Method for preparing (R)-styrene oxide based on epoxide hydrolase

Abstract

The invention discloses a method for preparing (R)-styrene oxide based on epoxide hydrolase, which comprises the following steps: carrying out enzyme catalysis reaction in a biphase system under the action of epoxide hydrolase by taking a racemic ethylene oxide compound as a substrate to obtain (R)-styrene oxide and (R)-1-phenyl-1, 2-ethylene glycol or (S)-1-phenyl-1, 2-ethylene glycol; the temperature of the enzyme catalytic reaction is controlled to be 5-50 DEG C, and the reaction time is 2-24 hours. According to the method, the racemic ethylene oxide compound can be efficiently split with high stereoselectivity, the enzyme catalysis effect is good, and the optical purity of the product is high.

Description

[0001] This application is a divisional application of the Chinese application with application number 2024103254427, application date 2024.03.21, and name “Chiral separation method of ethylene oxide compounds based on epoxide hydrolase”. Technical Field

[0002] The invention relates to the technical field of bioengineering, and in particular to a method for preparing (R)-epoxystyrene based on epoxide hydrolase. Background Art

[0003] Epoxide hydrolases (EH, EC 3.3.2.3) are widely present in microorganisms, plants, and animals. They can asymmetrically catalyze the hydrolysis of racemic or meso epoxides, selectively open the ring to generate chiral alcohol compounds and retain the required high-value single-configuration epoxides. For example, (R)-4-chlorophenylethylene glycol, a key synthon of neuroprotective drugs, can be prepared by hydrolyzing the corresponding epoxide by epoxide hydrolase. Similarly, indene ring epoxides can also be hydrolyzed by epoxide hydrolase to produce important intermediates of the anti-AIDS drug Ziv-23: (1R,2S)-epoxides and (1R,2R)-diols.

[0004] As an important organic synthesis intermediate, styrene oxide is widely used in the synthesis of levamisole hydrochloride, β-phenylethanol, ultraviolet absorber and epoxy resin diluent. At present, the chemical synthesis methods of styrene oxide on the market mainly include halogen alcohol method, oxygen epoxidation method, peroxide method, etc. The process of generating styrene oxide by oxygen method and peroxide method is similar. Oxygen epoxidation synthesizes styrene oxide at high temperature with 2-chloro-propanol as solvent and isopropylbenzene hydroperoxide as free radical initiator to obtain styrene oxide. After years of research and development, oxygen oxidation method has been widely used. The method can effectively reduce production costs and has the advantages of high selectivity, but the method is harsh and still faces bottlenecks and constraints in realizing industrial large-scale production. The peroxide method uses relatively expensive mesoporous titanium silicon molecular sieve as catalyst, making it difficult to industrialize large-scale production. In addition, although the halohydrin method has been developed for many years and its technology has become relatively mature and the production process is very simple, this method has high energy consumption and high cost, and the halide oxides used in the oxidation process will cause serious equipment corrosion and environmental pollution, which does not meet the social needs of low emissions and low pollution.

[0005] 1-Phenyl-1,2-ethylene glycol is an important chiral building block for the preparation of optically active medicines, pesticides and functional materials. For example, the R-enantiomer is an important intermediate for the synthesis of (R)-fluoxetine, (R)-norfluoxetine and β-lactam antibiotics. However, the chemical production method of chiral 1-phenyl-1,2-ethylene glycol generally requires the use of expensive chiral catalysts and the use of highly toxic osmium oxide, which has high production costs and severe environmental pollution, making it unfavorable for industrial production.

[0006] Although the chemical production method of styrene oxide is relatively mature, there are still many shortcomings. For example, in the process of synthesizing styrene oxide from epoxidation reaction, the target product selectivity is low, and the environmental pollution caused by a large number of by-products has been difficult to solve. Therefore, the green production of styrene oxide industry has attracted much attention. Epoxide hydrolase can hydrolyze racemic epoxides and retain the required configuration, and some of the generated alcohol compounds often have high added value, showing extremely high industrial application value. However, styrene oxide is highly toxic and will cause the enzyme to inactivate quickly. The catalytic effect of epoxide hydrolases found so far is poor, and there are generally problems such as low substrate concentration, poor enzyme catalytic effect, and low product optical purity, which limits its industrial application. At present, whether it is the preparation of (R)-styrene oxide, (S)-styrene oxide, or (R)-1-phenyl-1,2-ethylene glycol and (S)-1-phenyl-1,2-ethylene glycol, there is a lack of suitable epoxide hydrolases. Summary of the invention

[0007] The object of the present invention is to provide a method for preparing (R)-epoxystyrene based on epoxide hydrolase, which can efficiently and stereoselectively resolve racemic ethylene oxide compounds, has good enzyme catalytic effect, and has high product optical purity.

[0008] The technical solution adopted by the present invention to solve its technical problem is: A method for preparing (R)-epoxystyrene based on epoxide hydrolase, wherein racemic ethylene oxide compounds are used as substrates, and an enzyme-catalyzed reaction is carried out in a two-phase system under the action of epoxide hydrolase to obtain (R)-epoxystyrene and (R)-1-phenyl-1,2-ethylene glycol or (S)-1-phenyl-1,2-ethylene glycol; The temperature of the enzyme catalytic reaction is controlled at 5-50°C and the reaction time is 2-24 hours; The biphasic system is composed of 60%-80% aqueous phase and 20%-40% organic phase by volume fraction; the aqueous phase is a phosphate buffer with a pH of 6-10, and the organic phase is selected from one of isooctane, n-heptane and petroleum ether. The concentration of the phosphate buffer is preferably 200mM.

[0009] In the reaction system: the amount of substrate is 5-50g / L, and the epoxide hydrolase is added in the form of wet bacteria of genetically engineered bacteria producing epoxide hydrolase, and the amount of wet bacteria of genetically engineered bacteria producing epoxide hydrolase is 20-150g / L. Further, pure enzyme is directly added: in the reaction system: the amount of substrate is 5-50g / L, and the amount of epoxide hydrolase is 6-45g / L.

[0010] The epoxide hydrolase of the present invention can be a culture of the above-mentioned epoxide hydrolase genetically engineered recombinant bacteria, or can be bacterial cells obtained by centrifuging the culture medium or a processed product thereof. The processed product refers to an extract obtained from the bacterial cell, a crushed liquid, or an isolated product obtained by separating and / or purifying the nitrilase in the extract, or an immobilized product obtained by immobilizing the extract or the processed product.

[0011] The racemic oxirane compound is selected from racemic oxirane, racemic 2-(p-phenylmethyl)oxirane) Racemic 2-(p-chlorophenyl)oxirane Racemic benzyl glycidyl ether Racemic p-methyl-benzyl glycidyl ether One of them.

[0012] The epoxide hydrolase is selected from one of the following: EHs-3 derived from wild soybean Glycine soja, the encoding nucleotide sequence is shown in SEQ ID NO:5, and the amino acid sequence is shown in SEQ ID NO:6; EHs-5 derived from Aspergillus niger, the encoding nucleotide sequence is shown in SEQ ID NO:9, and the amino acid sequence is shown in SEQ ID NO:10; EHs-6 derived from Aspergillus usamii, the encoding nucleotide sequence is shown in SEQ ID NO:11, and the amino acid sequence is shown in SEQ ID NO:12; EHs-9 derived from Phaseolus vulgaris, the encoding nucleotide sequence is shown in SEQ ID NO:17, and the amino acid sequence is shown in SEQ ID NO:18; EHs-10 derived from Agromyces mediolanus, the encoding nucleotide sequence is shown in SEQ ID NO:19, and the amino acid sequence is shown in SEQ ID NO:20.

[0013] The genetic engineering bacteria producing epoxide hydrolase is obtained by inserting the coding nucleotide sequence of epoxide hydrolase into a plasmid vector to form a recombinant vector, and then transforming the recombinant vector into a host cell.

[0014] The plasmid vector refers to a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, an animal cell virus, a retrovirus or other vector well known in the art. The vector used in the present invention includes but is not limited to: the vector pET-28a expressed in Escherichia coli. As long as it can stably replicate and exist in the host cell, any vector can construct a recombinant expression vector. Preferably, the recombinant vector is a recombinant plasmid pET-28a-EHs obtained by inserting the epoxide hydrolase DNA fragment into the multiple cloning site of the pET-28a vector. Optionally, the cloning site is an NdeI restriction site and a SalI restriction site.

[0015] Host cells include prokaryotic cells, such as archaeal cells, bacterial cells, or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells, preferably recombinant engineered bacteria constructed by E. coli BL21.

[0016] As a preferred embodiment, the present invention uses racemic styrene oxide as a substrate and carries out an enzyme-catalyzed reaction in a two-phase system under the action of epoxide hydrolase to obtain (R)-styrene oxide and (R)-1-phenyl-1,2-ethylene glycol or (S)-1-phenyl-1,2-ethylene glycol.

[0017] Epoxide hydrolase can stereoselectively recognize and hydrolyze racemic epoxide to produce chiral 1-phenyl-1,2-ethanediol, while (R)- or (S)-epoxide is retained.

[0018] The ee value of the product (R)- or (S)-epoxystyrene is greater than 99%, which meets the requirement; the ee value of the product (R)- or (S)-1-phenyl-1,2-ethylene glycol is greater than 98%, which meets the requirement.

[0019] The epoxystyrene and 1-phenyl-1,2-ethylene glycol of the present invention can both produce R-type and S-type monomers, while CN105969837A can only produce S-epoxystyrene, and the number of enzymes used is single, which is too restrictive. The present invention utilizes multiple epoxide hydrolases from different sources for production, which has fewer restrictions.

[0020] The present invention selects epoxide hydrolases from different sources, respectively performs codon optimization and then fully synthesizes the corresponding sequences, adds corresponding restriction sites at both ends of the gene, constructs the synthesized genes into corresponding expression vectors, thereby constructing a series of expression vectors, transforming host cells to form recombinant engineering bacteria, and then fermenting and culturing them, and using them for the preparation of (R)- or (S)-epoxystyrene and (R)- or (S)-1-phenyl-1,2-ethylene glycol. The reaction equation is as follows: epoxide hydrolase hydrolyzes the S-type monomer in racemic epoxystyrene, and (R)-epoxystyrene is retained, while the epoxide hydrolase attacks the α and β carbon atoms in the ternary ring to generate 1-phenyl-1,2-ethylene glycol with different chirality; conversely, epoxide hydrolase hydrolyzes the R-type monomer in racemic epoxystyrene, and (S)-epoxystyrene is retained, and generates chiral 1-phenyl-1,2-ethylene glycol, and the reaction formula is as follows:

[0021] The beneficial effects of the present invention are: being able to efficiently and stereoselectively separate racemic ethylene oxide compounds, having good enzyme catalysis effect, having high product optical purity, having mild reaction conditions, having simple steps, using reagents with low toxicity, having low environmental pollution, and being suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is 1-phenyl-1,2-ethanediol H NMR spectrum Figure 2 This is the hydrogen NMR spectrum of styrene oxide. DETAILED DESCRIPTION

[0023] The technical solution of the present invention is further described in detail below through specific embodiments.

[0024] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the following embodiments, unless otherwise specified, are all conventional methods in the art.

[0025] Example 1 Construction of recombinant expression vector The synthesis of the whole gene was completed by Beijing Qingke Biotechnology Co., Ltd., and the genes of epoxide hydrolases EHs-1, EHs-2, EHs-3, EHs-4, EHs-5, EHs-6, EHs-7, EHs-8, EHs-9 and EHs-10 were codon optimized, and corresponding restriction sites were added at both ends of the genes and constructed into the corresponding vectors to obtain recombinant plasmid vectors of epoxide hydrolases. The obtained recombinant plasmid vectors were transferred into hosts such as Escherichia coli BL21 by conventional methods to obtain recombinant genetically engineered bacteria E. coli BL21 (DE3) / pET28a-EHs-1, E. coli BL21 (DE3) / pET28a-EHs-2, E. coli BL21 (DE3) / pET28a-EHs-3, E. coli BL21 (DE3) / pET28a-EHs-4, E. coli BL21(DE3) / pET28a-EHs-5, E. coli BL21(DE3) / pET28a-EHs-6, E. coli BL21(DE3) / pET28a-EHs-7, E. coli BL21(DE3) / pET28a-EHs-8, E. coli BL21(DE3) / pET28a-EHs-9 and E. coli BL21(DE3) / pET28a-EHs-10. The sources and amino acid sequences of the epoxide hydrolase of the present invention are shown in Table 1.

[0026] Epoxystyrene is highly toxic and can rapidly inactivate the enzyme, resulting in poor catalytic effect of epoxide hydrolase. The present invention solves the toxicity problem of epoxystyrene to the enzyme by optimizing the organic phase, and isooctane is most preferred.

[0027] Table 1 Sources of epoxide hydrolases

[0028] Example 2 Cultivation and induced expression of recombinant genetically engineered bacteria The 10 recombinant genetically engineered bacteria obtained in Example 1 were inoculated into LB liquid medium containing 50 μg / mL kanamycin, cultured at 37°C overnight, and then inoculated into LB liquid medium containing 50 μg / mL kanamycin at a 2% inoculum (v / v), and cultured at 37°C, 150 rpm until the bacterial concentration OD 600 = about 0.6, add IPTG with a final concentration of 0.1mM (addition of IPTG allows the expression of the target protein), induce culture at 28°C for 12h, collect the bacteria by centrifugation at 4°C and 12000rpm for 10min, wash the wet bacteria with 0.85% saline, and store at -20°C for later use (i.e., resting cells, used for hydrolysis reaction).

[0029] Example 3: Hydrolysis of racemic styrene oxide using recombinant E. coli containing epoxide hydrolases EHs-1, EHs-2, EHs-3, EHs-4, EHs-5, EHs-6, EHs-7, EHs-8, EHs-9, and EHs-10. Using the recombinant E. coli containing epoxide hydrolases EHs-1, EHs-2, EHs-3, EHs-4, EHs-5, EHs-6, EHs-7, EHs-8, EHs-9, and EHs-10 prepared in Example 2 as catalysts, hydrolysis reactions of racemic styrene oxide were carried out respectively. The reaction system is as follows: 14 mL of phosphate buffer (200 mM, pH 8.0), 6 mL of isooctane (epoxystyrene is highly toxic, in order to better carry out the catalytic reaction), 0.4 g of racemic epoxystyrene, 1.0 g of recombinant E. coli wet cells of EHs-1, EHs-2, EHs-3, EHs-4, EHs-5, EHs-6, EHs-7, EHs-8, EHs-9, and EHs-10. The catalytic reaction was reacted at 35 ° C for 12 hours, 1 mL of the sample was added with 10 μL of 6M HCl to terminate the reaction, and the layers were separated by centrifugation at 12000 rpm for 1 minute. The upper organic phase was dried with anhydrous sodium sulfate, and the ee value of epoxystyrene was determined by liquid chromatography. The lower aqueous phase was extracted with dichloromethane, dried with anhydrous sodium sulfate, and the ee value of 1-phenyl-1,2-ethylene glycol was determined by liquid chromatography. The specific results are shown in Table 2. The above results show that the (R)-epoxystyrene produced by EHs-2, EHs-3, EHs-5, EHs-6, EHs-9, and EHs-10 have high ee values, and the (S)-epoxystyrene produced by EHs-8 has high ee values, which can be used for the production of chiral epoxystyrene. In addition, the (R)-1-phenyl-1,2-ethylene glycol produced by EHs-2 and EHs-9 has high ee values, which can be used for the preparation of (R)-1-phenyl-1,2-ethylene glycol, and the (S)-1-phenyl-1,2-ethylene glycol produced by EHs-10 has high ee values, which can be used for the preparation of (S)-1-phenyl-1,2-ethylene glycol. Product verification: 1-phenyl-1,2-ethylene glycol H NMR spectrum, epoxystyrene H NMR spectrum, see Figure 1-2 .

[0030] Table 2 Data of racemic styrene oxide catalyzed by recombinant E. coli EHs-1, EHs-2, EHs-3, EHs-4, EHs-5, EHs-6, EHs-7, EHs-8, EHs-9, and EHs-10 Enzymes EE value of styrene oxide ee value of 1-phenyl-1,2-ethanediol EHS-1 48.9％(S) 6.7％(R) EHS-2 99.2％(R) 96.1％(R) EHS-3 98.4％(R) 81.5％(R) EHS-4 42.1％(R) 71.6％(R) EHS-5 99.4％(R) 15.8％(R) EHS-6 90.8％(R) 36.3％(R) EHS-7 71.9％(R) 75.7％(R) EHS-8 85.3％(S) 26.2％(S) EHS-9 92.7％(R) 87.1％(R) EHS-10 93.6％(R) 95.7％(S) .

[0031] Example 4 Preparation of (R)-epoxystyrene using recombinant E. coli EHs-2, EHs-3, EHs-5, EHs-6, EHs-9, EHs-10 containing epoxide hydrolase The recombinant E. coli containing epoxide hydrolase EHs-2, EHs-3, EHs-5, EHs-6, EHs-9, EHs-10 prepared in Example 2 were used as catalysts to prepare (R)-epoxystyrene. The reaction system was as follows: 700 mL of phosphate buffer (200 mM, pH 8.0), 300 mL of isooctane, 20 g of racemic epoxidestyrene, and 60 g of wet cells of recombinant E. coli EHs-2, EHs-3, EHs-5, EHs-6, EHs-9, EHs-10. The catalytic reaction was carried out at 35°C for 12-14h, and the reaction was stopped when (S)-epoxystyrene was completely hydrolyzed. The reaction was separated by centrifugation at 12000rpm for 10min, and the upper isooctane was taken out for rotary evaporation to obtain (R)-epoxystyrene. Petroleum ether was added to the aqueous phase to extract (R)-epoxystyrene. After extraction three times, the organic phase was rotary evaporated to obtain (R)-epoxystyrene with an ee value greater than 99%. The (R)-epoxystyrene prepared by the reaction of EHs-2, EHs-3, EHs-5, EHs-6, EHs-9, and EHs-10 were 8.32g, 6.14g, 2.26g, 3.67g, 7.36g, and 7.78g, respectively, and the product yields were 83.2%, 61.4%, 22.6%, 36.7%, 73.6%, and 77.8%, respectively.

[0032] Example 5 Preparation of (S)-epoxystyrene using recombinant Escherichia coli EHs-8 containing epoxide hydrolase The recombinant Escherichia coli containing epoxide hydrolase EHs-8 prepared in Example 2 was used as a catalyst to prepare (S)-epoxystyrene. The reaction system is as follows: 700 mL of phosphate buffer (200 mM, pH 8.0), 300 mL of isooctane, 20 g of racemic epoxystyrene, and 60 g of recombinant Escherichia coli wet cells. The catalytic reaction was reacted at 35 ° C for 12-14 h, and the reaction was stopped when (R)-epoxystyrene was completely hydrolyzed. The reaction was centrifuged at 12000 rpm for 10 min to separate the layers, and the upper isooctane was taken out for rotary evaporation. Petroleum ether was added to the aqueous phase to extract (S)-epoxystyrene. After extraction three times, the organic phase was rotary evaporated to obtain (S)-epoxystyrene with an ee value greater than 99%, a total of 3.15 g, and the product yield was 31.5%.

[0033] Example 6 Preparation of (R)-1-phenyl-1,2-ethylene glycol using recombinant E. coli EHs-2 and EHs-9 containing epoxide hydrolase The recombinant E. coli containing epoxide hydrolase EHs-2 and EHs-9 prepared in Example 2 was used as a catalyst to prepare (R)-1-phenyl-1,2-ethylene glycol. The reaction system is as follows: 700 mL of phosphate buffer (200 mM, pH 8.0), 300 mL of isooctane, 20 g of racemic styrene oxide, and 60 g of wet cells of recombinant E. coli EHs-2 and EHs-9. The catalytic reaction was reacted at 35 ° C for 10-12 h, centrifuged at 12000 rpm for 10 min, and the lower aqueous phase was extracted three times with petroleum ether, and then extracted three times with an equal volume of dichloromethane, followed by rotary evaporation of the organic phase to obtain the crude product of (R)-1-phenyl-1,2-ethylene glycol. The crude product was dissolved in methyl tert-butyl ether to saturation, and petroleum ether was slowly added dropwise until (R)-1-phenyl-1,2-ethylene glycol was precipitated, thereby obtaining (R)-1-phenyl-1,2-ethylene glycol with an ee value greater than 98%. 8.08 g and 5.54 g of EHs-2 and EHs-9 were harvested, and the product yields were 70.1% and 48.1%, respectively.

[0034] Example 7 Preparation of (S)-1-phenyl-1,2-ethylene glycol using recombinant Escherichia coli EHs-10 containing epoxide hydrolase The recombinant Escherichia coli containing epoxide hydrolase EHs-10 prepared in Example 2 was used as a catalyst to prepare (S)-1-phenyl-1,2-ethylene glycol. The reaction system is as follows: 700 mL of phosphate buffer (200 mM, pH 8.0), 300 mL of isooctane, 20 g of racemic ethylene oxide, and 60 g of recombinant Escherichia coli wet cells. The catalytic reaction was reacted at 35 ° C for 10-12 h, centrifuged at 12000 rpm for 10 min, and the lower aqueous phase was extracted three times with petroleum ether, and then extracted three times with an equal volume of dichloromethane, followed by rotary evaporation of the organic phase to obtain the crude product of (S)-1-phenyl-1,2-ethylene glycol. The crude product was dissolved in methyl tert-butyl ether to saturation, and petroleum ether was slowly added dropwise until (S)-1-phenyl-1,2-ethylene glycol was precipitated, thereby obtaining (S)-1-phenyl-1,2-ethylene glycol with an ee value greater than 98%, a total of 7.73 g, and a yield of 67.1%.

[0035] Example 8 Optimization of the biphasic reaction system of recombinant Escherichia coli EHs-2 containing epoxide hydrolase Since EHs-2 has a good effect in the preparation of epoxide styrene and 1-phenyl-1,2-ethylene glycol, this example uses the recombinant Escherichia coli containing epoxide hydrolase EHs-2 prepared in Example 2 as a catalyst to optimize the biphasic reaction system. The reaction system contains phosphate buffer (200mM, pH 8.0) and an organic phase totaling 1L, and the total organic phase types are isooctane, n-heptane, petroleum ether, methyl tert-butyl ether, ethyl acetate, etc., and 20g of racemic epoxide styrene and 60g of recombinant Escherichia coli wet cells are also added to the reaction. The catalytic reaction is reacted at 35°C for 5h, centrifuged at 12000rpm for 1min, and the upper organic phase is dried with anhydrous sodium sulfate, and the ee value of epoxide styrene is determined by liquid chromatography. The results are shown in Table 3. In the two-phase system, when the isooctane ratio is 10-50%, the catalytic activity of epoxide hydrolase EHs-2 varies greatly. After 5 hours of reaction, the catalytic reaction conversion rate corresponding to 10% isooctane is only 10.4%. With the increase of the isooctane ratio, the catalytic effect of epoxide hydrolase EHs-2 is significantly improved. The catalytic reaction effects corresponding to 30% isooctane and 40% isooctane are good. When the isooctane ratio is increased to 50%, the catalytic effect decreases. For cost considerations, 30% isooctane is selected as the reaction condition of the two-phase system. In addition, at the same 30% organic phase ratio, epoxide hydrolase EHs-2 shows a catalytic effect close to that of isooctane in n-heptane and petroleum ether. Therefore, 30% isooctane, petroleum ether, and n-heptane can be selected as the two-phase system conditions for epoxide hydrolase EHs-2 to prepare ethylene oxide and 1-phenyl-1,2-ethylene glycol, preferably 30% isooctane.

[0036] Table 3 Reaction results of different two-phase reaction systems Reaction organic phase and ratio EE value of styrene oxide (%) Conversion rate (%) 10% Isooctane 11.6(R) 10.4 20% Isooctane 67.8(R) 40.4 30% Isooctane 92.4(R) 48.0 40% Isooctane 94.1(R) 48.5 50% Isooctane 86.4(R) 46.3 30% n-heptane 93.8(R) 48.4 30% Ethyl acetate / / 30% Petroleum ether 96.6(R) 49.1 30% methyl tert-butyl ether 2.7(R) 2.6 .

[0037] Example 9 Optimization of the concentration of recombinant Escherichia coli EHs-2 containing epoxide hydrolase and substrate concentration The recombinant Escherichia coli containing epoxide hydrolase EHs-2 prepared in Example 2 was used as a catalyst to optimize the substrate and cell concentration in the reaction system. The reaction system is as follows: 700mL of phosphate buffer (200mM, pH 8.0), 300mL of isooctane, 10-50g of racemic styrene oxide, and 20-150g of recombinant Escherichia coli wet cells. The catalytic reaction was reacted at 35°C for 10-12h, centrifuged at 12000rpm for 1min, and the upper organic phase was dried with anhydrous sodium sulfate, and the ee value of styrene oxide was determined by liquid chromatography. The specific results are shown in Table 4. When the substrate concentration is 10g / L, only 20g / L of bacteria is needed for catalysis for about 12h. However, as the substrate concentration increases, the amount of bacteria concentration increases significantly. When the substrate concentration is 20g / L, 50g / L of bacteria is needed to completely split the racemate. When the substrate concentration is 40g / L, 150g / L of bacteria is needed to completely split the racemate. When the substrate concentration is 50g / L, 150g / L of bacteria also fails to completely split the racemate. The main reason may be that styrene oxide itself has strong toxicity. As the substrate concentration increases, the epoxide hydrolase is rapidly inactivated. Therefore, 20g / L substrate concentration and 50g / L bacteria concentration are selected as the reaction conditions for the preparation of subsequent products.

[0038] Table 4 Reaction results of different substrate concentrations and bacterial concentrations Substrate concentration (g / L) Bacteria concentration (g / L) EE value of styrene oxide (%) 5 20 99.6％ 10 20 99.5 20 30 76.5 20 40 91.8 20 50 99.6 40 100 66.2 40 150 99.2 50 100 40.1 50 150 92.3 .

[0039] Example 10 Using the recombinant E. coli EHs-2 containing epoxide hydrolase to carry out a 35L large reaction to prepare (R)-epoxystyrene and (R)-1-phenyl-1,2-ethylene glycol. The recombinant E. coli containing epoxide hydrolase EHs-2 prepared in Example 2 was used as a catalyst to carry out a 35L large reaction to prepare (R)-epoxystyrene and (R)-1-phenyl-1,2-ethylene glycol. The reaction system is as follows: 24.5L of phosphate buffer (200mM, pH 8.0), 10.5L of isooctane, 700g of racemic epoxidestyrene, and 1750g of wet cells of EHs-2 recombinant E. coli. The above reaction was carried out in 3 batches. The catalytic reaction was carried out at 35°C for 10-12h. The reaction was stopped when (S)-epoxystyrene was completely hydrolyzed. The mixture was centrifuged at 12000rpm for 10min to separate the layers. The upper isooctane was taken out and rotary evaporated to obtain (R)-epoxystyrene. Petroleum ether was added to the aqueous phase to extract (R)-epoxystyrene. After three extractions, the organic phase was rotary evaporated to obtain the remaining (R)-epoxystyrene. The lower aqueous phase was extracted three times with petroleum ether, and then extracted three times with an equal volume of dichloromethane. The organic phase was then rotary evaporated to obtain the crude product of (R)-1-phenyl-1,2-ethylene glycol. The crude product was dissolved in methyl tert-butyl ether to saturation, and petroleum ether was slowly added dropwise until (R)-1-phenyl-1,2-ethylene glycol was precipitated to obtain (R)-1-phenyl-1,2-ethylene glycol with an ee value greater than 98%. The three batches of large reactions yielded 287.3g, 295.1g and 297.8g of (R)-epoxystyrene, respectively, with ee values ​​and purity both greater than 99%, and yields of 82.1%, 84.3% and 85.1%, respectively; the (R)-1-phenyl-1,2-ethylene glycol fine product (ee value and purity both greater than 98%) yielded 291.2g, 284.7g and 288.4g, respectively, with yields of 72.4%, 70.7% and 71.6%, respectively.

[0040] Example 11 Preparation of a series of epoxides using recombinant Escherichia coli containing epoxide hydrolase The recombinant Escherichia coli containing epoxide hydrolase EHs-2, EHs-8 and EHs-10 prepared in Example 2 were used as catalysts to hydrolyze a series of racemic epoxides. The reaction system was as follows: 14 mL of phosphate buffer (200 mM, pH 8.0), 6 mL of isooctane, 0.4 g of racemic styrene oxide, and 2.0 g of recombinant Escherichia coli wet cells. The catalytic reaction was reacted at 35 ° C for 12 h, 1 mL of the sample was added to 10 μL of 6M HCl to terminate the reaction, and the ee values ​​of the substrate and product were determined. The catalytic substrates and specific results described in this example are shown in Table 5. The results show that EHs-2, EHs-8 and EHs-10 have good catalytic effects and stereoselectivity for styrene oxide-like compounds, and the stereoselectivity of other epoxides is general.

[0041] Table 5 Hydrolysis of various racemic epoxides catalyzed by epoxide hydrolases EHs-2, EHs-8 and EHs-10 recombinant Escherichia coli Note: Inactive or undetectable are indicated by / .

[0042] Sequence information: SEQ ID NO:5 SEQ ID NO:6 SEQ ID NO:9 SEQ ID NO:10 SEQ ID NO:11 SEQ ID NO:12 SEQ ID NO:17 SEQ ID NO:18 SEQ ID NO:19 SEQ ID NO:20

[0043] The above-described embodiment is only a preferred solution of the present invention and does not limit the present invention in any form. There are other variations and modifications without exceeding the technical solution described in the claims.

Claims

1. A method for preparing (R)-epoxystyrene based on epoxide hydrolase, characterized in that: Using racemic oxirane compounds as substrates, an enzyme-catalyzed reaction is carried out in a two-phase system under the action of oxirane hydrolase to obtain (R)-oxirane and (R)-1-phenyl-1,2-ethylene glycol or (S)-1-phenyl-1,2-ethylene glycol; The temperature of the enzyme catalytic reaction is controlled at 5-50°C and the reaction time is 2-24 hours; The two-phase system is composed of 60%-80% aqueous phase and 20%-40% organic phase by volume fraction; the aqueous phase is a phosphate buffer with a pH of 6-10, and the organic phase is selected from one of isooctane, normal heptane and petroleum ether.

2. The method according to claim 1, characterized in that In the reaction system: the dosage of substrate is 5-50 g / L, epoxide hydrolase is added in the form of wet bacterial cells of genetically engineered bacteria producing epoxide hydrolase, and the dosage of wet bacterial cells of genetically engineered bacteria producing epoxide hydrolase is 20-150 g / L.

3. The method according to claim 1, characterized in that The racemic ethylene oxide compound is selected from the group consisting of racemic styrene oxide, racemic 2-(p-phenylmethyl)ethylene oxide, racemic 2-(p-chlorophenyl)ethylene oxide, racemic benzyl glycidyl ether, and racemic p-methyl-benzyl glycidyl ether.

4. The method according to claim 1, characterized in that: The epoxide hydrolase is selected from one of the following: EHs-3 from wild soybean Glycine soja, the encoding nucleotide sequence is shown in SEQ ID NO:5, and the amino acid sequence is shown in SEQ ID NO:6; EHs-5 derived from Aspergillus niger, the encoding nucleotide sequence is shown in SEQ ID NO:9, and the amino acid sequence is shown in SEQ ID NO:10; EHs-6 derived from Aspergillus usamii, the encoding nucleotide sequence is shown in SEQ ID NO: 11, and the amino acid sequence is shown in SEQ ID NO: 12; EHs-9 from Phaseolus vulgaris, the encoding nucleotide sequence is shown in SEQ ID NO: 17, and the amino acid sequence is shown in SEQ ID NO: 18; EHs-10 is derived from Agromyces mediolanus, the encoding nucleotide sequence is shown in SEQ ID NO: 19, and the amino acid sequence is shown in SEQ ID NO:

20.

5. The method according to any one of claims 1 to 4, characterized in that: The genetic engineering bacteria producing epoxide hydrolase is obtained by inserting the coding nucleotide sequence of epoxide hydrolase into a plasmid vector to form a recombinant vector, and then transforming the recombinant vector into a host cell.

6. The method according to claim 1 or 2, characterized in that: With racemic styrene oxide as a substrate and under the action of epoxide hydrolase, an enzyme-catalyzed reaction is carried out in a two-phase system to obtain (R)-styrene oxide and (R)-1-phenyl-1,2-ethylene glycol or (S)-1-phenyl-1,2-ethylene glycol.

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