature program was: 220°C inlet, 250°C detector, 100°C for 0 min, then 2°C / min gradient to 200°C, and hold for 10 min.
[0052] The ee value is calculated as follows: (RS / R+S)×100%, where R is the peak area of one enantiomer and S is the peak area of the other enantiomer.
[0053] Yield calculation method: (actual separated product mass / theoretical product mass)×100%.
[0054] Turnover number (TON): The number of molecules of reactant converted per unit active center. TON = substrate conversion amount / enzyme dosage.
[0055] Example 1: P450BM3 protein engineering
[0056] Design and preparation of mutants:
[0057] (1) The gene with the sequence shown in SEQ ID NO.2 was ligated between the AcoI and AvrII sites of the vector pRSF-Duet1 to construct P450BM3-pRSF-Duet1, and the mutagenesis primers were designed using the constructed recombinant plasmid P450BM3-pRSF-Duet1 as a template.
[0058] Table 1 Mutation primers
[0059]
[0060] (2) Construction of mutants:
[0061] The D68V single mutant fragment was amplified by PCR using the recombinant plasmid P450BM3-pRSF-Duet1 as a template. The reaction system is shown in the following table:
[0062] Table 2 PCR amplification of W93V single mutant fragments
[0063]
[0064] PCR reaction program: denaturation at 98°C, 10 seconds; annealing at 58°C, 5 seconds; extension at 68°C, 5 seconds / kb.
[0065] The fragment obtained by the above amplification was then used as a primer to PCR amplify the W93V single mutant plasmid. The PCR system is as follows:
[0066] Table 3 PCR amplification of W93V single mutant plasmid
[0067]
[0068] PCR reaction program: denaturation at 98°C, 10 seconds; annealing at 58°C, 5 seconds; extension at 68°C, 5 seconds / kb.
[0069] Example 2: Expression and purification of P450BM3 mutant D68V
[0070] The expression plasmid pRSF-Duet1 containing the mutant D68V gene of P450BM3 was transformed into competent cells BL21 by chemical transformation. An appropriate amount of bacterial liquid was spread on a solid medium containing kanamycin and cultured at 37°C for 12-15h. A single colony containing the transformed recombinant plasmid was selected and placed in LB medium (containing 50 μg mL -1 Kanamycin) and cultured at 37°C overnight. Take 1% of the LB culture and inoculate it into TB medium (containing 50 μg mL -1 Kanamycin and 1 mM vitamin B1), then cultured at 37°C to the logarithmic growth phase, that is, the OD value was 0.6-0.8, 500 μM 5-aminolevulinic acid hydrochloride and 200 μM IPTG were added as inducers, and the expression was carried out at 25°C and 160 rpm for 18-20 h.
[0071] The expression bacteria were collected in a 4°C precooled centrifuge, washed twice with 100mM (pH=7.4) KH2PO4-K2HPO4 buffer solution containing 10% glycerol, then a small amount of lysozyme was added, quick-frozen in liquid nitrogen, thawed in an ice-water bath, ultrasonically broken, centrifuged at high speed, and purified by His-nickel column affinity purification (using a buffer solution containing 10% glycerol and 10mM imidazole to wash the impurities, and using a buffer solution containing 10% glycerol and 500mM imidazole to elute the target protein, and using a desalting column for desalting (the desalting buffer solution was a 100mM KH2PO4-K2HPO4 buffer solution containing 10% glycerol). The specific enzyme activity of the purified enzyme was determined to be 2560U / mg.
[0072] Example 3: Synthesis of (2S,3R)-3-phenoxyhexane-2-carboxylic acid methyl ester catalyzed by P450BM3 and its mutant D68V from methyl cinnamate
[0073] In a 300 μL reaction system, the final concentrations were: glucose concentration of 100 mM, substrate methyl cinnamate concentration of 10 mM, DMF (v / v, 1%), NADP + (200uM), the amount of the whole cell of the biocatalyst is about 10mg / mL, and the corresponding epoxide product is obtained by reacting at 30°C for 12h. 500uL of ethyl acetate is added to the reaction product for extraction and gas phase analysis.
[0074] like Figure 1 and Figure 2 As shown, mutant D68V catalyzes methyl cinnamate to obtain 8.3mM of the product (2S,3R)-3-phenoxyhexane-2-carboxylic acid methyl ester, which is greatly improved compared to the wild type. By calculating TON, the wild type (101TON) and the mutant (1043TON) increased by 10 times. By chiral gas column analysis, the wild type and mutant products can reach 99% ee value.
[0075] Example 4: Synthesis of (2S,3R)-3-(4-fluorophenyl)oxirane-2-carboxylic acid methyl ester catalyzed by P450BM3 mutant D68V from methyl p-fluorocinnamate
[0076] In a 300 μL reaction system, the final concentrations were: glucose concentration was 100 mM, substrate methyl 4-fluorocinnamate concentration was 10 mM, DMF (v / v, 1%), NADP + (200uM), the amount of the whole cell of the biocatalyst is about 10mg / mL, and the corresponding epoxide product is obtained by reacting at 30°C for 12h. 500uL of ethyl acetate is added to the reaction product for extraction and gas phase analysis. Figure 3
[0077] like Figure 3 , Figure 4 As shown, mutant D68V catalyzed the reaction of methyl p-fluorocinnamate to obtain 9.6 mM of product, and the ee value of the product reached 99% through chiral gas phase column analysis.
[0078] Example 5: Synthesis of (2S,3R)-3-(3-fluorophenyl)oxirane-2-carboxylic acid methyl ester catalyzed by P450BM3 mutant D68V from m-fluorocinnamic acid methyl ester
[0079] In a 300 μL reaction system, the final concentrations were: glucose concentration was 100 mM, substrate methyl 4-fluorocinnamate concentration was 10 mM, DMF (v / v, 1%), NADP + (200uM), the amount of the whole cell of the biocatalyst is about 10mg / mL, and the corresponding epoxide product is obtained by reacting at 30°C for 12h. 500uL of ethyl acetate is added to the reaction product for extraction and gas phase analysis.
[0080] like Figure 5 , Figure 6 As shown, mutant D68V catalyzed the reaction of methyl m-fluorocinnamate to obtain 6.2 mM of product, and the product ee value reached 99% through chiral gas phase column analysis.
[0081] Example 6: Synthesis of (2S,3R)-3-(2-fluorophenyl)oxirane-2-carboxylic acid methyl ester catalyzed by P450BM3 mutant D68V from methyl o-fluorocinnamate
[0082] In a 300 μL reaction system, the final concentrations were: glucose concentration was 100 mM, substrate methyl 4-fluorocinnamate concentration was 10 mM, DMF (v / v, 1%), NADP + (200uM), the amount of P450BM3 mutant D68V whole cells is about 10mg / mL, and the corresponding epoxide product is obtained by reaction at 30°C for 12h. 500uL of ethyl acetate is added to the reaction product for extraction and gas phase analysis.
[0083] like Figure 7 , Figure 8 As shown, mutant D68V catalyzed the reaction of methyl o-fluorocinnamate to obtain 4.3 mM of product, and the ee value of the product reached 99% through chiral gas phase column analysis.
[0084] Example 7: Preparation of (2S,3R)-3-phenoxyhexane-2-carboxylic acid methyl ester product by P450BM3 mutant D68V at gram scale
[0085] In a 30mL reaction system, the concentration of glucose was 100mM, the concentration of substrate methyl cinnamate was 20mM, DMF (v / v, 1%), NADP+ (200uM), the whole cells of the P450BM3 mutant D68V prepared in Example 2 were added to OD=100, 10mg / mL of GDH freeze-dried powder (enzyme activity 1000U / g), and the reaction was carried out at 30°C. A total of 10 bottles of reaction were performed, and the time gradient gas phase was monitored. The substrate was completely consumed in 7 hours, and ethyl acetate was added for extraction three times. Purification by silica gel column gave 1.043g, with a yield of 95% and an ee value of 99%. The NMR data are as follows:
[0086] [α]D 20 = +159.6 (c = 0.5, CHCl3). 1 H NMR (500MHz, CDCl3) δ7.38–7.34(m,3H),7.31–7.27(m,2H),4.10(s,1H),3.83(s,3H),3.52(s,1H). 13 C NMR (125MHz, CDCl3) δ168.6,134.8,129.0,128.6,125.7,57.9,56.6,52.6( Figure 9-10 ).
[0087] Example 8: Preparation of (2S,3R)-3-(4-fluorophenyl)oxirane-2-carboxylic acid methyl ester by P450BM3 mutant D68V
[0088] In a 30mL reaction system, the concentration of glucose is 100mM, the concentration of substrate methyl p-fluorocinnamate is 20mM, DMF (v / v, 1%), NADP+ (200uM), the amount of P450BM3 mutant D68V whole cell OD=100, GDH freeze-dried powder (enzyme activity 1000U) 10mg / mL, react at 30℃, a total of 10 bottles of reaction, time gradient gas phase monitoring, the substrate can be completely consumed in 5 hours, ethyl acetate is added for extraction three times, and 1.052g is obtained by silica gel column purification, with a yield of 97% and an ee value of 99%. The NMR data are as follows:
[0089] (2S,3R)-methyl 3-(4-fluorophenyl)oxirane-2-carboxylate(2b).[α]D 20 = +135.6 (c = 0.5, CHCl3), 1 H NMR (400MHz, CDCl3) δ7.31-7.27(m,2H),7.1-7.06m,2H),4.11(d,J=1.8Hz,1H),3.85(s,3H),3.50(d,J=1.7Hz,1H). 13 C NMR(100MHz,CDCl3)δ168.43,163.2(J F =250Hz),130.7(J F =3.0Hz),127.6(J F =8.0Hz),115.8(J F =22.0Hz),57.39,56.60,52.62. 19 F NMR (377MHz, CDCl3) δ-112.26( Figure 11-13 ).
[0090] Example 9: Preparation of (2S,3R)-3-(3-fluorophenyl)oxirane-2-carboxylic acid methyl ester by P450BM3 mutant D68V
[0091] In a 30 mL reaction system, the concentration of glucose was 100 mM, the concentration of substrate methyl m-fluorocinnamate was 20 mM, DMSO (v / v, 1%), NADP + (200uM), the added P450BM3 mutant D68V whole cell in the reaction system OD = 130, GDH freeze-dried powder (enzyme activity 1000U) 10mg / mL, react at 30℃, do 10 bottles of reaction in total, time gradient gas phase monitoring, the substrate can be completely consumed in 6 hours, add ethyl acetate extraction three times, and purify by silica gel column to obtain 1.034g, yield 95%, ee value 99%. NMR data are as follows:
[0092] (2S,3R)-methyl 3-(3-fluorophenyl)oxirane-2-carboxylate(2c).[α]D 20 = +172.2 (c = 0.5, CHCl3), 1H NMR (400MHz, CDCl3) δ7.36–7.30(m,1H),7.09(m,1H),7.06–7.01(m,1H),6.99 -6.96(m,1H),4.09(d,J=1.8Hz,1H),3.83(s,3H),3.47(d,J=1.8Hz,1H).[α]D 20=+166.2(c=0.5,CHCl3) 13 C NMR (100MHz, CDCl3) δ168.22, 163.0 (J = 240.0Hz), 137.6 (J = 8.0Hz), 130.4 (J = 8.0Hz), 121.6 (J = 3.0Hz), 116.0 (J F =11.0Hz),112.6(J F =23.0Hz),57.2(J F =2.0Hz),56.59,52.64. 19 F NMR (377MHz, CDCl3) δ-112.23( Figure 14-16 ).
[0093] Example 10: Preparation of (2S,3R)-3-(2-fluorophenyl)oxirane-2-carboxylic acid methyl ester by P450BM3 mutant D68V
[0094] In a 30 mL reaction system, the concentration of glucose was 100 mM, the concentration of substrate methyl o-fluorocinnamate was 20 mM, DMSO (v / v, 1%), NADP + (200uM), the amount of P450BM3 mutant D68V whole cell OD = 100, GDH freeze-dried powder (enzyme activity 1000U) 10mg / mL, reaction at 30℃, time gradient gas phase monitoring, the substrate can be completely consumed in 6 hours, extracted with ethyl acetate three times, purified by silica gel column to obtain 1.033g, yield 95%, ee value 99%. NMR data are as follows:
[0095] (2S,3R)-methyl 3-(2-fluorophenyl)oxirane-2-carboxylate(2d).[α]D 20 = +162.3 (c = 0.5, CHCl3), 1 H NMR (400MHz, CDCl3) δ7.35–7.28(m,1H),7.21–7.12(m,2H),7.09–7.04(m,1H),4.36(d,J=1.8Hz,1H),3.83(s,3H),3.52(d,J=1.8Hz,1H). 13C NMR(100MHz,CDCl3)δ168.36,161.6(J F =240.0Hz),130.4(J F =8.0Hz),126.2(J F =4.0Hz),124.5(J F =4.0Hz),122.5(J F =13.0Hz),115.5(J F =21.0Hz),55.9(J F =2.0Hz),52.75,52.69. 19 F NMR (377MHz, CDCl3) δ-120.27( Figure 17-Figure 19 ).
[0096] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.
Claims
1. A P450BM3 enzyme mutant, characterized in that The amino acid sequence of the mutant is shown in SEQ ID NO.
3.
2. A gene encoding the P450BM3 enzyme mutant according to claim 1.
3. An expression vector containing the gene according to claim 2.
4. A genetically engineered bacterium expressing the P450BM3 enzyme mutant according to claim 1.
5. A recombinant Escherichia coli, characterized in that Escherichia coli is used as a host and a pRSF-Duet1 series plasmid is used as a vector to express the P450BM3 enzyme mutant described in claim 1.
6. A cell catalyst containing the P450BM3 enzyme mutant according to claim 5.
7. A method for improving the efficiency of P450BM3 enzyme in the epoxidation reaction of methyl cinnamate olefin, characterized in that: Using the P450BM3 enzyme shown in SEQ ID NO.1 as the starting sequence, the aspartic acid at position 68 was mutated to valine.
8. A method for preparing the P450BM3 enzyme mutant according to claim 1, characterized in that: The genetically engineered bacteria according to claim 4 or the recombinant Escherichia coli according to claim 5 are cultured in a culture medium to collect P450BM3 enzyme mutants.
9. The method according to claim 8, characterized in that The culture process adopts IPTG induction, the final concentration of IPTG is 150-250 μM; the induction temperature is 22-28° C., and the induction time is 18-20 h.
10. Use of the P450BM3 enzyme mutant according to claim 1, or the genetically engineered bacteria according to claim 4, or the recombinant Escherichia coli according to claim 5, or the cell catalyst according to claim 6 in catalyzing methyl cinnamate or its derivatives, characterized in that: The derivative is methyl fluorocinnamate, methyl m-fluorocinnamate or methyl o-fluorocinnamate.