Styrene monooxygenase mutant and use thereof

Abstract

Provided are a styrene monooxygenase mutant and the use thereof. The styrene monooxygenase mutant comprises: (a) a protein having an amino acid sequence as shown in SEQ ID NO: 1; (b) a protein having an amino acid mutation on at least one of the following sites in the above amino acid sequence of (a): I206, S107, P54, N307, S178, L310, L275, L204, W86, E202, H392 or E221, and having the function of styrene monooxygenase; or (c) a protein having 80% or more homology to the amino acid sequence defined in either (a) or (b) and having the function of styrene monooxygenase. The styrene monooxygenase mutant can solve the problem of low activity of styrene monooxygenase in the prior art, has good properties such as high activity and / or wide range of substrate selection, has high economic value and broad application prospects, and is applicable in the field of enzymatic catalysis.

Description

Styrene monooxygenase mutants and uses thereof

[0001] Cross-reference to Related Applications

[0002] This application claims priority to Chinese Patent Application No. 202410602313.8, filed May 15, 2024, the disclosure of which is incorporated herein in its entirety by this reference as part of the present application. TECHNICAL FIELD

[0003] The present application relates to the field of enzyme catalysis, in particular, to a styrene monooxygenase mutant and uses thereof. BACKGROUND

[0004] The large steric hindrance chiral epoxide (a kind of compound containing benzene ring and / or three-membered ether structure, wherein the steric hindrance of the group on the benzene ring or on either side of the three-membered ether structure is greater than that of methyl) is a very important chiral synthesis building block in organic synthesis, and has important application value in the pharmaceutical, pesticide, perfume and fine chemical industries. For the epoxidation of olefin substrate, a noble metal catalyst and a crystalline titanium silicate catalyst with MFI structure are used as a double catalyst for preparation, but the chemical catalytic reaction conditions are harsh, the selectivity is low, there are many by-products, and the heavy metal catalyst used has high cost and is not environmentally friendly. At present, there are few reports on the synthesis of large steric hindrance chiral epoxide, and satisfactory results cannot be achieved. (Jaan Pesti, Chien-Kuang Chen, Lori Spangler, Albert J. DelMonte, Serge Benoit, Derek Berglund, Jeffrey Bien, Paul Brodfuehrer, Yeung Chan, Elisabeth Corbett, Carrie Costello, Paul DeMena, Robert P. Discordia, Wendel Doubleday, Zhinong Gao, Stephane Gingras, John Grosso, Oscar Haas, David Kacsur, Chiajen Lai, Simon Leung, Melanie Miller, Jale Muslehiddinoglu, Nina Nguyen, Jun Qiu, Martina Olzog, Emily Reiff, Dominique Thoraval, Michael Totleben, Dale Vanyo, Purushotham Vemishetti, John Wasylak, and Chenkou Wei. The Process Development of Ravuconazole: An Efficient Multikilogram Scale Preparation of an Antifungal Agent. Organic Process Research&Development. 2009, 13(4): 716-728) discloses a synthesis method of a large steric hindrance chiral epoxide intermediate, but the route is long, the active group needs to be protected and deprotected, the reaction conditions are harsh, the selectivity is poor and the process is complex.

[0005] The catalysis of synthesizing bulky chiral epoxides by styrene monooxygenase provides an economical, green and efficient synthesis idea. However, the catalysis of natural styrene monooxygenase on the substrate of bulky olefin is limited, and there is no styrene monooxygenase that can efficiently synthesize bulky chiral epoxides. Therefore, seeking a styrene monooxygenase with high activity and / or large substrate selection range is the key to the efficient synthesis of bulky chiral epoxides, and is also a technical problem to be solved urgently.

[0006] SUMMARY

[0007] The main purpose of the present application is to provide a styrene monooxygenase mutant and its application, so as to solve the problem of low activity of the styrene monooxygenase in the prior art.

[0008] In order to achieve the above-mentioned purpose, according to one aspect of the present application, a styrene monooxygenase mutant is provided, which includes (a) a protein having the amino acid sequence shown in SEQ ID NO: 1; (b) a protein having the amino acid sequence in (a) at least one of the following positions: I206, S107, P54, N307, S178, L310, L275, L204, W86, E202, H392, E221, R53, V83, A101, A102, R157, F180, I194, L218, A232, G273, Q277, Q313, V388 or A52, which is mutated by amino acid and has the function of styrene monooxygenase; or (c) a protein having more than 80% homology with the amino acid sequence defined in any one of (a) or (b) and having the function of styrene monooxygenase.

[0009] Further, the amino acid mutation of (b) is selected from at least one of the following positions: A52N or A52Q or A52F or A52H or A52P or A5S, R53N or R53G or R53H, P54L or P54S or P54F or P54K, V83S or V83A or V83G or V83F or V83P, W86F or W86Q or W86A or W86S or W86K or W86G or W86H or W86Y or W86M, A101 S or A101 R or A101 G or A101 D or A101 F or A101 M or A101 K, A102P or A102M or A102F, S107A or S107E or S107Y or S107K or S107G or S107V, R157F or R157Y or R157Q or R157W or R157N or R157C or R157P or R157D, S178F or S178N or S178W or S178C or S178I, F180Y or F180I or F180S or F180E, I194F or I194H or I194G or I194D or I194W, E202S or E202A or E202Q, L204C or L204A or L204S or L204F, I206V or I206A or I206L or I206S or I206Q or I206P or I206H, L218I or L218Q or L218S or L218G or L218W or L218D or L218A or L218H or L218E, E221N or E221A or E221F or E221C or E221G or E221I, A232Y or A232G or A232S or A232Q or A232V or A232C or A232M, G273S or G273A or G273H or G273F, L275F or L275A or L275T or L275Y or L275H or L275G or L275E or L275Q or L275M or L275S, Q277S or Q277G or Q277P or Q277F, N307C or N307D or N307W or N307Y, L310M or L310A or L310F or L310C or L310S or L310I or L310H or L310R or L310Q or L310V, Q313M or Q313A or Q313P or Q313K or Q313S, V388A or V388F or H392N or H392K or H392A or H392S or H392C or H392V; wherein the letter before the number represents the original amino acid and the letter after the number represents the mutated amino acid; preferably, the protein in (c) has 85% or more, preferably 90% or more, more preferably 95% or more, further preferably 99% or more homology to the amino acid sequence defined in (a) or (b) and has a styrene monooxygenase function.

[0010] Further, the mutation of the styrene monooxygenase mutant comprises any one of the following amino acid mutations: I206V, I206A, I206L, I206S, I206Q, I206P, I206H, S107A, S107E, S107Y, S107K, S107G, S107V, R157Q, R157C, R157P, R157D, R157Q, I194F, I194H, I194G, I194D, I194W, P54L, P54S, P54F, P54K, R157Q, R157Y, R157W, R157F, R157N, N307C, N307W, N307Y, N307D, L275F, L275A, L275T, S178F, S178N, S178W, R53N, R53G, R53H, F180Y, F180E, F180S, A52N, A52Q, A52F, A52H, A52P, G273S, G273A, G273H, G273F, I206V+S107A, I206V+S107E, I206V+S107K, I206V+R157Q, I206V+R157Y, I206V+R157W, I206V+R157F, I206V+I194F, I206V+I194D, I206V+I194W, I206V+P54L, I206V+P54S, I206V+P54F, I206V+R157Q, I206V+R157Y, I206V+R157F, I206V+N307C, I206V+N307W, I206V+N307Y, I206V+N307D, I206V+L275F, I206V+L275A, I206V+L275T, I206V+S178F, I206V+S178N, I206V+S178W, I206V+R53N, I206V+R53G, I206V+R53H, I206V+F180Y, I206V+F180E, I206V+F180S, I206V+A52N, I206V+A52Q, I206V+A52F, I206V+A52H, I206V+A52P, I206V+G273S, I206V+G273A, I206V+G273H, I206V+G273F, I206V+S107A+P54L, I206V+S107A+P54S, I206V+S107A+G273A, I206V+S107A+G273F, I206V+S107A+A52P, I206V+S107A+F180S, I206V+S107A+R53N, I206V+S107A+L275T,I206V + S107A + N307Y, I206V + S107A + R157F, I206V + S107A + I194D, I206V + S107A + P54L + N307C, I206V + S107A + P54L + N307W, I206V + S107A + P54L + L275A, I206V + S107A + P54L + L275F, I206V + S107A + P54L + I194W, I206V + S107A + P54L + R157F, I206V + S107A + P54L + I194D, I206V + S107A + P54L + L275T, I206V + S107A + P54L + A52P, I206V + S107A + P54L + R53H, I206V + S107A + P54L + N307D, I206V + S107A + P54L + N307C + S178F, I206V + S107A + P54L + N307C + S178C, I206V + S107A + P54L + N307C + S178I, I206V + S107A + P54L + N307C + R53N, I206V + S107A + P54L + N307C + F180Y, I206V + S107A + P54L + N307C + F180I, I206V + S107A + P54L + N307C + A52N, I206V + S107A + P54L + N307C + A52S, I206V + S107A + P54L + N307C + G273S, I206V + S107A + P54L + N307C + G273A, I206V + S107A + P54L + N307C + G273H, I206V + S107A + P54L + N307C + I194F, I206V + S107A + P54L + N307C + I194H, I206V + S107A + P54L + N307C + I194D, I206V + S107A + P54L + N307C + I194W, I206V + S107A + P54L + N307C + S178F, I206V + S107A + P54L + N307C + S178F + L310I, I206V + S107A + P54L + N307C + S178F + L310A, I206V + S107A + P54L + N307C + S178F + L310H, I206V + S107A + P54L + N307C + S178F + L310R, I206V + S107A + P54L + N307C + S178F + L310Q, I206V + S107A + P54L + N307C + S178F + L310M,I206V + S107A + P54L + N307C + S178F + L310V, I206V + S107A + P54L + N307C + S178F + L310C, I206V + S107A + P54L + N307C + S178F + L310F, I206V + S107A + P54L + N307C + S178F + Q313M, I206V + S107A + P54L + N307C + S178F + Q313M, I206V + S107A + P54L + N307C + S178F + Q313A, I206V + S107A + P54L + N307C + S178F + Q313P, I206V + S107A + P54L + N307C + S178F + Q313K, I206V + S107A + P54L + N307C + S178F + Q313S, I206V + S107A + P54L + N307C + S178F + L275Y, I206V + S107A + P54L + N307C + S178F + L275A, I206V + S107A + P54L + N307C + S178F + L275G, I206V + S107A + P54L + N307C + S178F + L275H, I206V + S107A + P54L + N307C + S178F + L275E, I206V + S107A + P54L + N307C + S178F + L310M, I206V + S107A + P54L + N307C + S178F + L310S, I206V + S107A + P54L + N307C + S178F + L310A, I206V + S107A + P54L + N307C + S178F + L310C, I206V + S107A + P54L + N307C + S178F + L310I + L275Y, I206V + S107A + P54L + N307C + S178F + L310I + L275Q, I206V + S107A + P54L + N307C + S178F + L310I + L275M, I206V + S107A + P54L + N307C + S178F + L310I + L275A, I206V + S107A + P54L + N307C + S178F + L310I + L275S, I206V + S107A + P54L + N307C + S178F + L310I + A232Y, I206V + S107A + P54L + N307C + S178F + L310I + A232G, I206V + S107A + P54L + N307C + S178F + L310I + A232S, I206V + S107A + P54L + N307C + S178F + L310I + A232Q,I206V + S107A + P54L + N307C + S178F + L310I + A232V, I206V + S107A + P54L + N307C + S178F + L310I + A232C, I206V + S107A + P54L + N307C + S178F + L310I + A232M, I206V + S107A + P54L + N307C + S178F + L310I + V388A, I206V + S107A + P54L + N307C + S178F + L310I + V388F, I206V + S107A + P54L + N307C + S178F + L310M + L275Y, I206V + S107A + P54L + N307C + S178F + L310S + L275Y, I206V + S107A + P54L + N307C + S178F + L310C + L275Y, I206V + S107A + P54L + N307C + S178F + L310F + L275Y, I206V + S107A + P54L + N307C + S178F + L310A + L275Y, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204A, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204S, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204F, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + W86F, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + W86Q, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + W86A, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + W86S, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + W86K, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + W86G, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86F, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86A,I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86Q, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86G, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86H, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86Y, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + L218Q, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + L218I, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + L218S, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + L218G, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + L218W, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + L218D, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218I, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218A, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218H, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218G, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218S, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218E,I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202A, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202Q, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + A101S, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + A101R, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + A101G, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218I + A101S, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218I + A101D, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218I + A101F, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218I + A101M, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + A101S, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + A101G, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + A101K, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392K, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392A,I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392S, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392C, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392V, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + E221N, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + E221A, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + E221F, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + E221C, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + E221G, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + E221I, I206V + S107A + P54L + N307C + S178F + L275Y + L204C + W86M + E202S + H392N + L310F, I206V + S107A + P54L + N307C + S178F + L275Y + L204C + W86M + E202S + H392N + L310A, I206V + S107A + P54L + N307C + S178F + L275Y + L204C + W86M + E202S + H392N + L310P, I206V + S107A + P54L + N307C + S178F + L275Y + L204C + W86M + E202S + H392N + L310R, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + Q277S,I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + Q277G, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + Q277P, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + Q277F, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + A102P, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + A102M, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + A102F, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + V83F, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + V83P, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + A102P + V83F, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + A102P + V83A, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + A102P + V83G, or I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + A102P + V83S.

[0011] To achieve the above object, according to a second aspect of the present application, there is provided a DNA molecule encoding the above-mentioned styrene monooxygenase mutant.

[0012] To achieve the above object, according to a third aspect of the present application, there is provided a recombinant plasmid which is linked to the above DNA molecule.

[0013] To achieve the above object, according to a fourth aspect of the present application, there is provided a host cell which is transformed with the above recombinant plasmid.

[0014] To achieve the above object, according to a fifth aspect of the present application, there is provided a method for preparing a chiral epoxide, which comprises catalyzing a reaction of a styrene monooxygenase mutant with an olefin substrate represented by Formula I to obtain the above chiral epoxide represented by Formula II. wherein R1 is selected from F, Cl, Br, methoxy or substituted or unsubstituted C1-C4 alkyl, and when substituted, the substituent is selected from hydroxyl or AcO-; R2 is selected from H, phenyl, C3-C6 heteroalkyl or substituted or unsubstituted C1-C6 alkyl, and when substituted, the substituent is selected from wherein "*" represents the connecting position of R2 in the above olefin substrate or the above chiral epoxide; R3 is selected from H, C3-C6 heteroalkyl, substituted or unsubstituted C1-C6 alkyl, wherein "*" represents the connecting position of R3 in the above olefin substrate or the above chiral epoxide.

[0015] Further, the above olefin substrate is selected from:

[0016] Further, the above reaction system for catalyzing a reaction comprises the above styrene monooxygenase mutant, the above olefin substrate, reductase StyB, glucose dehydrogenase, glucose, FAD, NAD + and 50-200 mM phosphate buffer with pH 7-9.

[0017] Further, the mass ratio of the above olefin substrate to the above styrene monooxygenase in the above reaction system is 1:10-50 in g:g; preferably, the amount of the above styrene monooxygenase is 0.02 g-10 g.

[0018] Further, the reaction temperature for catalyzing a reaction is 20°C-40°C, preferably 25°C-35°C.

[0019] The technical scheme of the present application is used to take styrene monooxygenase StyA (SEQ ID NO: 1) derived from Streptomyces exfoliatus as the mother, and perform protein engineering modification such as single-point site-directed mutation, saturation mutation or combination mutation, to obtain a styrene monooxygenase mutant with high activity and capable of efficiently producing a highly hindered chiral epoxide. DETAILED DESCRIPTION

[0020] The present application is further illustrated by the following examples, but any example or combination thereof should not be interpreted as limiting the scope or examples of the present application. Those skilled in the art can clearly understand the scope of the claims in combination with the present specification and general knowledge in the art.

[0021] As mentioned in the background, the organic synthesis of highly hindered chiral epoxide has the disadvantages of harsh catalytic reaction conditions, low selectivity, many by-products, high cost of heavy metal catalysts used, and environmental unfriendliness, and the use of enzyme method to synthesize highly hindered chiral epoxide has the advantages of economy, greenness and high efficiency, and becomes the preferred method for synthesizing the material. However, the wild-type styrene monooxygenase for biological synthesis has the problems of low activity and / or narrow substrate range. Therefore, the inventors of the present application attempt to modify the styrene monooxygenase by the method of directed evolution, and then improve various properties of the enzyme, so that it can be used in the technical scheme of industrialized production of highly hindered chiral epoxide, and thus a series of protection schemes of the present application are proposed. The specific modification includes:

[0022] By the method of homology modeling, the substrate 1 in the examples is subjected to molecular docking according to the model structure, the docking results are analyzed, and 26 residues near the active center that may affect the catalytic properties of the protein are selected, including I206, S107, P54, N307, S178, L310, L275, L204, W86, E202, H392, E221, R53, V83, A101, A102, R157, F180, I194, L218, A232, G273, Q277, Q313, V388 or A52. The residues are subjected to saturation mutation and site-directed mutation by the method of whole plasmid PCR.

[0023] Among them, the saturation mutation is a method of obtaining a mutant in which the target site amino acid is replaced by other 19 amino acids in a short time by modifying the coding gene of the target protein. This method is not only a powerful tool for protein directed modification, but also an important means for studying the structure-function relationship of proteins. Saturation mutation often obtains more ideal evolution than single-point mutation. For these problems that cannot be solved by site-directed mutation method, saturation mutation method is good at these unique places. The mutants obtained by saturation mutation are identified by sequencing, and their activities on different substrates and tolerances at high temperature are tested.

[0024] Site-directed mutation: refers to introducing the required changes (usually changes in the direction of characterization) into the target DNA fragment (which can be genomic or plasmid) by polymerase chain reaction (PCR) and other methods, including addition, deletion, point mutation, etc. of bases. Site-directed mutation can quickly and efficiently improve the traits and characterization of the target protein expressed by DNA, and is a very useful means in gene research work. The method of introducing site-directed mutation by whole plasmid PCR is simple and effective, and is currently used more. The principle is that a pair of primers (forward and reverse) containing the mutation site and the template plasmid are annealed and then extended by polymerase (so-called cycle extension refers to that the polymerase extends the primer according to the template, and after one cycle, it returns to the 5' end of the primer to terminate, and then repeats the cycle of heating, annealing and extension. This reaction is different from rolling circle amplification, and multiple tandem copies are not formed. The extension products of the forward and reverse primers are annealed to form a notched open plasmid. The extension product is cut by Dpn I enzyme, and the original template plasmid derived from conventional E. coli is dam methylated and modified, and is sensitive to Dpn I and is cut into pieces, while the in vitro synthesized plasmid with mutant sequence is not methylated and is not cut, so it can be successfully transformed in the subsequent transformation, that is, the mutant plasmid clone can be obtained.

[0025] The mutants screened in the mutant library are sequenced, and appropriate mutants are selected according to the sequencing results, and the activity of the amplification reaction is verified. After several rounds of evolution, the inventors obtained a series of styrene monooxygenase mutants, and the activities and selectivities of these mutants on a variety of highly hindered olefin substrates were greatly improved. These mutants can be used for industrial production, and the catalytic efficiency is greatly improved.

[0026] On the basis of the above-mentioned modification results, the applicant proposes a series of protection schemes of the present application. In the first typical embodiment of the present application, a styrene monooxygenase mutant is provided, including (a) a protein having the amino acid sequence shown in SEQ ID NO: 1; or (b) a protein having the amino acid sequence in (a) mutated at at least one of the following positions: I206, S107, P54, N307, S178, L310, L275, L204, W86, E202, H392, E221, R53, V83, A101, A102, R157, F180, I194, L218, A232, G273, Q277, Q313, V388 or A52, and having the function of a styrene monooxygenase; (c) a protein having more than 80% homology with the amino acid sequence defined in any one of (a) and (b) and having the function of a styrene monooxygenase.

[0027] It should be noted that the homology in the present application refers to the "sequence identity" between two amino acid sequences, i.e. the percentage of identical amino acids between the sequences. Methods for assessing the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, the degree of amino acid sequence identity is usually measured using sequence analysis software. For example, it can be determined by the BLAST program of the NCBI database. For determination of sequence identity, see, for example: Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.

[0028] The above-mentioned protein having more than 70%, 75%, 80%, 85%, 90%, 95%, 99% homology with the mutant shown in SEQ ID NO: 1 and having the function of a styrene monooxygenase, the active site, active pocket, active mechanism, protein structure, etc. of which are probably the same as those of the protein provided in (a) of the corresponding protein.

[0029] Amino acid residues can be represented according to the standard three-letter or one-letter amino acid code as is well known and conventional in the art. Herein, amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0030] Conservative amino acid substitutions or replacements are well known in the art, for example, conservative amino acid substitutions are preferably substitution of one amino acid residue by another amino acid residue within the same group (1) - (5) as follows: (1) smaller aliphatic nonpolar or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (2) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gin; (3) polar positively charged residues: His, Arg, and Lys; (4) larger aliphatic nonpolar residues: Met, Leu, Ile, Val, and Cys; and (5) aromatic residues: Phe, Tyr, and Trp. Particularly preferred conservative amino acid substitutions are as follows: Ala is substituted by Gly or Ser; Arg is substituted by Lys; Asn is substituted by Gin or His; Asp is substituted by Glu; Cys is substituted by Ser; Gin is substituted by Asn; Glu is substituted by Asp; Gly is substituted by Ala or Pro; His is substituted by Asn or Gin; Ile is substituted by Leu or Val; Leu is substituted by Ile or Val; Lys is substituted by Arg, Gin, or Glu; Met is substituted by Leu, Tyr, or Ile; Phe is substituted by Met, Leu, or Tyr; Ser is substituted by Thr; Thr is substituted by Ser; Trp is substituted by Tyr; Tyr is substituted by Trp or Phe; and Val is substituted by Ile or Leu.

[0031] Conservative amino acid substitutions can also be made according to the rules for amino acid substitution well known to those skilled in the art, such as the "blosum62 score matrix" in the art.

[0032] The following examples were subjected to site-directed mutagenesis of the styrene monooxygenase (amino acid sequence shown in SEQ ID NO: 1, nucleotide sequence shown in SEQ ID NO: 2) to obtain various variants.

[0033] The amino acid sequence shown in SEQ ID NO: 1 is as follows:

[0034] The nucleotide sequence shown in SEQ ID NO: 2 is as follows:

[0035] The amino acid sequence shown in SEQ ID NO: 1 is the amino acid sequence of styrene monooxygenase StyA derived from Streptomyces exfoliatus. By homology modeling of the amino acid sequence and using computer simulation to analyze the model structure and molecular docking results of (bulky) olefin substrate 1, 26 amino acid residues including I206, S107, P54, N307, S178, L310, L275, L204, W86, E202, H392, E221, R53, V83, A101, A102, R157, F180, I194, L218, A232, G273, Q277, Q313, V388 or A52 were found to be located near the active center and to have the potential to affect the catalytic properties of the protein. By mutating the above-mentioned amino acid sites, a protein having styrene monooxygenase function or even enhanced styrene monooxygenase function can be obtained. For the protein obtained above, changes can be made at non-critical mutation sites and active sites, and a protein having more than 80% homology with the above-mentioned amino acid sequence and having styrene monooxygenase function can be obtained.

[0036] In a preferred embodiment, the amino acid mutations of (b) are each independently selected from the group consisting of: A52N or A52Q or A52F or A52H or A52P or A5S, R53N or R53G or R53H, P54L or P54S or P54F or P54K, V83S or V83A or V83G or V83F or V83P, W86F or W86Q or W86A or W86S or W86K or W86G or W86H or W86Y or W86M, A101S or A101R or A101G or A101D or A101F or A101M or A101K, A102P or A102M or A102F, S107A or S107E or S107Y or S107K or S107G or S107V, R157F or R157Y or R157Q or R157W or R157N or R157C or R157P or R157D, S178F or S178N or S178W or S178C or S178I, F180Y or F180I or F180S or F180E, I194F or I194H or I194G or I194D or I194W, E202S or E202A or E202Q, L204C or L204A or L204S or L204F, I206V or I206A or I206L or I206S or I206Q or I206P or I206H, L218I or L218Q or L218S or L218G or L218W or L218D or L218A or L218H or L218E, E221N or E221A or E221F or E221C or E221G or E221I, A232Y or A232G or A232S or A232Q or A232V or A232C or A232M, G273S or G273A or G273H or G273F, L275F or L275A or L275T or L275Y or L275H or L275G or L275E or L275Q or L275M or L275S, Q277S or Q277G or Q277P or Q277F, N307C or N307D or N307W or N307Y, L310M or L310A or L310F or L310C or L310S or L310I or L310H or L310R or L310Q or L310V, Q313M or Q313A or Q313P or Q313K or Q313S, V388A or V388F or H392N or H392K or H392A or H392S or H392C or H392V; wherein the letter before the number represents the original amino acid and the letter after the number represents the mutated amino acid; preferably, the protein of (c) has 85% or more, preferably 90% or more, more preferably 95% or more, further preferably 99% or more homology to the amino acid sequence defined in (a) or (b) and has stilbene mono-oxygenase function.

[0037] It should be noted that only part of the sources of styrene monooxygenase have the function of catalyzing the synthesis of (large steric hindrance) chiral epoxide from (large steric hindrance) olefin substrate. Therefore, in a preferred embodiment, the inventors screened the substrates 1 (see the example part for details) of styrene monooxygenase from five different sources (Marinobacterium litorale DSM 23545, Sphingopyxis fribergensis, Streptomyces exfoliatus, Bradyrhizobium sp. ORS 375 and Amycolatopsis albispora), and the styrene monooxygenase from Streptomyces exfoliatus showed relatively good activity for the substrate with large steric hindrance, which could catalyze the generation of chiral epoxide.

[0038] In the present application, the applicant continues to explore the active site, and finds that the mutation of the active site to different amino acids has different activities of the corresponding protein, and a specific mutation can enhance the activity of the styrene monooxygenase. Through experimental exploration, it is found that the above-mentioned specific mutation to the active site can obtain the protein with enhanced activity. For the amino acid mutation site of the enzyme protein, the above-mentioned mutation can be flexibly selected and combined.

[0039] In a preferred embodiment, the mutation of the styrene monooxygenase mutant comprises any one of the following amino acid mutations: I206V, I206A, I206L, I206S, I206Q, I206P, I206H, S107A, S107E, S107Y, S107K, S107G, S107V, R157Q, R157C, R157P, R157D, R157Q, I194F, I194H, I194G, I194D, I194W, P54L, P54S, P54F, P54K, R157Q, R157Y, R157W, R157F, R157N, N307C, N307W, N307Y, N307D, L275F, L275A, L275T, S178F, S178N, S178W, R53N, R53G, R53H, F180Y, F180E, F180S, A52N, A52Q, A52F, A52H, A52P, G273S, G273A, G273H, G273F, I206V+S107A, I206V+S107E, I206V+S107K, I206V+R157Q, I206V+R157Y, I206V+R157W, I206V+R157F, I206V+I194F, I206V+I194D, I206V+I194W, I206V+P54L, I206V+P54S, I206V+P54F, I206V+R157Q, I206V+R157Y, I206V+R157F, I206V+N307C, I206V+N307W, I206V+N307Y, I206V+N307D, I206V+L275F, I206V+L275A, I206V+L275T, I206V+S178F, I206V+S178N, I206V+S178W, I206V+R53N, I206V+R53G, I206V+R53H, I206V+F180Y, I206V+F180E, I206V+F180S, I206V+A52N, I206V+A52Q, I206V+A52F, I206V+A52H, I206V+A52P, I206V+G273S, I206V+G273A, I206V+G273H, I206V+G273F, I206V+S107A+P54L, I206V+S107A+P54S, I206V+S107A+G273A, I206V+S107A+G273F, I206V+S107A+A52P, I206V+S107A+F180S, I206V+S107A+R53N, I206V+S107A+L275T,I206V + S107A + N307Y, I206V + S107A + R157F, I206V + S107A + I194D, I206V + S107A + P54L + N307C, I206V + S107A + P54L + N307W, I206V + S107A + P54L + L275A, I206V + S107A + P54L + L275F, I206V + S107A + P54L + I194W, I206V + S107A + P54L + R157F, I206V + S107A + P54L + I194D, I206V + S107A + P54L + L275T, I206V + S107A + P54L + A52P, I206V + S107A + P54L + R53H, I206V + S107A + P54L + N307D, I206V + S107A + P54L + N307C + S178F, I206V + S107A + P54L + N307C + S178C, I206V + S107A + P54L + N307C + S178I, I206V + S107A + P54L + N307C + R53N, I206V + S107A + P54L + N307C + F180Y, I206V + S107A + P54L + N307C + F180I, I206V + S107A + P54L + N307C + A52N, I206V + S107A + P54L + N307C + A52S, I206V + S107A + P54L + N307C + G273S, I206V + S107A + P54L + N307C + G273A, I206V + S107A + P54L + N307C + G273H, I206V + S107A + P54L + N307C + I194F, I206V + S107A + P54L + N307C + I194H, I206V + S107A + P54L + N307C + I194D, I206V + S107A + P54L + N307C + I194W, I206V + S107A + P54L + N307C + S178F, I206V + S107A + P54L + N307C + S178F + L310I, I206V + S107A + P54L + N307C + S178F + L310A, I206V + S107A + P54L + N307C + S178F + L310H, I206V + S107A + P54L + N307C + S178F + L310R, I206V + S107A + P54L + N307C + S178F + L310Q, I206V + S107A + P54L + N307C + S178F + L310M,I206V + S107A + P54L + N307C + S178F + L310V, I206V + S107A + P54L + N307C + S178F + L310C, I206V + S107A + P54L + N307C + S178F + L310F, I206V + S107A + P54L + N307C + S178F + Q313M, I206V + S107A + P54L + N307C + S178F + Q313M, I206V + S107A + P54L + N307C + S178F + Q313A, I206V + S107A + P54L + N307C + S178F + Q313P, I206V + S107A + P54L + N307C + S178F + Q313K, I206V + S107A + P54L + N307C + S178F + Q313S, I206V + S107A + P54L + N307C + S178F + L275Y, I206V + S107A + P54L + N307C + S178F + L275A, I206V + S107A + P54L + N307C + S178F + L275G, I206V + S107A + P54L + N307C + S178F + L275H, I206V + S107A + P54L + N307C + S178F + L275E, I206V + S107A + P54L + N307C + S178F + L310M, I206V + S107A + P54L + N307C + S178F + L310S, I206V + S107A + P54L + N307C + S178F + L310A, I206V + S107A + P54L + N307C + S178F + L310C, I206V + S107A + P54L + N307C + S178F + L310I + L275Y, I206V + S107A + P54L + N307C + S178F + L310I + L275Q, I206V + S107A + P54L + N307C + S178F + L310I + L275M, I206V + S107A + P54L + N307C + S178F + L310I + L275A, I206V + S107A + P54L + N307C + S178F + L310I + L275S, I206V + S107A + P54L + N307C + S178F + L310I + A232Y, I206V + S107A + P54L + N307C + S178F + L310I + A232G, I206V + S107A + P54L + N307C + S178F + L310I + A232S, I206V + S107A + P54L + N307C + S178F + L310I + A232Q,I206V + S107A + P54L + N307C + S178F + L310I + A232V, I206V + S107A + P54L + N307C + S178F + L310I + A232C, I206V + S107A + P54L + N307C + S178F + L310I + A232M, I206V + S107A + P54L + N307C + S178F + L310I + V388A, I206V + S107A + P54L + N307C + S178F + L310I + V388F, I206V + S107A + P54L + N307C + S178F + L310M + L275Y, I206V + S107A + P54L + N307C + S178F + L310S + L275Y, I206V + S107A + P54L + N307C + S178F + L310C + L275Y, I206V + S107A + P54L + N307C + S178F + L310F + L275Y, I206V + S107A + P54L + N307C + S178F + L310A + L275Y, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204A, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204S, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204F, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + W86F, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + W86Q, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + W86A, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + W86S, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + W86K, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + W86G, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86F, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86A,I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86Q, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86G, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86H, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86Y, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + L218Q, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + L218I, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + L218S, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + L218G, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + L218W, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + L218D, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218I, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218A, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218H, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218G, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218S, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218E,I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202A, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202Q, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + A101S, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + A101R, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + A101G, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218I + A101S, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218I + A101D, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218I + A101F, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218I + A101M, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + A101S, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + A101G, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + A101K, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392K, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392A,I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392S, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392C, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392V, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + E221N, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + E221A, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + E221F, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + E221C, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + E221G, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + E221I, I206V + S107A + P54L + N307C + S178F + L275Y + L204C + W86M + E202S + H392N + L310F, I206V + S107A + P54L + N307C + S178F + L275Y + L204C + W86M + E202S + H392N + L310A, I206V + S107A + P54L + N307C + S178F + L275Y + L204C + W86M + E202S + H392N + L310P, I206V + S107A + P54L + N307C + S178F + L275Y + L204C + W86M + E202S + H392N + L310R, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + Q277S,I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + Q277G, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + Q277P, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + Q277F, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + A102P, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + A102M, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + A102F, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + V83F, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + V83P, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + A102P + V83F, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + A102P + V83A, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + A102P + V83G, or I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + A102P + V83S.

[0040] The styrene monooxygenase mutants with the above-mentioned amino acid mutations were tested in the embodiments of the present application, and the results showed that all of them had styrene monooxygenase activity, and compared with the parent having the amino acid sequence shown in SEQ ID NO: 1, they had the advantages of high enzyme activity and / or high selectivity.

[0041] In a second exemplary embodiment of the present application, a DNA molecule is provided, which encodes the styrene monooxygenase mutant described above.

[0042] In a third exemplary embodiment of the present application, a recombinant plasmid is provided, which is linked with the DNA molecule described above. The DNA described above can encode the styrene monooxygenase mutant described above and can be linked to the recombinant plasmid to form a circular DNA. Both the DNA and the recombinant plasmid can be transcribed and translated under the action of RNA polymerase, ribosome and tRNA, etc. to obtain the styrene monooxygenase mutant described above.

[0043] In a fourth exemplary embodiment of the present application, a host cell is provided, which is transformed with the recombinant plasmid described above. By using the host cell, the replication of the recombinant plasmid in the host cell can be carried out, and the DNA molecule carried by the recombinant plasmid can be transcribed and translated to obtain a large amount of styrene monooxygenase mutant. By using the prior art, the styrene monooxygenase mutant can be obtained by crushing the host cell for protein purification, catalyzing the crude enzyme after crushing or other ways, and the subsequent catalytic synthesis of the large steric hindered chiral epoxide can be carried out.

[0044] In a fifth exemplary embodiment of the present application, a preparation method of a (large steric hindered) chiral epoxide is provided, which comprises catalyzing the reaction of the styrene monooxygenase mutant described above and a (large steric hindered) olefin substrate shown in formula I to obtain a (large steric hindered) chiral epoxide shown in formula II;

[0045] wherein R1 is selected from F, Cl, Br, methoxy or substituted or unsubstituted C1-C4 alkyl, and when substituted, the substituent is selected from hydroxyl or AcO-; R2 is selected from H, phenyl, C3-C6 heteroalkyl or substituted or unsubstituted C1-C6 alkyl, and when substituted, the substituent is selected from wherein “*” represents the connection position of R2 in the (large steric hindered) olefin substrate or the (large steric hindered) chiral epoxide; R3 is selected from H, C3-C6 heteroalkyl, substituted or unsubstituted C1-C6 alkyl, wherein “*” represents the connection position of R3 in the (large steric hindered) olefin substrate or the (large steric hindered) chiral epoxide.

[0046] It should be noted that the heteroalkyl contains nitrogen, oxygen or sulfur atoms.

[0047] In some preferred embodiments, the (large steric hindered) olefin substrate is selected from: In some preferred embodiments, the (large steric hindered) olefin substrate is selected from:

[0048] The preparation method of the above-mentioned chiral epoxide provided by the present application, in a specific catalytic reaction system, in addition to the styrene monooxygenase mutant and the above-mentioned large steric hindrance olefin substrate of the present application, the rest of the components (such as buffer and coenzyme, etc.) are similar to those in the existing enzyme method for synthesizing chiral epoxide. The specific dosage and catalytic reaction conditions can be optimized and matched according to the activity of the enzyme and the substrate. In a preferred embodiment of the present application, the reaction system of the above-mentioned catalytic reaction includes: the above-mentioned styrene monooxygenase mutant, the above-mentioned (large steric hindrance) olefin substrate, the reductase StyB, the glucose dehydrogenase, the glucose, the FAD, the NAD + and 50-200 mM phosphate buffer at pH 7-9. Among them, the mass ratio of the above-mentioned (large steric hindrance) olefin substrate to the above-mentioned styrene monooxygenase is 1:10-50 by g:g; preferably, the dosage of the above-mentioned styrene monooxygenase is 0.02 g-10 g. The reaction temperature of the above-mentioned catalytic reaction is 20°C-40°C, preferably 25°C-35°C.

[0049] Based on the above-mentioned preparation method, using the above-mentioned styrene monooxygenase mutant in the reaction of the provided formula I (large steric hindrance) olefin substrate, the (large steric hindrance) olefin substrate represented by formula I can be reacted to obtain (large steric hindrance) chiral epoxide. The above-mentioned styrene monooxygenase mutant can catalyze the above-mentioned (large steric hindrance) olefin substrate to synthesize the required (large steric hindrance) chiral epoxide (reaction formula is shown below). The greatly improved activity and selectivity of the styrene monooxygenase can improve the production efficiency, reduce the industrial production cost, and be more suitable for industrial production.

[0050] The present application will be further described in detail in conjunction with specific examples, which cannot be understood as limiting the scope of the present application. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. The reductase StyB and glucose dehydrogenase GDH used in the following examples can be commercially available products or can be constructed and expressed by oneself. In the present application, the reductase StyB and glucose dehydrogenase GDH are respectively derived from Pseudomonas sp. (Gene ID: ABB03728.1) and Bacillus subtilis (Gene ID: ABR21558.1), and the applicant expresses and prepares them by himself.

[0051] Example 1 Enzyme screening

[0052] The embodiment selects hundreds of oxidases in the enzyme library of Kayla Ying Company to carry out enzyme screening of substrate 1, and the screening system is as follows: 1 mL of reaction system includes 1 mg of substrate 1, 50 mg of styrene monooxygenase StyA enzyme powder, 10 mg of reductase StyB enzyme powder, 7.5 mg of glucose dehydrogenase (GDH) enzyme powder, 5 mg of glucose, 25 μM of FAD, 5 mM of NAD + , 100 mM of phosphate buffer at pH 7.5, 35 °C reaction for 16 h. After the reaction is completed, 2 mL of acetonitrile is added to terminate the reaction, and after being fully shaken and mixed, 8000 rpm centrifugation is carried out for 1 min to obtain the supernatant water phase, which is sent to HPLC for detection of conversion rate. The calculation formula of the conversion rate is: product HPLC Area% / (product HPLC Area%+substrate HPLC Area%) * 100%.

[0053] The results show that most of the enzymes in the enzyme library do not detect product generation on HPLC, but a small number of enzymes have product generation, and the highest conversion rate is about 5%, and these enzymes include the following sequences, as shown in Table 1:

[0054] Table 1

[0055] Note: In the above table, * represents that the conversion rate is less than 1%, and ** represents that the conversion rate is greater than or equal to 1% and less than 5%.

[0056] Example 2 First round of enzyme evolution

[0057] The styrene monooxygenase StyA derived from Streptomyces exfoliatus which has better activity on substrate 1 is selected for enzyme evolution. Through protein three-dimensional structure simulation, sites that may affect enzyme activity are selected for mutation, and full-plasmid PCR method is used for site / saturation mutation. The following reaction conditions are used to screen the mutants.

[0058] 1 mL of reaction system includes 2 mg of substrate 1, 50 mg of styrene monooxygenase StyA enzyme powder, 10 mg of reductase StyB enzyme powder, 7.5 mg of glucose dehydrogenase (GDH) enzyme powder, 5 mg of glucose, 25 μM of FAD, 5 mM of NAD + , 100 mM of phosphate buffer at pH 7.5, 35 °C reaction for 16 h. After the reaction is completed, 2 mL of acetonitrile is added to terminate the reaction, and after being fully shaken and mixed, 8000 rpm centrifugation is carried out for 1 min to obtain the supernatant water phase, which is sent to HPLC for detection of conversion rate.

[0059] After the primary screening and the secondary screening, the following mutants show improved activity, and the specific results are shown in Table 2:

[0060] Table 2

[0061] Note: In the above table, * represents the conversion rate greater than 1% and less than 5%, ** represents the conversion rate greater than or equal to 5% and less than 10%, and *** represents the conversion rate greater than or equal to 10% and less than 15%.

[0062] Example 3

[0063] Based on the mutants of Example 2, combined mutations were carried out, and the combined mutants were tested for activity under the same reaction conditions as in Example 2, with the results shown in Table 3:

[0064] Table 3

[0065] Note: In the above table, *** represents the conversion rate greater than or equal to 10% and less than 15%, **** represents the conversion rate greater than or equal to 15% and less than 25%, ***** represents the conversion rate greater than or equal to 25% and less than 35%, ****** represents the conversion rate greater than or equal to 35% and less than 45%, and ******* represents the conversion rate greater than or equal to 55% and less than 65%.

[0066] Example 4

[0067] Based on the mutants of Example 3, saturation mutations were carried out, and due to the increased enzyme activity at this stage, the amount of StyA enzyme was reduced to 30 mg during screening, and the mutants were tested for activity under the following reaction conditions:

[0068] 1 mL of the reaction system included 2 mg of substrate 1, 30 mg of phenylethylene monooxygenase StyA enzyme powder, 10 mg of reductase StyB enzyme powder, 7.5 mg of glucose dehydrogenase (GDH) enzyme powder, 5 mg of glucose, 25 μM FAD, 5 mM NAD + , 100 mM phosphate buffer at pH 7.5, and the reaction was carried out at 35°C for 16 h. After the reaction was completed, 2 mL of acetonitrile was added to terminate the reaction, and after thorough shaking and mixing, centrifugation was carried out at 8000 rpm for 1 min to obtain the supernatant aqueous phase, which was sent for HPLC detection of the conversion rate.

[0069] Through primary screening and secondary screening, the following mutants showed improved activity, with the specific results shown in Table 4:

[0070] Table 4

[0071] Note: In the above table, * represents a conversion rate of 10% or more and less than 15%, ** represents a conversion rate of 10% or more and less than 15%, *** represents a conversion rate of 25% or more and less than 35%, **** represents a conversion rate of 35% or more and less than 45%, ***** represents a conversion rate of 55% or more and less than 65%, and ****** represents a conversion rate of 55% or more and less than 65%.

[0072] Example 5

[0073] Based on the mutants of Example 4, further improvement of enzyme activity was achieved in this stage, and the amount of StyA enzyme was reduced to 20 mg in screening, and the mutants were tested for activity under the following reaction conditions:

[0074] 1 mL reaction system included 2 mg of substrate 1, 20 mg of styrene monooxygenase StyA enzyme powder, 10 mg of reductase StyB enzyme powder, 7.5 mg of glucose dehydrogenase (GDH) enzyme powder, 5 mg of glucose, 25 μM FAD, 5 mM NAD + , 100 mM phosphate buffer at pH 7.5, 35°C for 16 h. After the reaction was completed, 2 mL of acetonitrile was added to terminate the reaction, and after thorough shaking and mixing, centrifugation was performed at 8000 rpm for 1 min to obtain the supernatant aqueous phase, which was sent for HPLC detection of conversion rate.

[0075] Through primary screening and secondary screening, the following mutants showed improved activity, and the specific results are shown in Table 5:

[0076] Table 5

[0077] Note: In the above table, * represents a conversion rate of 10% or more and less than 15%, ** represents a conversion rate of 10% or more and less than 15%, *** represents a conversion rate of 25% or more and less than 35%, **** represents a conversion rate of 35% or more and less than 45%, ***** represents a conversion rate of 55% or more and less than 65%, and ****** represents a conversion rate of 55% or more and less than 65%.

[0078] Example 6

[0079] 500 mL of 100 mM phosphate buffer (pH 7.5), 2.5 g of glucose, 25 μM FAD, 5 mM NAD + were added to a 2 L four-necked flask at room temperature, and the pH was adjusted to 7.0-7.5. Then 1 g Stirring evenly, then add 10 g of styrene monooxygenase mutant (I206V+S107A+P54L+N307C+S178F+L310M+L275Y+L204C+W86M+E202S+H392N+E221N) enzyme powder based on SEQ ID NO: 1, 20 g of reductase StyB enzyme powder, 3.7 g of glucose dehydrogenase (GDH) enzyme powder, adjust pH = 7.0-7.5. Warm up to 35°C and stir overnight. After the reaction is complete, adjust the system to pH = 2-3, denature the protein. After filtration, the filtrate is extracted with 500 mL of methyl tert-butyl ether three times. The combined organic phase is dried with anhydrous magnesium sulfate and concentrated to no fraction under the condition of T < 40°C, P ≤-0.06 Mpa. The target product is obtained

[0080] After HPLC and GC detection, the purity is >98%, the de value is >99%, and the yield is 81%.

[0081] Example 7

[0082] At room temperature, add 500 mL of 100 mM phosphate buffer (pH 7.5), 2.5 g of glucose, 25 μM FAD, 5 mM NAD + to a 2L four-necked flask, adjust pH = 7.0-7.5. Then add 1 g of Stir evenly, then add 10 g of styrene monooxygenase mutant (I206V+S107A+P54L+N307C+S178F+L310M+L275Y+L204C+W86M+E202S+H392N+E221N) enzyme powder based on SEQ ID NO: 1, 20 g of reductase StyB enzyme powder, 3.7 g of glucose dehydrogenase (GDH) enzyme powder, adjust pH = 7.0-7.5. Warm up to 35°C and stir overnight. After the reaction is complete, adjust the system to pH = 2-3, denature the protein. After filtration, the filtrate is extracted with 500 mL of methyl tert-butyl ether three times. The combined organic phase is dried with anhydrous magnesium sulfate and concentrated to no fraction under the condition of T < 40°C, P ≤-0.06 Mpa. The target product is obtained

[0083] After HPLC and GC detection, the purity is >98%, the de value is >99%, and the yield is 83%.

[0084] Example 8

[0085] At room temperature, add 500 mL of 100 mM phosphate buffer (pH 7.5), 2.5 g of glucose, 25 μM FAD, 5 mM NAD + to a 2L four-necked flask, adjust pH = 7.0-7.5. Then add 1 g of Stirring evenly, then add 10 g of the mutant of styrene monooxygenase based on SEQ ID NO: 1 (I206V+S107A+P54L+N307C+S178F+L310M+L275Y+L204C+W86M+E202S+H392N+E221N) enzyme powder, 20 g of reductase StyB enzyme powder, 3.7 g of glucose dehydrogenase (GDH) enzyme powder, adjust pH = 7.0-7.5. Warm up to 35°C and stir overnight. After the reaction is complete, adjust the system to pH = 2-3, denature the protein. After filtration, the filtrate is extracted with 500 mL of methyl tert-butyl ether three times. Dry the combined organic phase with anhydrous magnesium sulfate, then concentrate to no fraction under the condition of T < 40°C, P ≤ -0.06 Mpa. The target product is obtained

[0086] After HPLC and GC detection, the purity is > 98%, the de value is > 99%, and the yield is 79%.

[0087] Example 9

[0088] At room temperature, add 500 mL of 100 mM phosphate buffer (pH 7.5), 2.5 g of glucose, 25 μM FAD, 5 mM NAD+ to a 2 L four-necked flask, adjust pH = 7.0-7.5. Then add 1 g of the mutant of styrene monooxygenase based on SEQ ID NO: 1 (I206V+S107A+P54L+N307C+S178F+L310M+L275Y+L204C+W86M+E202S+H392N+E221N) enzyme powder, 20 g of reductase StyB enzyme powder, 3.7 g of glucose dehydrogenase (GDH) enzyme powder, adjust pH = 7.0-7.5. Warm up to 35°C and stir overnight. After the reaction is complete, adjust the system to pH = 2-3, denature the protein. After filtration, the filtrate is extracted with 500 mL of methyl tert-butyl ether three times. Dry the combined organic phase with anhydrous magnesium sulfate, then concentrate to no fraction under the condition of T < 40°C, P ≤ -0.06 Mpa. The target product is obtained Stirring evenly, then add 10 g of the mutant of styrene monooxygenase based on SEQ ID NO: 1 (I206V+S107A+P54L+N307C+S178F+L310M+L275Y+L204C+W86M+E202S+H392N+E221N) enzyme powder, 20 g of reductase StyB enzyme powder, 3.7 g of glucose dehydrogenase (GDH) enzyme powder, adjust pH = 7.0-7.5. Warm up to 35°C and stir overnight. After the reaction is complete, adjust the system to pH = 2-3, denature the protein. After filtration, the filtrate is extracted with 500 mL of methyl tert-butyl ether three times. Dry the combined organic phase with anhydrous magnesium sulfate, then concentrate to no fraction under the condition of T < 40°C, P ≤ -0.06 Mpa. The target product is obtained

[0089] After HPLC and GC detection, the purity is > 98%, the de value is > 99%, and the yield is 79%.

[0090] Example 10

[0091] The styrene monooxygenase mutant (I206V+S107A+P54L+N307C+S178F+L310M+L275Y+L204C+W86M+E202S+H392N+E221N) based on SEQ ID NO: 1 was used to catalyze the reaction of the substrate 5-10 according to the catalytic synthesis steps of Reference Examples 6 to 9, and the results are shown in Table 6:

[0092] Table 6

[0093] In summary, the above-mentioned embodiments of the present application achieve the following technical effects: the above-mentioned styrene monooxygenase mutant of the present application has high activity and / or a wide range of substrate selection, etc. excellent performance, high economic value, wide application prospect. The high activity of the enzyme makes it only need a small amount in actual use to obtain a large amount of target product, which greatly reduces the cost of enterprise production of target product and increases its profit. The large substrate selection range of the enzyme enables it to efficiently catalyze the synthesis of different series of large steric hindrance epoxides, which helps to increase its application fields and application scenarios, and lays an important foundation for the development and income of the enzyme. Compared with traditional chemical method, enzyme catalytic synthesis of large steric hindrance chiral epoxide is an economical, green and efficient synthesis method. It does not need complex process and steps, and the operation is more simple and mild, and does not need heavy metal, which helps to reduce the three wastes, reduce the cost and realize green production. In general, the research and construction of the styrene monooxygenase mutant will bring great benefits to the subsequent downstream application.

[0094] The above examples only describe the preferred embodiments of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by ordinary engineering technicians in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A styrene monooxygenase mutant, characterized in that, comprising (a) a protein having an amino acid sequence shown in SEQ ID NO: 1; (b) a protein having an amino acid sequence shown in SEQ ID NO: 1, in which at least one of the amino acid residues at positions I206, S107, P54, N307, S178, L310, L275, L204, W86, E202, H392, E221, R53, V83, A101, A102, R157, F180, I194, L218, A232, G273, Q277, Q313, V388, or A52 is mutated and the protein has a p-menthane monooxygenase function; or (c) a protein having 80% or more homology with the amino acid sequence defined in any one of (a) or (b) and the protein has a p-menthane monooxygenase function.

2. The styrene monooxygenase mutant of claim 1, wherein, the amino acid mutation of the (b) is selected from at least one of the following positions: A52N or A52Q or A52F or A52H or A52P or A52S, R53N or R53G or R53H, P54L or P54S or P54F or P54K, V83S or V83A or V83G or V83F or V83P, W86F or W86Q or W86A or W86S or W86K or W86G or W86H or W86Y or W86M, A101S or A101R or A101G or A101D or A101F or A101M or A101K, A102P or A102M or A102F, S107A or S107E or S107Y or S107K or S107G or S107V, R157F or R157Y or R157Q or R157W or R157N or R157C or R157P or R157D, S178F or S178N or S178W or S178C or S178I, F180Y or F180I or F180S or F180E, I194F or I194H or I194G or I194D or I194W, E202S or E202A or E202Q, L204C or L204A or L204S or L204F, I206V or I206A or I206L or I206S or I206Q or I206P or I206H, L218I or L218Q or L218S or L218G or L218W or L218D or L218A or L218H or L218E, E221N or E221A or E221F or E221C or E221G or E221I, A232Y or A232G or A232S or A232Q or A232V or A232C or A232M, G273S or G273A or G273H or G273F, L275F or L275A or L275T or L275Y or L275H or L275G or L275E or L275Q or L275M or L275S, Q277S or Q277G or Q277P or Q277F, N307C or N307D or N307W or N307Y, L310M or L310A or L310F or L310C or L310S or L310I or L310H or L310R or L310Q or L310V, Q313M or Q313A or Q313P or Q313K or Q313S, V388A or V388F or H392N or H392K or H392A or H392S or H392C or H392V; wherein the letter before the number represents the original amino acid and the letter after the number represents the mutated amino acid.

3. The styrene monooxygenase mutant of claim 2, wherein, The mutation of the styrene monooxygenase mutant comprises any one of the following amino acid mutations: I206V, I206A, I206L, I206S, I206Q, I206P, I206H, S107A, S107E, S107Y, S107K, S107G, S107V, R157Q, R157C, R157P, R157D, R157Q, I194F, I194H, I194G, I194D, I194W, P54L, P54S, P54F, P54K, R157Q, R157Y, R157W, R157F, R157N, N307C, N307W, N307Y, N307D, L275F, L275A, L275T, S178F, S178N, S178W, R53N, R53G, R53H, F180Y, F180E, F180S, A52N, A52Q, A52F, A52H, A52P, G273S, G273A, G273H, G273F, I206V+S107A, I206V+S107E, I206V+S107K, I206V+R157Q, I206V+R157Y, I206V+R157W, I206V+R157F, I206V+I194F, I206V+I194D, I206V+I194W, I206V+P54L, I206V+P54S, I206V+P54F, I206V+R157Q, I206V+R157Y, I206V+R157F, I206V+N307C, I206V+N307W, I206V+N307Y, I206V+N307D, I206V+L275F, I206V+L275A, I206V+L275T, I206V+S178F, I206V+S178N, I206V+S178W, I206V+R53N, I206V+R53G, I206V+R53H, I206V+F180Y, I206V+F180E, I206V+F180S, I206V+A52N, I206V+A52Q, I206V+A52F, I206V+A52H, I206V+A52P, I206V+G273S, I206V+G273A, I206V+G273H, I206V+G273F, I206V+S107A+P54L, I206V+S107A+P54S, I206V+S107A+G273A, I206V+S107A+G273F, I206V+S107A+A52P, I206V+S107A+F180S, I206V+S107A+R53N, I206V+S107A+L275T, I206V+S107A+N307Y, I206V+S107A+R157F, I206V+S107A+I194D, I206V+S107A+P54L+N307C, I206V+S107A+P54L+N307W, I206V+S107A+P54L+L275A, I206V+S107A+P54L+L275F, I206V+S107A+P54L+I194W, I206V+S107A+P54L+R157F, I206V+S107A+P54L+I194D, I206V+S107A+P54L+L275T, I206V+S107A+P54L+A52P, I206V+S107A+P54L+R53H, I206V+S107A+P54L+N307D, I206V+S107A+P54L+N307C+S178F,I206V + S107A + P54L + N307C + S178C, I206V + S107A + P54L + N307C + S178I, I206V + S107A + P54L + N307C + R53N, I206V + S107A + P54L + N307C + F180Y, I206V + S107A + P54L + N307C + F180I, I206V + S107A + P54L + N307C + A52N, I206V + S107A + P54L + N307C + A52S, I206V + S107A + P54L + N307C + G273S, I206V + S107A + P54L + N307C + G273A, I206V + S107A + P54L + N307C + G273H, I206V + S107A + P54L + N307C + I194F, I206V + S107A + P54L + N307C + I194H, I206V + S107A + P54L + N307C + I194D, I206V + S107A + P54L + N307C + I194W, I206V + S107A + P54L + N307C + S178F, I206V + S107A + P54L + N307C + S178F + L310I, I206V + S107A + P54L + N307C + S178F + L310A, I206V + S107A + P54L + N307C + S178F + L310H, I206V + S107A + P54L + N307C + S178F + L310R, I206V + S107A + P54L + N307C + S178F + L310Q, I206V + S107A + P54L + N307C + S178F + L310M, I206V + S107A + P54L + N307C + S178F + L310V, I206V + S107A + P54L + N307C + S178F + L310C, I206V + S107A + P54L + N307C + S178F + L310F, I206V + S107A + P54L + N307C + S178F + Q313M, I206V + S107A + P54L + N307C + S178F + Q313M, I206V + S107A + P54L + N307C + S178F + Q313A, I206V + S107A + P54L + N307C + S178F + Q313P, I206V + S107A + P54L + N307C + S178F + Q313K, I206V + S107A + P54L + N307C + S178F + Q313S, I206V + S107A + P54L + N307C + S178F + L275Y, I206V + S107A + P54L + N307C + S178F + L275A, I206V + S107A + P54L + N307C + S178F + L275G, I206V + S107A + P54L + N307C + S178F + L275H, I206V + S107A + P54L + N307C + S178F + L275E, I206V + S107A + P54L + N307C + S178F + L310M, I206V + S107A + P54L + N307C + S178F + L310S, I206V + S107A + P54L + N307C + S178F + L310A, I206V + S107A + P54L + N307C + S178F + L310C, I206V + S107A + P54L + N307C + S178F + L310I + L275Y, I206V + S107A + P54L + N307C + S178F + L310I + L275Q, I206V + S107A + P54L + N307C + S178F + L310I + L275M, I206V + S107A + P54L + N307C + S178F + L310I + L275A, I206V + S107A + P54L + N307C + S178F + L310I + L275S, I206V + S107A + P54L + N307C + S178F + L310I + A232Y, I206V + S107A + P54L + N307C + S178F + L310I + A232G, I206V + S107A + P54L + N307C + S178F + L310I + A232S, I206V + S107A + P54L + N307C + S178F + L310I + A232Q, I206V + S107A + P54L + N307C + S178F + L310I + A232V, I206V + S107A + P54L + N307C + S178F + L310I + A232C, I206V + S107A + P54L + N307C + S178F + L310I + A232M, I206V + S107A + P54L + N307C + S178F + L310I + V388A, I206V + S107A + P54L + N307C + S178F + L310I + V388F, I206V + S107A + P54L + N307C + S178F + L310M + L275Y,I206V + S107A + P54L + N307C + S178F + L310S + L275Y, I206V + S107A + P54L + N307C + S178F + L310C + L275Y, I206V + S107A + P54L + N307C + S178F + L310F + L275Y, I206V + S107A + P54L + N307C + S178F + L310A + L275Y, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204A, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204S, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204F, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + W86F, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + W86Q, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + W86A, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + W86S, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218A, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218H, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218D, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218W, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218G, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218S, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218I, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218Q, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218T, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218V, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218L, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218K, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218E, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218P, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218F, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218Y, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218N, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218Q + L218A, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218Q + L218H, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218Q + L218D, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218Q + L218W, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218Q + L218G, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218Q + L218S, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218Q + L218I, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218Q + L218T, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218Q + L218V, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218Q + L218L, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218Q + L218K, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218Q + L218E, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218Q + L218P, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218Q + L218F, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218Q + L218Y, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218Q + L218N,I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218G, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218S, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218E, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202A, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202Q, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + A101S, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + A101R, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + A101G, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218I + A101S, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218I + A101D, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218I + A101F, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + L218I + A101M, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + A101S, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + A101G, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + A101K,I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392K, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392A, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392S, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392C, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392V, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + E221N, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + E221A, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + E221F, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + E221C, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + E221G, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + E221I, I206V + S107A + P54L + N307C + S178F + L275Y + L204C + W86M + E202S + H392N + L310F, I206V + S107A + P54L + N307C + S178F + L275Y + L204C + W86M + E202S + H392N + L310A, I206V + S107A + P54L + N307C + S178F + L275Y + L204C + W86M + E202S + H392N + L310P, I206V + S107A + P54L + N307C + S178F + L275Y + L204C + W86M + E202S + H392N + L310R, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + Q277S,I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + Q277G, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + Q277P, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + Q277F, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + A102P, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + A102M, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + A102F, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + V83F, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + V83P, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + A102P + V83F, I206V + S107A + P54L + N307C + S178F + L310M + L275Y + L204C + W86M + E202S + H392N + A102P + V83A, I206V+S107A+P54L+N307C+S178F+L310M+L275Y+L204C+W86M+E202S+H392N+A102P+V83G or I206V+S107A+P54L+N307C+S178F+L310M+L275Y+L204C+W86M+E202S+H392N+A102P+V83S.

4. A DNA molecule, characterized in that, The DNA molecule encodes the styrene monooxygenase mutant of any one of claims 1 to 3.

5. A recombinant plasmid, characterized in that, The recombinant plasmid is linked with the DNA molecule of claim 4.

6. A host cell, characterized in that, The host cell is transformed with the recombinant plasmid of claim 5.

7. A process for the preparation of a chiral epoxide, characterized in that, The preparation method comprises catalyzing a styrene monooxygenase mutant according to any one of claims 1 to 3 and an olefin substrate shown in formula I to obtain a chiral epoxide shown in formula II. R1 is selected from F, Cl, Br, methoxy or substituted or unsubstituted C1-C4 alkyl, and when substituted, the substituent is selected from hydroxyl or AcO-; R2is selected from H, phenyl, C3-C6heteroalkyl or substituted or unsubstituted C1-C6alkyl, when and where substituted, with one or more substituents selected from wherein "*" indicates the attachment position of R2 in the olefin substrate or the chiral epoxide; said R3 is selected from H, C3-C6 heteroalkyl, substituted or non-substituted C1-C6 alkyl, wherein "*" indicates the attachment position of R3 in the olefin substrate or the chiral epoxide.

8. The preparation method according to claim 7, characterized in that, The olefin substrate is selected from:

9. The preparation method according to claim 7, characterized in that, The reaction system for catalyzing the reaction comprises: the styrene monooxygenase mutant, the olefin substrate, the reductase StyB, glucose dehydrogenase, glucose, FAD, NAD + and 50-200 mM phosphate buffer with pH 7.0-9.

0.

10. The method of claim 9, wherein, The mass ratio of the olefin substrate to the styrene monooxygenase mutant in the reaction system is 1:10-50 by g:g.

11. The method of claim 10, wherein, The amount of the styrene monooxygenase is 0.02g-10g.

12. The method of claim 7, wherein, The reaction temperature for the catalytic reaction is 20-40℃.

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