Non-specific peroxygenase mutant and application thereof

Abstract

The invention discloses a non-specific peroxygenase mutant and an application thereof. According to the method, Collariella virescens UPO of which the protein structure is analyzed is rationally designed by utilizing a multiple computer-aided strategy, and through functional verification, a UPO mutant library which has rich enantioselectivity to various substrates and is improved in activity is successfully obtained, so that a solid foundation is laid for researching and developing a green and efficient chiral compound production method. Compared with the prior art, the non-specific peroxygenase mutant provided by the invention shows high catalytic activity in selective oxidation of carbon-hydrogen bonds of various substrates, the selectivity is remarkably improved or overturned, the obtained reaction product is high in optical purity, the reaction efficiency is improved, the separation process is simplified, the cost is reduced, and the non-specific peroxygenase mutant is suitable for industrial production. Good industrial application prospects are realized.

Description

[0001] This application is a divisional application of Chinese invention patent application No. "202510801527.2" entitled "Non-specific peroxyase mutant and its application". Technical Field

[0002] This invention belongs to the fields of molecular biology and bioengineering technology, and specifically relates to a non-specific peroxygenase mutant and its application. Background Technology

[0003] The selective oxidation of inert C-H bonds is of great significance in the synthetic chemistry of chiral drug molecules. Compared with chemical methods, which typically require high temperature, high pressure, and multiple steps of functional group protection and deprotection, enzymatic oxidation of C-H bonds offers advantages such as mild reaction conditions, high selectivity, and environmental friendliness, making it an important supporting technology for the development of green manufacturing. Cytochrome P450 and unspecific peroxygenase (UPO) have attracted widespread attention as biocatalysts for the oxidation of C-H bonds in the asymmetric synthesis of chiral alcohols. Compared with P450, UPO can utilize inexpensive H2O2 to initiate the reaction, eliminating the need for expensive co-reactants and cofactors; moreover, its electron transport mechanism is relatively simple, promising to provide a simple and economical synthetic route for the synthesis of chiral alcohols.

[0004] Existing UPO research focuses on Aae UPO and its mutants Aae Regarding the modification and application of UPO-PaDa (Nature Communications, 2024, 15, 831; Angew. Chem. Int. Ed. 2023, 62, e202214759; Biotechnology Advances, 2021, 51, 107615; Current Opinion in Green and Sustainable Chemistry 2023, 41, 100786). Miguel Alcalde et al. previously obtained improved expression and activity through directed evolution. Aae The UPO mutant PaDa (Appl Environ Microbiol. 2014, 80, 11, 3496-3507) was then used as a starting point to rationally design and obtain the variant pada-1_A77L with enhanced catalytic activity for fatty acids (Angew. Chem. Int. Ed. 2023, e202217372). Recently, this research group has also obtained a variant with significantly improved enantioselectivity and activity through combinatorial mutations. AaeThe UPO_PaDa mutant (J. Am. Chem. Soc. 2023, 145, 6, 3443-3453). Zhang Wuyuan's team, however, used a semi-rational design to improve... Aae The ability of UPO to catalyze sterols (ACS Catal. 2025, 15, 1952–1960). However, the catalytic selectivity and activity of UPO reported so far are still very limited and far from meeting the requirements of industrial production. Therefore, improving the activity and / or selectivity of UPO is of great application value for establishing efficient synthetic systems for chiral alcohols. Summary of the Invention

[0005] In view of the problems existing in the current non-specific peroxygenases, the primary objective of the present invention is to provide a non-specific peroxygenase mutant.

[0006] Another object of the present invention is to provide the application of the above-mentioned nonspecific peroxygenase mutant.

[0007] The objective of this invention is achieved through the following technical solution: A nonspecific peroxyase mutant, abbreviated as Cvi The UPO mutant, M9, is a nonspecific peroxygenase whose amino acid sequence, as shown in SEQ ID NO.1, has lysine at position 165 replaced with alanine.

[0008] A nucleic acid molecule that encodes the aforementioned nonspecific peroxygenase mutant.

[0009] A recombinant expression vector containing the aforementioned nucleic acid molecules.

[0010] The backbone of the recombinant expression vector is preferably derived from a prokaryotic expression vector; more preferably from the pET series vectors; and most preferably from pET28a.

[0011] An expression cell containing the above-mentioned recombinant expression vector.

[0012] The starting cell for the expression cell is preferably a prokaryotic cell; more preferably, it is Escherichia coli.

[0013] The application of the above-mentioned nonspecific peroxygenase mutant in the catalytic synthesis of chiral compounds preferably includes the following steps: preparing a reaction system: mixing the substrate, the above-mentioned nonspecific peroxygenase mutant, an organic solvent for solubilization, hydrogen peroxide and a buffer solution to obtain a reaction system; reacting the obtained reaction system at 25-35°C, supplementing hydrogen peroxide during the reaction according to the product formation rate to obtain a chiral compound.

[0014] The substrate is an aliphatic hydrocarbon, cycloalkanes, or aromatic hydrocarbon compound; preferably a compound as shown in Formula I or Formula II: ; Wherein, R1 is an alkyl, alkenyl, or ester group; R2 is hydrogen or halogen; R is hydrogen or halogen; X is hydrogen or oxygen; n = 1 or 2.

[0015] Preferably, R1 is an alkyl group and R2 is hydrogen or bromine; R1 is an ester group and R2 is hydrogen; R is hydrogen and X is hydrogen, n=1 or 2; R is hydrogen or bromine and X is oxygen, n=2.

[0016] More preferably, R1 is an ester group and R2 is hydrogen; R is hydrogen and X is hydrogen, n=1; R is hydrogen or bromine and X is oxygen, n=2.

[0017] The preferred substrates are ethylbenzene, styrene, tetrahydronaphthalene, p-bromoethylbenzene, o-bromoethylbenzene, m-bromoethylbenzene, p-chlorostyrene, m-chlorostyrene, o-chlorostyrene, indene, 3,4-dihydrobenzopyran, and 6-bromobenzopyran.

[0018] The concentration of the substrate in the reaction system is preferably 1 to 10 mmol / L.

[0019] The concentration of the nonspecific peroxygenase mutant in the reaction system is preferably 5–10 mg / L; more preferably 8 mg / L.

[0020] The organic solvent is preferably acetonitrile.

[0021] The concentration of the organic solvent in the reaction system is 5-30% by volume; more preferably 5-10% by volume.

[0022] The buffer solution is preferably a disodium hydrogen phosphate-citric acid buffer, a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, a Tris-HCl buffer, or a glycine-sodium hydroxide buffer; more preferably a disodium hydrogen phosphate-citric acid buffer with pH 5-6, a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer with pH 6-7, a Tris-HCl buffer with pH 7-8, or a glycine-sodium hydroxide buffer with pH 8-9.

[0023] The concentration of the buffer solution in the reaction system is preferably 40–60 mmol / L; more preferably 50 mmol / L.

[0024] The pH of the reaction system is preferably 3-9; more preferably 5-9; further preferably 6-8; and most preferably 7.

[0025] The concentration of hydrogen peroxide in the reaction system is preferably 1 to 3 mmol / L.

[0026] The preferred temperature for the reaction is 30°C.

[0027] The reaction time is preferably 1 to 10 h; more preferably 2 to 10 h.

[0028] The hydrogen peroxide is preferably added at a rate of 0.5 to 5 mM / h during the reaction.

[0029] The chiral compound is preferably a chiral alcohol or a chiral epoxide.

[0030] When the substrate is 6-bromobenzopyran, the chiral compound is a chiral alcohol, specifically 6-bromotrypan-4-ol.

[0031] When the substrate is ethylbenzene, the chiral compound is a chiral alcohol, specifically 1-phenylethanol.

[0032] The substrate is styrene, and the chiral compound is a chiral epoxide, specifically epoxide phenyl ethane.

[0033] When the substrate is tetrahydronaphthalene, the chiral compound is a chiral alcohol, specifically α-tetrahydronaphthaleneol.

[0034] When the substrate is p-bromoethylbenzene, the chiral compound is a chiral alcohol, specifically 1-(4-bromophenyl)-1-ethanol.

[0035] When the substrate is o-bromoethylbenzene, the chiral compound is a chiral alcohol, specifically 1-(2-bromophenyl)-1-ethanol.

[0036] When the substrate is m-bromoethylbenzene, the chiral compound is a chiral alcohol, specifically 1-(3-bromophenyl)-1-ethanol.

[0037] When the substrate is p-chlorostyrene, the chiral compound is a chiral epoxide, specifically 4-chlorostyrene epoxide.

[0038] When the substrate is m-chlorostyrene, the chiral compound is a chiral epoxide, specifically 3-chlorostyrene epoxide.

[0039] When the substrate is o-chlorostyrene, the chiral compound is a chiral epoxide, specifically 2-chlorostyrene epoxide.

[0040] When the substrate is indane, the chiral compound is a chiral alcohol, specifically 1-indane alcohol.

[0041] The substrate is 3,4-dihydrobenzopyran, and the chiral compound is a chiral alcohol, specifically 4-diacetyl alcohol.

[0042] The present invention has the following advantages and effects compared with the prior art: The applicant used multiple computer-aided strategies to analyze the resolved protein structures. Collariella virescens UPO ( Cvi Through rational design and functional verification, a library of UPO mutants with rich enantioselectivity to a variety of substrates and enhanced activity was successfully obtained, laying a solid foundation for the research and development of green and efficient methods for producing chiral compounds.

[0043] Compared with existing technologies, the non-specific peroxygenase mutant provided by this invention exhibits high catalytic activity, significantly improved or reversed selectivity in the selective oxidation of C-H bonds of various substrates, and the resulting reaction products have high optical purity, thus improving reaction efficiency, simplifying the separation process, reducing costs, and showing good prospects for industrial application. Attached Figure Description

[0044] Figure 1 This is a schematic diagram of the recombinant expression plasmid pET28a-UPO.

[0045] Figure 2 This is an SDS-PAGE electrophoresis image of wild-type nonspecific peroxygenases; where: lane M is the protein marker; lane 1 is the protein loading flow-through buffer; lane 2 is 40 mM imidazole elution buffer; lane 3 is 90 mM imidazole elution buffer; lane 4 is 125 mM imidazole elution buffer; lane 5 is 175 mM imidazole elution buffer; lane 6 is 200 mM imidazole elution buffer; and lane 7 is 250 mM imidazole elution buffer.

[0046] Figure 3 The image shows the SDS-PAGE electrophoresis of the purified enzymes of the nonspecific peroxyase mutants; where lane M is the protein marker; lanes 1-6 in a are mutants M1-M6 respectively; lanes 1-4 in b are mutants M7-M10 respectively.

[0047] Figure 4 This is a substrate range diagram for the asymmetric oxidation catalyzed by wild-type and high-performance mutant nonspecific peroxyases.

[0048] Figure 5 The graph shows the yield of chiral alcohols produced by the mutant nonspecific peroxygenase under optimal conditions. Detailed Implementation

[0049] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to specific related embodiments. However, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0050] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0051] Example 1. Preparation of wild-type nonspecific peroxyase The gene carrying the wild-type nonspecific peroxygenase gene (derived from) Collariella virescens (The nucleotide sequence is shown in SEQ ID NO. 6, and the amino acid sequence is shown in SEQ ID NO. 1) EcoR I and Sac I. The restriction site was cloned into pET28a to obtain the following: Figure 1 The recombinant plasmid pET28a-UPO is shown. The recombinant plasmid pET28a-UPO was transformed into Escherichia coli C43(DE3) (UBO Biotechnology, Changsha) to obtain recombinant bacteria.

[0052] Recombinant bacteria were inoculated into 40 mL of LB liquid medium containing 50 mg / L kanamycin and cultured at 37°C and 220 rpm for 12 hours. They were then transferred to 200 mL of ZYM-5052 medium containing 50 mg / L kanamycin and cultured at 16°C and 180 rpm for 72 hours. The bacterial cells were collected by centrifugation, the supernatant was discarded, and the cells were resuspended in phosphate buffer (pH 7.4) containing 40 mM imidazole and 500 mM NaCl. The cells were lysed using lysozyme and sonication, followed by centrifugation at 12000 rpm for 30 min. The supernatant was collected and injected into a nickel column. Elution was performed sequentially with phosphate buffers containing 40, 90, 125, 250, and 500 mM imidazole, collecting the eluent at a concentration of 250 mM imidazole. The eluent was desalted and concentrated by ultrafiltration until the remaining volume was approximately 1 mL. The purified protein was analyzed by SDS-PAGE, and the results are shown below. Figure 2 As shown. Protein concentration was quantified using a protein concentration assay kit from Shanghai Bioengineering Co., Ltd.

[0053] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.2, sterilized at 121℃ for 20 min; solid medium is supplemented with 20 g / L agar powder.

[0054] ZYM-5052 culture medium: 1% tryptone, 0.5% yeast extract, 25 mM disodium hydrogen phosphate, 25 mM potassium dihydrogen phosphate, 50 mM ammonium chloride, 5 mM sodium sulfate, 2 mM magnesium sulfate, 0.5% glycerol, 0.05% glucose, 0.2% α-lactose, 200 μM heme chloride. Except for glycerol, glucose, and α-lactose, the medium was sterilized at 121℃ for 20 min. Glycerol, glucose, and α-lactose were sterilized by filtration to obtain a sterile liquid.

[0055] The solvent for the culture medium is deionized water.

[0056] Example 2: Design of a nonspecific peroxyase mutant library Using the wild-type nonspecific peroxygenase amino acid sequence or crystal structure (PDB ID 7zcl) as input, M1 and M2 were first obtained through rationally designed site-directed mutagenesis. Then, key amino acid sites were determined using hotSpot wizard, CAVER, molecular dynamics simulation, and molecular docking. Subsequently, the key amino acid sites were input into FuncLib for combinatorial mutagenesis calculations to obtain combinatorial mutants with M1 and M2 as the parent genes, respectively.

[0057] The M1 mutant is formed by replacing lysine at position 165 of the wild-type nonspecific peroxygenase with leucine (M is the amino acid corresponding to ATG, which is not counted in the numbering, the same below). Its amino acid sequence is shown in SEQ ID NO.2, and its encoding nucleotide sequence is shown in SEQ ID NO.3. The M2 mutant is formed by replacing methionine at position 210 of the wild-type nonspecific peroxygenase with leucine. Its amino acid sequence is shown in SEQ ID NO.4, and its encoding nucleotide sequence is shown in SEQ ID NO.5.

[0058] Example 3 Construction of a nonspecific peroxyase mutant library Using the pET28a recombinant plasmid containing the wild-type UPO gene (i.e., pET28a-UPO) as a template, site-directed mutagenesis was performed at the above-mentioned sites. Primers were designed using snapgene software (as shown in Table 1, synthesized by Shanghai Bioengineering Co., Ltd.), and then PCR site-directed mutagenesis was performed. The PCR reaction system was as follows: 1 μL template, 12.5 μL DNA polymerase (Takara), 1 μL F primer, 1 μL R primer, and ddH2O to a final volume of 25 μL. The PCR reaction conditions were as follows: preheating at 98℃ for 3 min; denaturation at 98℃ for 10 s, annealing at 62℃ for 30 s, extension at 72℃ for 90 s, for 28 cycles; and a final extension at 72℃ for 5 min.

[0059] Table 1. Primer sequences

[0060] PCR products were detected by 1% agarose gel electrophoresis, then recovered using a Shanghai Sangon Biotech nucleic acid purification kit, and ligated after concentration determination.

[0061] The seamless cloning procedure is as follows: The linearized vector and the target fragment are mixed at a molar ratio of 2:6–9, and water is added to bring the volume to 10 μL. Then, 10 μL of HB-infusion is added, and ligation is carried out at 50℃ for 30–40 min. The mixture is then transformed into *E. coli* C43(DE3) competent cells, plated on LB agar plates (containing 50 μg / mL kanamycin), and incubated overnight at 37℃. The next day, positive transformants are screened using universal primers T7-F (taatacgactcactatagg) and T7-R (gctagttattgctcagcgg), and recombinant plasmids are extracted and sent to Shanghai Bioengineering Co., Ltd. for sequencing verification.

[0062] Using recombinant plasmid pET28a-UPO as a template, PCR was performed with primer pairs 165L-F / UPO-R and UPO-F / 165L-R to obtain target fragment A and target fragment B, respectively. PCR was then performed with 28a-F and 28a-R to obtain linearized vector A. Target fragment A, target fragment B, and linearized vector A were mixed in a molar ratio of 3:3:2, and seamless cloning was performed according to the above steps to obtain pET28a-M1.

[0063] Using recombinant plasmid pET28a-UPO as a template, PCR was performed with primer pairs M210L-F / UPO-R and UPO-F / M210L-R to obtain target fragment C and target fragment D, respectively. PCR was then performed with 28a-F and 28a-R to obtain linearized vector A. Target fragment C, target fragment D, and linearized vector A were mixed in a molar ratio of 3:3:2, and seamless cloning was performed according to the above steps to obtain pET28a-M2.

[0064] Using pET28a-M1 as a template, PCR was performed using primer pairs L64Q-F / Q154L_I157K_T158L_165L-R, UPO-F / L64Q-R, and Q154L_I157K_T158L_165L-F / UPO-R to obtain target fragments E, F, and G, respectively; PCR was performed using 28a-F and 28a-R to obtain linearized vector A. Target fragments E, F, and G were mixed with linearized vector A at a molar ratio of 3:3:3:2, and seamless cloning was performed according to the above steps to obtain pET28a-M3.

[0065] Using pET28a-M1 as a template, PCR was performed using primers L64Q-F / Q154L_T158L_165L-R, UPO-F / L64Q-R, and Q154L_T158L_165L-F / UPO-R to obtain target fragments H, I, and J, respectively; PCR was performed using 28a-F and 28a-R to obtain linearized vector A. Target fragments H, I, and J were mixed with linearized vector A at a molar ratio of 3:3:3:2, and seamless cloning was performed according to the above steps to obtain pET28a-M4.

[0066] Using pET28a-M2 as a template, PCR was performed using primers F88A-F / Q154L_I157L_T158L_G161S-R, UPO-F / F88A-R, and Q154L_I157L_T158L_G161S-F / UPO-R to obtain target fragments K, L, and M, respectively; PCR was performed using 28a-F and 28a-R to obtain linearized vector A. Target fragments K, L, and M were mixed with linearized vector A at a molar ratio of 3:3:3:2, and seamless cloning was performed according to the above steps to obtain pET28a-M5.

[0067] Using pET28a-M2 as a template, PCR was performed using primers F88A-F / I157L_T158L_G161S-R, UPO-F / F88A-R, and I157L_T158L_G161S-F / UPO-R to obtain the target fragments N, O, and P, respectively. PCR was then performed using 28a-F and 28a-R to obtain the linearized vector A. The target fragments N, O, and P were mixed with the linearized vector A at a molar ratio of 3:3:3:2, and seamless cloning was performed according to the above steps to obtain pET28a-M6.

[0068] Using pET28a-M2 as a template, PCR was performed using primers L87V_F88I-F / Q154T_T158L-R, UPO-F / L87V_F88I-R, and Q154T_T158L-F / UPO-R to obtain the target fragments Q, R, and S, respectively. PCR was then performed using 28a-F and 28a-R to obtain the linearized vector A. The target fragments Q, R, and S were mixed with the linearized vector A at a molar ratio of 3:3:3:2, and seamless cloning was performed according to the above steps to obtain pET28a-M7.

[0069] Using pET28a-M2 as a template, PCR was performed using primers Q154K_I157N_T158I_165F-F / UPO-R and UPO-F / Q154K_I157N_T158I_165F-R to obtain the target fragments T and U, respectively; PCR was performed using 28a-F and 28a-R to obtain the linearized vector A. The target fragments T and U were mixed with the linearized vector A at a molar ratio of 3:3:2, and seamless cloning was performed according to the above steps to obtain pET28a-M8.

[0070] Using pET28a-M1 as a template, PCR was performed using primers 165A-F / UPO-R and UPO-F / 165A-R to obtain target fragments V and W, respectively; PCR was performed using primers 28a-F and 28a-R to obtain linearized vector A. Target fragment V, target fragment W, and linearized vector A were mixed in a molar ratio of 3:3:2, and seamless cloning was performed according to the above steps to obtain pET28a-M9.

[0071] Using pET28a-M1 as a template, PCR was performed using primers 165G-F / UPO-R and UPO-F / 165G-R to obtain target fragments X and Y, respectively; PCR was performed using primers 28a-F and 28a-R to obtain linearized vector A. Target fragment X, target fragment Y, and linearized vector A were mixed in a molar ratio of 3:3:2, and seamless cloning was performed according to the above steps to obtain pET28a-M10.

[0072] Using pET28a-M1 as a template, PCR was performed using primers UPO-F / L64M-R, L64M-F / Q154L_I157E_T158L_165M-R, and Q154L_I157E_T158L_165M-F / UPO-R to obtain target fragments B2, B3, and B4, respectively; PCR was performed using 28a-F and 28a-R to obtain linearized vector A. Target fragments B2, B3, and B4 were mixed with linearized vector A at a molar ratio of 3:3:3:2, and seamless cloning was performed according to the above steps to obtain pET28a-M11.

[0073] Using pET28a-M1 as a template, PCR was performed using primers UPO-F / T60A-R, T60A-F / Q154T_I157N_T158F_165M-R, and Q154T_I157N_T158F_165M-F / UPO-R to obtain target fragments C2, C3, and C4, respectively; PCR was performed using 28a-F and 28a-R to obtain linearized vector A. Target fragments C2, C3, and C4 were mixed with linearized vector A at a molar ratio of 3:3:3:2, and seamless cloning was performed according to the above steps to obtain pET28a-M12.

[0074] Using pET28a-M2 as a template, PCR was performed using primers UPO-F / L87V-R, L87V-F / Q154K_I157K_T158I-R, and Q154K_I157K_T158I-F / UPO-R to obtain target fragments D2, D3, and D4, respectively; PCR was performed using 28a-F and 28a-R to obtain linearized vector A. Target fragments D2, D3, and D4 were mixed with linearized vector A at a molar ratio of 3:3:3:2, and seamless cloning was performed according to the above steps to obtain pET28a-M13.

[0075] Example 4: Detection of catalytic efficiency and selectivity of wild-type and mutant nonspecific peroxyases Wild-type and mutant proteins were purified according to Example 1. Figure 3 ), and then catalytic efficiency and selectivity tests were conducted.

[0076] The reaction system contained 50 mM buffer solutions with pH 5.0-9.0 (specifically, disodium hydrogen phosphate-citric acid buffer at pH 5-6, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer at pH 6-7, Tris-HCl buffer at pH 7-8, and glycine-sodium hydroxide buffer at pH 8-9). Substrate (ethylbenzene, styrene, tetrahydronaphthalene, p-bromoethylbenzene, o-bromoethylbenzene, m-bromoethylbenzene, p-chlorostyrene, m-chlorostyrene, o-chlorostyrene, indane, 3,4-dihydrobenzopyran, 6-bromobenzopyran) was added to a final concentration of 1 mM, along with acetonitrile (10% v / v), 8 mg / L purified enzyme, and 1 mM H2O2. Water was then added to bring the total volume to 1 mL to obtain the reaction solution.

[0077] Wild-type and mutant purified enzymes of equal concentrations, with a final concentration of 1 mM substrate (containing 10% acetonitrile), 1 mM hydrogen peroxide, pH=7, and 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, were reacted in a metal bath at 30°C for 2 hours (rxn. Time=2h). After the reaction, ethyl acetate was added at a volume ratio of 1:1 to terminate the reaction. The sample was vortexed for 30 s, followed by centrifugation at 12000 rpm for 1 min. The upper organic phase was collected, dried with anhydrous sodium sulfate, and the content of the corresponding chiral alcohol was detected by gas chromatography (chiral capillary column CP-Chirasil-Dex-CB). The detection conditions were: nitrogen as carrier gas, rate 1.0 mL / min, 100°C for 2 min, increased to 180°C at a rate of 10°C / min and held for 5 min, followed by increased to 200°C at a rate of 2°C / min and held for 3 min. TON refers to the product amount divided by the enzyme concentration. Therefore, given the same enzyme concentration and reaction time, it reflects the amount of product generated, i.e., the magnitude of catalytic activity. For example, when ethylbenzene is used as a substrate, the TON of M7 (TON = 173) is 5.2 times that of the wild type (TON = 33). ee The value reflects the preference for configuration in the product. ee The higher the value, the purer the product. ee ( R )or ee ( S Most of them are fine, because the product is a chiral alcohol. R or S Both configurations can serve as building blocks for chirality. For example, when p-bromoethylbenzene is used as a substrate, M5 not only shows a significant increase in activity, but also... ee The activity values ​​were also significantly improved compared to the wild type. M3, in particular, showed increased activity and a reversal of selectivity compared to the wild type, which is very advantageous. Gas chromatography analysis revealed that the mutants M1-M10 exhibited 1-78.3 times the activity of the wild type, with improved selectivity or chiral reversal. ee The value can reach up to 99% ( Figure 4 However, not all mutations can improve or reverse selectivity while maintaining activity. For example, when using ethylbenzene as a substrate, the activities of mutants M11 and M12 are 63% and 84% of the wild type, respectively. When using styrene as a substrate, the activity of mutant M13 is only 44% of the wild type. The mutants M1-M10 we provide, however, can improve or reverse selectivity while maintaining activity greater than or equal to that of the wild type.

[0078] Example 5: Application of nonspecific peroxygenase mutants in chiral alcohol production Using the mutant enzyme obtained in Example 3 as a catalyst, the reaction system contained 50 mM buffer solution at pH 5.0-9.0 (specifically, disodium hydrogen phosphate-citric acid buffer at pH 5-6, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer at pH 6-7, Tris-HCl buffer at pH 7-8, and glycine-sodium hydroxide buffer at pH 8-9), 10 mM substrate (Formula I and Formula II were used as substrates), and 5%-30% acetonitrile, and the chiral alcohol was produced at 30°C.

[0079] Preferably, 6-bromobenzopyran was used as the substrate (final concentration 10 mM), the final concentration of the mutant M3 enzyme was 8 mg / L, the final concentration of acetonitrile was 5%, the initial concentration of hydrogen peroxide in the reaction system was 2 mM, and the pH was 5 (disodium hydrogen phosphate-citrate buffer, final concentration 50 mM). 1-3 mM was added every 2 hours (after periodic sampling and testing, the addition was based on the product formation rate to maintain a relatively constant value), and the reaction was carried out at 30°C for 10 h. Finally, an equal volume of ethyl acetate was added to terminate the reaction. The remaining substrate and product (6-bromotryptane-4-ol(R)) content were detected using gas chromatography (chiral capillary column CP-Chirasil-Dex-CB). Analysis of the gas chromatography results showed that the final conversion rate could reach 60%. Figure 5 ).

[0080] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A nonspecific peroxygenase mutant, characterized in that: The nonspecific peroxygenase mutant is mutant M9, which is formed by replacing lysine at position 165 in the nonspecific peroxygenase shown in SEQ ID NO.1 with alanine.

2. A nucleic acid molecule, characterized in that: It is the encoding nucleic acid of the nonspecific peroxyase mutant as described in claim 1.

3. A recombinant expression vector, characterized in that: It contains the nucleic acid molecule as described in claim 2.

4. An expression cell, characterized in that: Contains the recombinant expression vector as described in claim 3.

5. The application of the nonspecific peroxygenase mutant of claim 1 in the catalytic synthesis of chiral compounds.

6. The application according to claim 5, characterized in that... The process includes the following steps: preparing the reaction system: mixing the substrate, the non-specific peroxygenase mutant of claim 1, the organic solvent for solubilization, hydrogen peroxide, and buffer solution to obtain the reaction system; reacting the obtained reaction system at 25–35°C, supplementing hydrogen peroxide during the reaction according to the product formation rate to obtain the chiral compound; The substrate is an aliphatic hydrocarbon, cycloalkanes, or aromatic hydrocarbon compound; The pH of the reaction system is 3 to 9.

7. The application according to claim 6, characterized in that: The substrate is a compound as shown in Formula I or Formula II: ； Wherein, R1 is an alkyl, alkenyl, or ester group; R2 is hydrogen or halogen; R is hydrogen or halogen; X is hydrogen or oxygen; n = 1 or 2; The organic solvent is acetonitrile; The buffer solution is a disodium hydrogen phosphate-citric acid buffer, a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, a Tris-HCl buffer, or a glycine-sodium hydroxide buffer.

8. The application according to claim 6, characterized in that: The concentration of the substrate in the reaction system is 1–10 mmol / L; The concentration of the nonspecific peroxygenase mutant in the reaction system is 5–10 mg / L; The concentration of the organic solvent in the reaction system is 5-30% by volume. The concentration of the buffer solution in the reaction system is 40–60 mmol / L; The pH of the reaction system is 5–9; The concentration of hydrogen peroxide in the reaction system is 1–3 mmol / L; The hydrogen peroxide is added at a rate of 0.5-5 mM / h during the reaction.

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