Cytochrome P450 monooxygenase and application thereof

Abstract

The invention discloses cytochrome P450 monooxygenase and application thereof, and belongs to the technical field of enzyme engineering. The amino acid sequence of the cytochrome P450 monooxygenase provided by the invention is as shown in SEQ ID NO. 1. The invention also provides an application of the cytochrome P450 monooxygenase in synthesis of chiral tertiary alcohol ester compounds. The monooxygenase can effectively solve the problem that in the prior art, chiral tertiary alcohol ester needs to be indirectly synthesized through a precursor structure, and a tertiary alcohol ester compound with enantioselectivity cannot be synthesized through direct hydroxylation of methyl ester racemate.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme engineering, and particularly relates to a cytochrome P450 monooxygenase and its application. Background Art

[0002] Chiral tertiary alcohol ester compounds are important intermediates in the synthesis of drugs, pesticides, and fine chemicals, and their stereoconfiguration directly affects biological activity and drug efficacy. For example, the active molecules of many antiviral drugs and cardiovascular drugs contain chiral tertiary alcohol ester structural units.

[0003] Traditional methods for synthesizing such compounds mainly include chemical catalytic asymmetric synthesis and racemate resolution, but such methods have significant limitations. Chemical catalytic methods mainly rely on precious metal catalysts (such as ruthenium and rhodium complexes), which are costly and pose a risk of heavy metal residues. Their reaction conditions are harsh, with high energy consumption and many side reactions, and it is extremely easy to cause instability of product stereoselectivity. At the same time, the direct hydroxylation ability of precious metal catalysts for inert C-H bonds is insufficient, and multiple steps are required to construct the tertiary alcohol structure, resulting in low atom economy. For the existing racemate resolution technology, the maximum theoretical yield of kinetic resolution is 50%. The resolution process requires additional steps to separate isomers, with high costs and cumbersome steps. The chemical resolving agents used in the reaction process are likely to cause environmental pollution, which does not conform to the development trend of green chemistry.

[0004] Compared with chemical methods, biocatalytic methods are simple to operate, have mild reaction conditions, and strong chemo-selectivity, regioselectivity, and stereoselectivity. However, the existing oxidases used in biocatalytic methods are difficult to efficiently activate the inert C-H bonds of methyl ester substrates, resulting in low catalytic efficiency. Their enzyme-catalyzed reaction substrate spectrum is narrow, and they have poor adaptability to tertiary alcohol ester precursors with complex steric hindrance. At the same time, there are also problems of strong dependence on cofactors (such as NADPH), the need to add or construct a regeneration system additionally, and the complication of the process. Summary of the Invention

[0005] The purpose of the present invention is to provide a cytochrome P450 monooxygenase and its application, so as to solve the problem in the prior art that chiral tertiary alcohol esters need to be indirectly synthesized through precursor structures, and it is impossible to directly hydroxylate methyl ester racemates to synthesize enantioselective tertiary alcohol ester compounds.

[0006] The technical solution adopted to achieve the above purpose is: According to the first aspect of the present invention, a cytochrome P450 monooxygenase is provided, and the amino acid sequence of the cytochrome P450 monooxygenase is as shown in SEQ ID NO.1.

[0007] Preferably, the nucleotide sequence of the cytochrome P450 monooxygenase is as shown in SEQ ID NO.2.

[0008] According to the second aspect of the present invention, there is provided the use of the above cytochrome P450 monooxygenase in the synthesis of chiral tertiary alcohol esters.

[0009] Preferably, the cytochrome P450 monooxygenase synthesizes enantioselective tertiary alcohol esters by catalyzing the asymmetric hydroxylation of the C-H bond of methyl ester racemates; the synthesis route is as follows: ; In the formula, R 1 is thiophene, -C 6 H 4 Br, -C 6 H 4 Cl, -C 6 H 4 F, -C 6 H 5 , -C 6 H 4 CH 3 , -C 6 H 4 OCH 3 or -C 6 H 4 CF 3 , R 2 is -H or -CH 3 .

[0010] Preferably, the catalysis is carried out under the condition that the pH is 7.5 - 8.5.

[0011] More preferably, the catalysis is carried out under the condition that the pH is 8.5.

[0012] More preferably, the catalysis is carried out in a phosphate buffer system; the phosphate concentration in the phosphate buffer system is 0.03 - 0.08 mol / L.

[0013] More preferably, the phosphate concentration in the phosphate buffer system is 0.05 mol / L.

[0014] Preferably, the reaction concentration of the methyl ester racemate is 0.03 - 0.08 mol / L.

[0015] More preferably, the reaction concentration of the methyl ester racemate is 0.05 mol / L.

[0016] Preferably, the addition form of the cytochrome P450 monooxygenase is a recombinant cell containing the cytochrome P450 monooxygenase.

[0017] More preferably, the cell dry weight concentration of the recombinant cell containing the cytochrome P450 monooxygenase is 10 - 20 g / L.

[0018] More preferably, the dry cell weight concentration of the recombinant cell containing cytochrome P450 monooxygenase is 15 g / L.

[0019] According to the third aspect of the present invention, there is provided a recombinant cell expressing cytochrome P450 monooxygenase.

[0020] According to the fourth aspect of the present invention, there is provided a vector carrying a gene encoding cytochrome P450 monooxygenase.

[0021] According to the fifth aspect of the present invention, there is provided a strain expressing cytochrome P450 monooxygenase.

[0022] The present invention has the following beneficial effects: (1) In the present invention, an engineered recombinant cell expressing cytochrome P450 monooxygenase is used as a biocatalyst, and in a 0.05 mol / L phosphate buffer system at pH 8.5, the efficient synthesis of chiral tertiary alcohol products is successfully achieved. The highest yield of the target product can reach 49%, and the catalytic system has a significant conversion efficiency for the substrate. At the same time, the enantiomeric excess value (ee value) of the product reaches up to 99.8%, with strong stereocontrol ability, and a target molecule with a single absolute configuration can be directly obtained.

[0023] (2) The catalytic system of the present invention has excellent stereospecificity and precise control ability of molecular modification sites. The enzyme-catalyzed reaction can accurately achieve the selective substitution of the hydroxyl group at the tertiary carbon site, effectively avoiding the generation of by-products such as benzene rings or other chain structures. Compared with traditional methods, the present technology uses an aqueous reaction system at normal temperature and pressure, which not only ensures the structural stability of thermosensitive substrates and products but also significantly reduces the energy consumption and operation risk during the reaction process, providing basic conditions for industrial scale-up production.

[0024] (3) In the present invention, no toxic by-products are generated during the whole reaction process, the cofactor requirement is significantly lower than that of conventional enzyme-catalyzed systems, and the high cost problem of using pure enzymes is effectively avoided through the whole-cell catalysis mode. At the same time, the reaction method of the present invention has low dependence on complex synthesis equipment and simple operation parameter regulation, opening up a new path for the industrial production of chiral compounds. Description of the Drawings

[0025] Figure 1 is the liquid chromatography chart of methyl (R)-2-phenyl-2-hydroxy-propionate prepared in Example 3; Figure 2 is the liquid chromatography chart of methyl (R)-2-(2-fluoro-phenyl)-2-hydroxy-propionate prepared in Example 4; Figure 3 is the liquid chromatography chart of methyl (R)-2-(3-fluoro-phenyl)-2-hydroxy-propionate prepared in Example 5; Figure 4 is the liquid chromatogram of methyl (R)-2-(4-fluorophenyl)-2-hydroxypropionate prepared in Example 6; Figure 5 is the liquid chromatogram of methyl (R)-2-(2-chlorophenyl)-2-hydroxypropionate prepared in Example 7; Figure 6 is the liquid chromatogram of methyl (R)-2-(3-bromophenyl)-2-hydroxypropionate prepared in Example 8; Figure 7 is the liquid chromatogram of methyl (S)-2-(α-thienyl)-2-hydroxypropionate prepared in Example 9. Detailed implementation manners

[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Apparently, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention. In the following embodiments, unless otherwise specified, the technical means used are all conventional means well known to those skilled in the art.

[0027] Example 1 Preparation of a cytochrome P450 monooxygenase plasmid transformant In order to optimize the amino acid sequence of SEQ ID NO.1 to make it more suitable for expression in Escherichia coli, the nucleotide sequence was optimized according to the codon preference of Escherichia coli, and the optimized nucleotide sequence SEQ ID NO.2 was obtained. The optimized gene SEQ ID NO.2 was artificially synthesized in full length and ligated to the Escherichia coli expression vector pET 28b to construct the recombinant expression plasmid pET 28b-P450. The specific steps are as follows: (1) Gene optimization and synthesis: According to the codon preference of Escherichia coli, the amino acid sequence of SEQ ID NO.1 was optimized, and the optimized gene SEQ ID NO.2 was synthesized by the method of full-length artificial gene synthesis; (2) Plasmid construction: The synthesized gene fragment of SEQ ID NO.2 was inserted into the Escherichia coli expression vector pET 28b to obtain the recombinant expression plasmid pET 28b-P450; (3) Transformation and screening: The constructed recombinant expression plasmid pET 28b-P450 was transformed into competent cells of Escherichia coli E.coli BL21(DE3). The transformed solution was spread on a TB plate containing kanamycin and streptomycin, sealed with a sealing film, and cultured overnight at 37°C in an inverted manner to screen out resistant colonies; (4) Cloning verification: The selected colonies were initially screened by colony PCR, and the positive clones were selected. Gene sequencing verification was carried out on the positive clones to verify the correct insertion and expression of the recombinant plasmid pET 28b-P450 in E. coli BL21(DE3); Amino acid sequence SEQ ID NO.1 of cytochrome P450 monooxygenase: Nucleotide sequence of cytochrome P450 monooxygenase SEQ ID NO.2:

[0028] Example 2 Expression of Cytochrome P450 Monooxygenase The recombinant Escherichia coli E. coli BL21(DE3) / pET 28b-P450 obtained in Example 1 was cultured and induced for expression according to the following steps: (1) Seed culture: The recombinant Escherichia coli was inoculated into TB medium containing kanamycin and streptomycin at a concentration of 50 μg / mL. The composition of the TB medium was: peptone 12 g / L, yeast extract 24 g / L, pH 7.0. It was cultured overnight with shaking at 37 °C and 250 rpm to allow the bacterial solution to grow fully; (2) Scale-up culture: The seed solution cultured overnight was inoculated into 100 mL of TB medium containing kanamycin and streptomycin at a concentration of 50 μg / mL at an inoculation amount of 1% (v / v). The above TB medium was placed in a 500 mL Erlenmeyer flask and continuously cultured with shaking at 37 °C and 250 rpm until the OD 600 value reached about 0.6, and the time was 2 - 3 h; (3) Induction expression: When the OD 600 value reached 0.6, isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.1 mmol / L was added to the culture solution as an inducer to induce the expression of P450 monooxygenase. The induced culture system was continuously cultured with shaking at 25 °C and 250 rpm for 12 h to allow the recombinant protein to be fully expressed; (4) Bacterial cell collection: After the protein was fully expressed, the culture solution was centrifuged to collect the bacteria. The centrifugation conditions were set at 4 °C and 5000 rpm, and the centrifugation time was 10 min. After centrifugation, the supernatant was discarded, and the precipitated recombinant bacterial cells were collected. These cells contained the expressed P450 monooxygenase protein; (5) Preservation and application: The collected recombinant bacterial cells can be directly used for subsequent biocatalytic reactions, or further processed as needed, such as freeze-drying preservation or preparation of crude enzyme solution.

[0029] Through the above steps, recombinant Escherichia coli cells containing P450 monooxygenase were successfully cultured and induced for expression, providing an efficient catalyst source for the biocatalytic synthesis of enantioselective tertiary alcohol esters. The synthesis route is as follows: ; In the formula, R 1 is thiophene, -C 6 H 4 Br, -C 6 H 4 Cl, -C 6 H 4 F, -C6 H 5 , -C 6 H 4 CH 3 , -C 6 H 4 OCH 3 or -C 6 H 4 CF 3 , R 2 -H or -CH 3 .

[0030] Example 3 Synthesis of (R)-2-phenyl-2-hydroxy-propionic acid methyl ester from 2-phenyl-1-propionic acid methyl ester catalyzed by cytochrome P450 monooxygenase recombinant cells The synthesis of (R)-2-phenyl-2-hydroxy-propionic acid methyl ester from 2-phenyl-1-propionic acid methyl ester catalyzed by cytochrome P450 monooxygenase recombinant cells comprises the following steps:

[0031] (1) Add 100 mL of 50 mM phosphate buffer at pH 8.5 to a 500 mL conical flask, and add recombinant cells at a cell dry weight concentration of 15 g / L to the buffer, wherein the recombinant cells contain expressed cytochrome P450 monooxygenase for catalyzing the target reaction; (2) adding 2-phenyl-1-propionic acid methyl ester racemate with a final concentration of 50 mM as a reaction substrate to the system, and placing it on a constant temperature shaker to react at 30° C. and 250 rpm for 24 h. During the reaction, ensure sufficient oxygen supply and uniform mixing between the reactants and the buffer, and ensure that the substrate is fully converted into the target product; (3) After the reaction is completed, the reaction liquid is taken out and extracted with ethyl acetate of the same volume as the reaction liquid for 3 times. After each extraction, the mixture is fully shaken and mixed, and then the mixture is allowed to stand for separation. The ethyl acetate layer after each extraction is taken and dried with anhydrous sodium sulfate to absorb the moisture in the organic phase. After drying, the extract is concentrated under reduced pressure distillation to remove the ethyl acetate solvent. The concentrated target product (R)-2-phenyl-2-hydroxy-propionic acid methyl ester is weighed to obtain an isolated yield of 43% for the product. The purity and composition of the single-configuration (R)-2-phenyl-2-hydroxy-propionic acid methyl ester prepared by the reaction are verified by liquid chromatography (HPLC). The results are as follows: Figure 1 As shown, the optical purity (enantiomeric excess, ee value) of the target product (R)-2-phenyl-2-hydroxy-propionic acid methyl ester is 99.4%, which means that the product has high stereoselectivity.

[0032] Example 4: Synthesis of (R)-2-(2-fluorophenyl)-2-hydroxypropionate methyl ester by cytochrome P450 monooxygenase recombinant cells from methyl 2-(2-fluorophenyl)propionate The synthesis of (R)-2-(2-fluorophenyl)-2-hydroxypropionate methyl ester by cytochrome P450 monooxygenase recombinant cells from methyl 2-(2-fluorophenyl)propionate comprises the following steps:

[0033] (1) Add 100 mL of phosphate buffer with a concentration of 50 mM and a pH of 8.5 to a 500 mL Erlenmeyer flask, and add recombinant cells with a dry cell weight concentration of 15 g / L to this buffer. The recombinant cells contain the expressed cytochrome P450 monooxygenase for catalyzing the target reaction; (2) Then add the racemate of methyl 2-(2-fluorophenyl)propionate with a final concentration of 50 mM as the reaction substrate to this system, and place it on a constant temperature shaker. React at 30 °C and 250 rpm for 24 h. During the reaction process, ensure sufficient oxygen supply and uniform mixing between the reactants and the buffer to ensure that the substrate is fully converted into the target product; (3) After the reaction is completed, take out the reaction solution, extract it 3 times with ethyl acetate with the same volume as the reaction solution. After each extraction, shake it well and let it stand for stratification; Take the ethyl acetate layer after each extraction and dry it with anhydrous sodium sulfate to adsorb the water in the organic phase; After drying, concentrate the extract under reduced pressure distillation to remove the ethyl acetate solvent; Weigh the concentrated target product (R)-2-(2-fluorophenyl)-2-hydroxypropionate methyl ester, and the separation yield of this product is 49%; And verify the purity and composition of the single configuration (R)-2-(2-fluorophenyl)-2-hydroxypropionate methyl ester prepared by the reaction through liquid chromatography (HPLC). The results are as Figure 2 shown. The optical purity (enantiomeric excess, ee value) of the target product (R)-2-(2-fluorophenyl)-2-hydroxypropionate methyl ester is 99.8%, indicating that the product has high stereoselectivity.

[0034] Example 5: Synthesis of (R)-2-(3-fluorophenyl)-2-hydroxypropionate methyl ester by cytochrome P450 monooxygenase recombinant cells from methyl 2-(3-fluorophenyl)propionate The synthesis of (R)-2-(3-fluorophenyl)-2-hydroxypropionate methyl ester by cytochrome P450 monooxygenase recombinant cells from methyl 2-(3-fluorophenyl)propionate comprises the following steps:

[0035] (1) Add 100 mL of phosphate buffer with a concentration of 50 mM and a pH of 8.5 to a 500 mL Erlenmeyer flask. Add recombinant cells with a dry cell weight concentration of 15 g / L to this buffer. The recombinant cells contain the expressed cytochrome P450 monooxygenase for catalyzing the target reaction; (2) Add the racemate of methyl 2-(3-fluorophenyl)propionate with a final concentration of 50 mM to this system as the reaction substrate, and place it on a thermostatic shaker. React for 24 h at 30 °C and 250 rpm. During the reaction, ensure sufficient oxygen supply and uniform mixing between the reactants and the buffer to ensure that the substrate is fully converted into the target product; (3) After the reaction is completed, take out the reaction solution and extract it 3 times with an equal volume of ethyl acetate. After each extraction, shake it well and let it stand for layering; Take the ethyl acetate layer after each extraction and dry it with anhydrous sodium sulfate to adsorb the water in the organic phase; After drying, concentrate the extract under reduced pressure distillation to remove the ethyl acetate solvent; Weigh the concentrated target product, (R)-methyl 2-(3-fluorophenyl)-2-hydroxypropionate, and obtain the separation yield of this product as 46%; And verify the purity and composition of the single configuration (R)-methyl 2-(3-fluorophenyl)-2-hydroxypropionate prepared by the reaction through liquid chromatography (HPLC). The results are as Figure 3 shown. The optical purity (enantiomeric excess, ee value) of the target product, (R)-methyl 2-(3-fluorophenyl)-2-hydroxypropionate, is 93.3%, indicating that the product has high stereoselectivity.

[0036] Example 6 Synthesis of (R)-methyl 2-(4-fluorophenyl)-2-hydroxypropionate by cytochrome P450 monooxygenase recombinant cells catalyzing methyl 2-(4-fluorophenyl)propionate The synthesis of (R)-methyl 2-(4-fluorophenyl)-2-hydroxypropionate by cytochrome P450 monooxygenase recombinant cells catalyzing methyl 2-(4-fluorophenyl)propionate includes the following steps:

[0037] (1) Add 100 mL of phosphate buffer with a concentration of 50 mM and a pH of 8.5 to a 500 mL Erlenmeyer flask. Add recombinant cells with a dry cell weight concentration of 15 g / L to this buffer. The recombinant cells contain the expressed cytochrome P450 monooxygenase for catalyzing the target reaction; (2) Add the racemate of methyl 2-(4-fluorophenyl)propionate with a final concentration of 50 mM to this system as the reaction substrate, and place it on a thermostatic shaker. React for 24 h under the conditions of 30 °C and 250 rpm. During the reaction process, ensure sufficient oxygen supply and uniform mixing between the reactants and the buffer solution to ensure that the substrate is fully converted into the target product; (3) After the reaction is completed, take out the reaction solution and extract it 3 times with ethyl acetate with the same volume as the reaction solution. After each extraction, shake and mix well and then let it stand for stratification; Take the ethyl acetate layer after each extraction and dry it with anhydrous sodium sulfate to adsorb the water in the organic phase; After drying, concentrate the extract under reduced pressure distillation to remove the ethyl acetate solvent; Weigh the concentrated target product (R)-methyl 2-(4-fluorophenyl)-2-hydroxypropionate to obtain a separation yield of 42% for this product; And verify the purity and composition of the single configuration (R)-methyl 2-(4-fluorophenyl)-2-hydroxypropionate prepared by the reaction through liquid chromatography (HPLC). The results are as Figure 4 shown. The optical purity (enantiomeric excess, ee value) of the target product (R)-methyl 2-(4-fluorophenyl)-2-hydroxypropionate is 98.1%, indicating that the product has high stereoselectivity.

[0038] Example 7 Synthesis of (R)-methyl 2-(2-chlorophenyl)-2-hydroxypropionate by recombinant cells of cytochrome P450 monooxygenase The synthesis of (R)-methyl 2-(2-chlorophenyl)-2-hydroxypropionate by recombinant cells of cytochrome P450 monooxygenase includes the following steps:

[0039] (1) Add 100 mL of phosphate buffer with a concentration of 50 mM and a pH of 8.5 to a 500 mL Erlenmeyer flask. Add recombinant cells with a dry cell weight concentration of 15 g / L to this buffer. The recombinant cells contain the expressed cytochrome P450 monooxygenase for catalyzing the target reaction; (2) Add the racemate of methyl 2-(2-chlorophenyl)propionate with a final concentration of 50 mM to this system as the reaction substrate, and place it on a thermostatic shaker. React for 24 h under the conditions of 30 °C and 250 rpm. During the reaction process, ensure sufficient oxygen supply and uniform mixing between the reactants and the buffer solution to ensure that the substrate is fully converted into the target product; (3) After the reaction was completed, the reaction solution was taken out and extracted 3 times with ethyl acetate of the same volume as the reaction solution. Each time, after sufficient shaking and mixing, the layers were allowed to separate; the ethyl acetate layer after each extraction was taken and dried with anhydrous sodium sulfate to adsorb the water in the organic phase; after drying, the extract was concentrated under reduced pressure distillation to remove the ethyl acetate solvent; the concentrated target product methyl (R)-2-(2-chlorophenyl)-2-hydroxypropionate was weighed, and the separation yield of this product was 49%; and the purity and composition of the single configuration methyl (R)-2-(2-chlorophenyl)-2-hydroxypropionate prepared by the reaction were verified by liquid chromatography (HPLC) analysis. The results are as Figure 5 shown. The optical purity (enantiomeric excess, ee value) of the target product methyl (R)-2-(2-chlorophenyl)-2-hydroxypropionate was 97.8%, indicating that the product had a high stereoselectivity.

[0040] Example 8 Synthesis of methyl (R)-2-(3-bromophenyl)-2-hydroxypropionate by cytochrome P450 monooxygenase recombinant cells catalyzing methyl 2-(3-bromophenyl)propionate The synthesis of methyl (R)-2-(3-bromophenyl)-2-hydroxypropionate by cytochrome P450 monooxygenase recombinant cells catalyzing methyl 2-(3-bromophenyl)propionate includes the following steps:

[0041] (1) Add 100 mL of phosphate buffer with a concentration of 50 mM and pH 8.5 to a 500 mL Erlenmeyer flask, and add recombinant cells with a dry cell weight concentration of 15 g / L to this buffer. The recombinant cells contain the expressed cytochrome P450 monooxygenase for catalyzing the target reaction. (2) Add the racemate of methyl 2-(3-bromophenyl)propionate with a final concentration of 50 mM as the reaction substrate to this system, and place it on a thermostatic shaker. React at 30 °C and 250 rpm for 24 h. During the reaction, ensure sufficient oxygen supply and uniform mixing between the reactants and the buffer to ensure that the substrate is fully converted into the target product. (3) After the reaction was completed, the reaction solution was taken out and extracted three times with ethyl acetate of the same volume as the reaction solution. Each time, after sufficient shaking and mixing, the layers were allowed to separate by standing; the ethyl acetate layer after each extraction was taken and dried with anhydrous sodium sulfate to adsorb the water in the organic phase; after drying, the extract was concentrated under reduced pressure distillation to remove the ethyl acetate solvent; the concentrated target product methyl (R)-2-(3-bromophenyl)-2-hydroxypropionate was weighed, and the isolated yield of this product was obtained as 48%; and the purity and composition of the single configuration methyl (R)-2-(3-bromophenyl)-2-hydroxypropionate prepared by the reaction were verified by liquid chromatography (HPLC) analysis. The results are as Figure 6 shown. The optical purity (enantiomeric excess, ee value) of the target product methyl (R)-2-(3-bromophenyl)-2-hydroxypropionate was 98.3%, indicating that the product had a high stereoselectivity.

[0042] Example 9 Synthesis of methyl (S)-2-(α-thienyl)-2-hydroxypropionate by recombinant cells of cytochrome P450 monooxygenase catalyzing methyl 2-(α-thienyl)-propionate The synthesis of methyl (S)-2-(α-thienyl)-2-hydroxypropionate by recombinant cells of cytochrome P450 monooxygenase catalyzing methyl 2-(α-thienyl)-propionate includes the following steps:

[0043] (1) Add 100 mL of phosphate buffer with a concentration of 50 mM and a pH of 8.5 to a 500 mL Erlenmeyer flask, and add recombinant cells with a dry cell weight concentration of 15 g / L to this buffer. The recombinant cells contain the expressed cytochrome P450 monooxygenase for catalyzing the target reaction. (2) Add methyl 2-(α-thienyl)-propionate with a final concentration of 50 mM to this system as the reaction substrate, and place it on a constant temperature shaker. React at 30 °C and 250 rpm for 24 h. During the reaction, sufficient oxygen supply and uniform mixing between the reactants and the buffer should be ensured to ensure that the substrate is fully converted into the target product. (3) After the reaction was completed, the reaction solution was taken out and extracted three times with ethyl acetate of the same volume as the reaction solution. Each time, after sufficient shaking and mixing, the layers were allowed to separate by standing; the ethyl acetate layer after each extraction was taken and dried with anhydrous sodium sulfate to adsorb the water in the organic phase; after drying, the extract was concentrated under reduced pressure distillation to remove the ethyl acetate solvent; the concentrated target product methyl (S)-2-(α-thienyl)-2-hydroxypropionate was weighed, and the isolated yield of this product was obtained as 41%; and the purity and composition of the single configuration methyl (S)-2-(α-thienyl)-2-hydroxypropionate prepared by the reaction were verified by liquid chromatography (HPLC) analysis. The results are as Figure 7As shown, the optical purity (enantiomeric excess, ee value) of the target product methyl (S)-2-(α-thienyl)-2-hydroxypropionate is 82.4%, indicating that the product has high stereoselectivity.

[0044] Comparative Example 1 To optimize the amino acid sequence of SEQ ID NO.3 to make it more suitable for expression in Escherichia coli, the nucleotide sequence was optimized according to the codon preference of Escherichia coli, and the optimized nucleotide sequence was artificially synthesized in full gene and ligated to the Escherichia coli expression vector pET 28b to construct the recombinant expression plasmid pET 28b-P450-2. The specific steps were the same as those in Example 1; The cytochrome P450 monooxygenase with the amino acid sequence of SEQ ID NO.3 was obtained, denoted as cytochrome P450 monooxygenase-2, and the amino acid sequence SEQ ID NO.3: And recombinant cells expressing the P450 monooxygenase-2 with the amino acid sequence SEQ ID NO.3.

[0045] Comparative Example 2 The cytochrome P450 monooxygenase-2 recombinant cells of Comparative Example 1 were used to catalyze the synthesis of methyl (R)-2-phenyl-2-hydroxypropionate from methyl 2-phenyl-1-propionate; the difference between the synthesis steps of this comparative example and those of Example 3 was only the cytochrome P450 monooxygenase recombinant cells, and the remaining steps and parameters were the same as those of Example 3.

[0046] The concentrated target product, methyl (R)-2-phenyl-2-hydroxypropionate, was weighed, and the isolated yield of this product was 7%, with a relatively low yield, indicating that the recombinant cells expressing the cytochrome P450 monooxygenase-2 with the amino acid sequence SEQ ID NO.3 are not suitable for catalyzing the C-H bond asymmetric hydroxylation of methyl ester racemates to synthesize enantioselective tertiary alcohol esters.

[0047] Comparative Example 3 The cytochrome P450 monooxygenase-2 recombinant cells of Comparative Example 1 were used to catalyze the synthesis of methyl (R)-2-(2-fluorophenyl)-2-hydroxypropionate from methyl 2-(2-fluorophenyl)propionate; the difference between the synthesis steps of this comparative example and those of Example 4 was only the cytochrome P450 monooxygenase recombinant cells, and the remaining steps and parameters were the same as those of Example 4.

[0048] The concentrated target product, methyl (R)-2-(2-fluorophenyl)-2-hydroxypropionate, was weighed, and the isolated yield of this product was 5%, with a relatively low yield, indicating that the recombinant cells expressing the cytochrome P450 monooxygenase-2 with the amino acid sequence SEQ ID NO.3 are not suitable for catalyzing the C-H bond asymmetric hydroxylation of methyl ester racemates to synthesize enantioselective tertiary alcohol esters.

[0049] Comparative Example 4 The cytochrome P450 monooxygenase-2 recombinant cells of Comparative Example 1 were used to catalyze the synthesis of methyl (R)-2-(3-fluorophenyl)-2-hydroxypropionate from methyl 2-(3-fluorophenyl)propionate; the difference between the synthesis steps of this comparative example and those of Example 5 was only the cytochrome P450 monooxygenase recombinant cells, and the remaining steps and parameters were the same as those of Example 5.

[0050] The concentrated target product, methyl (R)-2-(3-fluorophenyl)-2-hydroxypropionate, was weighed, and the isolated yield of this product was 8%, which was relatively low, indicating that the recombinant cells expressing cytochrome P450 monooxygenase-2 with the amino acid sequence SEQ ID NO.3 were not suitable for catalyzing the asymmetric hydroxylation of the C-H bond of methyl ester racemates to synthesize enantioselective tertiary alcohol esters.

[0051] Comparative Example 5 The recombinant cells of cytochrome P450 monooxygenase-2 in Comparative Example 1 were used to catalyze the synthesis of chiral (R)-2-(4-fluorophenyl)-2-hydroxypropionate from methyl 2-(4-fluorophenyl)propionate; the difference between the synthesis steps of this comparative example and those of Example 6 was only the different recombinant cells of cytochrome P450 monooxygenase, and the remaining steps and parameters were the same as those of Example 6.

[0052] The concentrated target product, methyl (R)-2-(4-fluorophenyl)-2-hydroxypropionate, was weighed, and the isolated yield of this product was 6%, which was relatively low, indicating that the recombinant cells expressing cytochrome P450 monooxygenase-2 with the amino acid sequence SEQ ID NO.3 were not suitable for catalyzing the asymmetric hydroxylation of the C-H bond of methyl ester racemates to synthesize enantioselective tertiary alcohol esters.

[0053] Comparative Example 6 The recombinant cells of cytochrome P450 monooxygenase-2 in Comparative Example 1 were used to catalyze the synthesis of chiral (R)-2-(2-chlorophenyl)-2-hydroxypropionate from methyl 2-(2-chlorophenyl)propionate; the difference between the synthesis steps of this comparative example and those of Example 7 was only the different recombinant cells of cytochrome P450 monooxygenase, and the remaining steps and parameters were the same as those of Example 7.

[0054] The concentrated target product, methyl (R)-2-(2-chlorophenyl)-2-hydroxypropionate, was weighed, and the isolated yield of this product was 3%, which was relatively low, indicating that the recombinant cells expressing cytochrome P450 monooxygenase-2 with the amino acid sequence SEQ ID NO.3 were not suitable for catalyzing the asymmetric hydroxylation of the C-H bond of methyl ester racemates to synthesize enantioselective tertiary alcohol esters.

[0055] Comparative Example 7 The recombinant cells of cytochrome P450 monooxygenase-2 in Comparative Example 1 were used to catalyze the synthesis of chiral (R)-2-(2-bromophenyl)-2-hydroxypropionate from methyl 2-(2-bromophenyl)propionate; the difference between the synthesis steps of this comparative example and those of Example 8 was only the different recombinant cells of cytochrome P450 monooxygenase, and the remaining steps and parameters were the same as those of Example 8.

[0056] The concentrated target product, methyl (R)-2-(2-bromophenyl)-2-hydroxypropionate, was weighed, and the isolated yield of this product was 9%, which was relatively low. This indicates that the recombinant cells expressing cytochrome P450 monooxygenase-2 with the amino acid sequence SEQ ID NO.3 are not suitable for catalyzing the C-H bond asymmetric hydroxylation of methyl ester racemates to synthesize enantioselective tertiary alcohol esters.

[0057] Comparative Example 8 The recombinant cells of cytochrome P450 monooxygenase-2 in Comparative Example 1 were used to catalyze the synthesis of chiral (S)-2-(α-thienyl)-2-hydroxypropionate methyl ester from methyl 2-(α-thienyl)propionate; the difference between the synthesis steps of this comparative example and those of Example 9 was only the different recombinant cells of cytochrome P450 monooxygenase, and the remaining steps and parameters were the same as those of Example 9.

[0058] The concentrated target product, methyl (S)-2-(α-thienyl)-2-hydroxypropionate, was weighed, and the isolated yield of this product was 8%, which was relatively low. This indicates that the recombinant cells expressing cytochrome P450 monooxygenase-2 with the amino acid sequence SEQ ID NO.3 are not suitable for catalyzing the C-H bond asymmetric hydroxylation of methyl ester racemates to synthesize enantioselective tertiary alcohol esters.

[0059] The present invention has been described according to the above embodiments. It should be understood that the above embodiments do not limit the present invention in any form. Any technical solutions obtained by equivalent substitution or equivalent transformation fall within the scope of the present invention.

Claims

1. A cytochrome P450 monooxygenase, characterized in that The amino acid sequence of the cytochrome P450 monooxygenase is shown in SEQ ID NO.

1.

2. Use of the cytochrome P450 monooxygenase according to claim 1 in the synthesis of chiral tertiary alcohol ester compounds.

3. The use according to claim 2, characterized in that The cytochrome P450 monooxygenase synthesizes tertiary alcohol ester compounds with enantioselectivity by catalyzing the asymmetric hydroxylation of the CH bond of the methyl ester racemate; the synthesis route is as follows: ； In the formula, R1 is thiophene, -C6H4Br, -C6H4Cl, -C6H4F, -C6H5, -C6H4CH3, -C6H4OCH3 or -C6H4CF3, and R2 is -H or -CH3.

4. The use according to claim 3, characterized in that The catalysis is carried out at a pH of 7.5 to 8.

5.

5. The use according to claim 3, characterized in that The reaction concentration of the methyl ester racemate is 0.03-0.08 mol / L.

6. The use according to claim 3, characterized in that The cytochrome P450 monooxygenase is added in the form of recombinant cells containing cytochrome P450 monooxygenase.

7. The use according to claim 6, characterized in that The cell dry weight concentration of the recombinant cells containing cytochrome P450 monooxygenase is 10-20 g / L.

8. A recombinant cell expressing the cytochrome P450 monooxygenase according to claim 1.

9. A vector carrying the gene encoding cytochrome P450 monooxygenase according to claim 1.

10. A strain expressing the cytochrome P450 monooxygenase according to claim 1.

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