Non-specific peroxygenase mutant and application thereof

Abstract

The invention discloses a non-specific peroxygenase mutant and application of the non-specific peroxygenase mutant. 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

TECHNICAL FIELD

[0001] The present application belongs to the field of molecular biology and bioengineering technology, and particularly relates to a non-specific peroxygenase mutant and application thereof. BACKGROUND

[0002] The selective oxidation of inert carbon-hydrogen bonds has important significance in synthetic chemistry of chiral drug molecules. Compared with the synthesis steps in chemical method which usually need to use high temperature and high pressure and multi-step functional group protection and deprotection, the enzyme catalytic oxidation of carbon-hydrogen bond has the advantages of mild reaction condition, good selectivity, environmental friendliness and the like, and is an important supporting technology for the development of green intelligent manufacturing. Cytochrome P450 and non-specific peroxygenase (unspecific peroxygenase, UPO) as biological catalysts capable of catalyzing the oxidation of carbon-hydrogen bond have been widely concerned in the asymmetric synthesis of chiral alcohol. Compared with P450, UPO can utilize cheap H2O2 to start the reaction, and does not need expensive co-reactants and cofactors; and the electron transfer mechanism is relatively simple, which is expected to provide a simple and economical synthesis path for the synthesis of chiral alcohol.

[0003] Existing UPO research focuses on the modification and application of AaeUPO and its mutant AaeUPO-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 used directed evolution to obtain an AaeUPO mutant, PaDa, with improved expression and activity (Appl Environ Microbiol. 2014, 80, 11, 3496-3507). Using this mutant as a starting point, they then used rational design to obtain a variant, pada-1_A77L, with improved fatty acid catalytic activity (Angew. Chem. Int. Ed. 2023, e202217372). Recently, the same group used combinatorial mutagenesis to obtain an AaeUPO_PaDa mutant with significantly improved enantioselectivity and activity (J. Am. Chem. Soc. 2023, 145, 6, 3443-3453). Zhang Wuyuan's team used semi-rational design to improve the sterol catalytic activity of AaeUPO (ACS Catal. 2025, 15, 1952-1960). However, the catalytic selectivity and activity of UPOs reported so far remain very limited, far from meeting industrial production requirements. Therefore, improving the activity and / or selectivity of UPO has important application value in establishing an efficient synthesis system for chiral alcohols. Summary of the Invention

[0004] In view of the problems existing in existing non-specific peroxygenases, the primary purpose of the present invention is to provide a non-specific peroxygenase mutant.

[0005] Another object of the present invention is to provide the use of the non-specific peroxygenase mutant.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A nonspecific peroxygenase mutant, referred to as a CviUPO mutant, is obtained by mutating at least one of the amino acids 64, 87, 88, 154, 157, 158, 161, 165, and 210 in the nonspecific peroxygenase with an amino acid sequence as shown in SEQ ID NO. 1; the mutant has higher catalytic performance than the nonspecific peroxygenase with an amino acid sequence as shown in SEQ ID NO. 1.

[0008] Preferably, the 64th is mutated from leucine to glutamine.

[0009] Preferably, the 87th is mutated from leucine to valine.

[0010] Preferably, the 88th is mutated from phenylalanine to alanine or isoleucine.

[0011] Preferably, the 154th is mutated from glutamine to leucine or threonine.

[0012] Preferably, the 157th is mutated from isoleucine to lysine, leucine or asparagine.

[0013] Preferably, the 158th is mutated from threonine to leucine or isoleucine.

[0014] Preferably, the 161st is mutated from glycine to serine.

[0015] Preferably, the 165th is mutated from lysine to leucine, phenylalanine, alanine or glycine.

[0016] Preferably, the 210th is mutated from methionine to leucine.

[0017] Preferably, the non-specific peroxygenase mutant is any one of the following mutants:

[0018] (1) the lysine at the 165th in the non-specific peroxygenase having the amino acid sequence shown in SEQ ID NO. 1 is replaced by leucine, which is mutant M1;

[0019] (2) the methionine at the 210th in the non-specific peroxygenase having the amino acid sequence shown in SEQ ID NO. 1 is replaced by leucine, which is mutant M2;

[0020] (3) the leucine at the 64th in the mutant M1 is replaced by glutamine, the glutamine at the 154th is replaced by leucine, the isoleucine at the 157th is replaced by lysine, and the threonine at the 158th is replaced by leucine, which is mutant M3;

[0021] (4) the leucine at the 64th in the mutant M1 is replaced by glutamine, and the glutamine at the 154th is replaced by leucine, which is mutant M4;

[0022] (5) the phenylalanine at the 88th in the mutant M2 is replaced by alanine, the glutamine at the 154th is replaced by leucine, the isoleucine at the 157th is replaced by leucine, the threonine at the 158th is replaced by leucine, and the glycine at the 161st is replaced by serine, which is mutant M5;

[0023] (6) the phenylalanine at position 88, the isoleucine at position 157, the threonine at position 158, and the glycine at position 161 in mutant M2 are replaced by alanine, leucine, leucine, and serine, respectively, to obtain mutant M6;

[0024] (7) the leucine at position 87, the phenylalanine at position 88, the glutamine at position 154, and the threonine at position 158 in mutant M2 are replaced by valine, isoleucine, threonine, and leucine, respectively, to obtain mutant M7;

[0025] (8) the glutamine at position 154, the isoleucine at position 157, the threonine at position 158, and the lysine at position 165 in mutant M2 are replaced by lysine, asparagine, isoleucine, and phenylalanine, respectively, to obtain mutant M8;

[0026] (9) the lysine at position 165 in the non-specific peroxygenase having the amino acid sequence shown in SEQ ID NO. 1 is replaced by alanine to obtain mutant M9;

[0027] (10) the lysine at position 165 in the non-specific peroxygenase having the amino acid sequence shown in SEQ ID NO. 1 is replaced by glycine to obtain mutant M10.

[0028] The amino acid sequence of the mutant M1 is shown in SEQ ID NO. 2.

[0029] The amino acid sequence of the mutant M2 is shown in SEQ ID NO. 4.

[0030] A nucleic acid molecule is a coding nucleic acid of the above-mentioned non-specific peroxygenase mutant.

[0031] The nucleotide sequence of the coding nucleic acid of the mutant M1 is shown in SEQ ID NO. 3.

[0032] The nucleotide sequence of the coding nucleic acid of the mutant M2 is shown in SEQ ID NO. 5.

[0033] A recombinant expression vector contains the above-mentioned nucleic acid molecule.

[0034] The backbone of the recombinant expression vector is preferably derived from a prokaryotic expression vector; more preferably, it is a pET series vector; most preferably, it is pET28a.

[0035] An expression cell contains the above-mentioned recombinant expression vector.

[0036] The starting cell of the expression cell is preferably a prokaryotic cell; more preferably, it is Escherichia coli.

[0037] The use of the above-mentioned nonspecific peroxygenase mutant in the catalytic synthesis of chiral compounds preferably includes the following steps: preparing a reaction system: mixing a substrate, the above-mentioned nonspecific peroxygenase mutant, an organic solvent for promoting dissolution, hydrogen peroxide and a buffer 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.

[0038] The substrate is an aliphatic hydrocarbon, a cycloalkane or an aromatic hydrocarbon compound; preferably a compound as shown in Formula I or Formula II:

[0039]

[0040] Wherein, R1 is an alkyl group, an alkenyl group or an ester group;

[0041] R2 is hydrogen or halogen;

[0042] R is hydrogen or halogen;

[0043] X is hydrogen or oxygen;

[0044] n=1 or 2.

[0045] Preferably, R1 is an alkyl group, R2 is hydrogen or bromine; R1 is an ester group, R2 is hydrogen; R is hydrogen, X is hydrogen, n=1 or 2; R is hydrogen or bromine, X is oxygen, n=2.

[0046] More preferably, R1 is an ester group, R2 is hydrogen; R is hydrogen, X is hydrogen, and n=1; R is hydrogen or bromine, X is oxygen, and n=2.

[0047] The substrate is most preferably ethylbenzene, styrene, tetralin, p-bromoethylbenzene, o-bromoethylbenzene, m-bromoethylbenzene, p-chlorostyrene, m-chlorostyrene, o-chlorostyrene, indane, 3,4-dihydrobenzopyran, and 6-bromobenzopyran.

[0048] The concentration of the substrate in the reaction system is preferably 1 to 10 mmol / L.

[0049] The concentration of the non-specific peroxygenase mutant in the reaction system is preferably 5-10 mg / L, more preferably 8 mg / L.

[0050] The organic solvent is preferably acetonitrile.

[0051] The concentration of the organic solvent in the reaction system is 5 to 30% by volume, more preferably 5 to 10% by volume.

[0052] The buffer is preferably sodium hydrogen phosphate-citric acid buffer, sodium hydrogen phosphate-sodium dihydrogen phosphate buffer, Tris-HCl buffer or glycine-sodium hydroxide buffer; more preferably sodium hydrogen phosphate-citric acid buffer of pH 5-6, sodium hydrogen phosphate-sodium dihydrogen phosphate buffer of pH 6-7, Tris-HCl buffer of pH 7-8, or glycine-sodium hydroxide buffer of pH 8-9.

[0053] The concentration of the buffer solution in the reaction system is preferably 40 to 60 mmol / L, more preferably 50 mmol / L.

[0054] The pH of the reaction system is preferably 3-9; more preferably 5-9; further preferably 6-8; and most preferably 7.

[0055] The concentration of the hydrogen peroxide in the reaction system is preferably 1 to 3 mmol / L.

[0056] The reaction temperature is preferably 30°C.

[0057] The reaction time is preferably 1 to 10 hours, more preferably 2 to 10 hours.

[0058] The amount of hydrogen peroxide added during the reaction is preferably at a rate of 0.5 to 5 mM / h.

[0059] The chiral compound is preferably a chiral alcohol or a chiral epoxide.

[0060] When the substrate is 6-bromobenzopyran, the chiral compound is a chiral alcohol, specifically 6-bromochromen-4-ol.

[0061] When the substrate is ethylbenzene, the chiral compound is a chiral alcohol, specifically 1-phenylethanol.

[0062] The substrate is styrene, and the chiral compound is a chiral epoxide, specifically styrene oxide.

[0063] When the substrate is tetralin, the chiral compound is a chiral alcohol, specifically α-tetralinol.

[0064] When the substrate is p-bromoethylbenzene, the chiral compound is a chiral alcohol, specifically 1-(4-bromophenyl)-1-ethanol.

[0065] When the substrate is o-bromoethylbenzene, the chiral compound is a chiral alcohol, specifically 1-(2-bromophenyl)-1-ethanol.

[0066] When the substrate is m-bromoethylbenzene, the chiral compound is a chiral alcohol, specifically 1-(3-bromophenyl)-1-ethanol.

[0067] When the substrate is p-chlorostyrene, the chiral compound is a chiral epoxide, specifically 4-chlorostyrene epoxide.

[0068] When the substrate is m-chlorostyrene, the chiral compound is a chiral epoxide, specifically 3-chlorostyrene epoxide.

[0069] When the substrate is o-chlorostyrene, the chiral compound is a chiral epoxide, specifically 2-chlorostyrene epoxide.

[0070] When the substrate is indane, the chiral compound is a chiral alcohol, specifically 1-indanol.

[0071] The substrate is 3,4-dihydrobenzopyran, and the chiral compound is a chiral alcohol, specifically 4-diacetyl alcohol.

[0072] The present invention has the following advantages and effects compared to the prior art:

[0073] The applicant used multiple computer-assisted strategies to rationally design the solved protein structure of Collariella virescens UPO (CviUPO). After functional verification, they successfully obtained a library of UPO mutants with rich enantioselectivity and enhanced activity for multiple substrates, laying a solid foundation for the research and development of green and efficient methods for the production of chiral compounds.

[0074] Compared with the prior art, the nonspecific peroxygenase mutant provided by the present invention exhibits high catalytic activity and significantly improved or reversed selectivity in the selective oxidation of carbon-hydrogen bonds of various substrates. The obtained reaction products have high optical purity, which improves reaction efficiency, simplifies the separation process, reduces costs, and has good industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 Schematic diagram of the recombinant expression plasmid pET28a-UPO.

[0076] Figure 2 The figure is the SDS-PAGE protein electrophoresis diagram of wild-type nonspecific peroxygenase; wherein: lane M is the protein marker; lane 1 is the protein loading flow-through; lane 2 is the 40mM imidazole eluent; lane 3 is the 90mM imidazole eluent; lane 4 is the 125mM imidazole eluent; lane 5 is the 175mM imidazole eluent; lane 6 is the 200mM imidazole eluent; lane 7 is the 250mM imidazole eluent.

[0077] Figure 3The SDS-PAGE electrophoresis diagram of the pure enzyme of the nonspecific peroxygenase mutants; wherein: 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.

[0078] Figure 4 Figure 3. Substrate range diagram for asymmetric oxidation catalyzed by wild-type and elite peroxygenase mutants.

[0079] Figure 5 The graph shows the yield of chiral alcohol produced by mutant nonspecific peroxygenase under optimal conditions. DETAILED DESCRIPTION

[0080] The technical solutions in the embodiments of the present invention are clearly and completely described below in conjunction with specific relevant embodiments. However, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0081] The experimental methods used in the following examples are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0082] Example 1. Preparation of wild-type nonspecific peroxygenase

[0083] The wild-type nonspecific peroxygenase gene (derived from Collariella virescens, nucleotide sequence as shown in SEQ ID NO.6) was cloned into pET28a through the EcoRI and SacⅠ restriction sites to obtain Figure 1 The recombinant plasmid pET28a-UPO was transformed into Escherichia coli C43 (DE3) (Ubao Biotechnology, Changsha) to obtain recombinant bacteria.

[0084] The recombinant bacteria were inoculated into 40 mL of LB liquid culture 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 culture medium containing 50 mg / L kanamycin and cultured at 16°C and 180 rpm for 72 hours. The bacteria were collected by centrifugation, the supernatant was discarded, and an appropriate amount of phosphate buffer (pH 7.4) containing 40 mM imidazole and 500 mM NaCl was added to resuspend the bacteria. The cells were broken by lysozyme and ultrasound, and centrifuged at 12000 rpm for 30 minutes. The supernatant was collected and injected into the nickel column. The phosphate buffer containing 40, 90, 125, 250 and 500 mM imidazole was used for elution in sequence, and the eluate at a concentration of 250 mM imidazole was collected. The eluate was desalted and concentrated by ultrafiltration until the residual volume reached about 1 mL. The purified protein was analyzed by SDS-PAGE, and the results were as shown below. Figure 2 The protein concentration was quantified using a protein concentration test kit from Shanghai Bioengineering Company.

[0085] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.2, high-temperature sterilization at 121°C for 20 min; solid medium plus 20 g / L agar powder.

[0086] ZYM-5052 medium: 1% tryptone, 0.5% yeast extract, 25 mM sodium phosphate (disodium hydrogen), 25 mM potassium phosphate (monobasic), 50 mM ammonium chloride, 5 mM sodium sulfate, 2 mM magnesium sulfate, 0.5% glycerol, 0.05% glucose, 0.2% α-lactose, 200 μM hemin. All except glycerol, glucose, and α-lactose were sterilized by autoclaving at 121°C for 20 min; glycerol, glucose, and α-lactose were sterilized by filtration to obtain a sterile liquid.

[0087] The solvent of the culture medium is deionized water.

[0088] Example 2 Design of non-specific peroxygenase mutant library

[0089] Using the wild-type nonspecific peroxygenase amino acid sequence or crystal structure (PDB ID 7zcl) as input, M1 and M2 were first obtained through rational site-directed mutagenesis. Key amino acid positions were then identified using the hotSpot wizard, CAVER, molecular dynamics simulations, and molecular docking. These key amino acid positions were then input into FuncLib for combinatorial mutagenesis calculations, resulting in combinatorial mutants using M1 and M2 as parental constructs.

[0090] The M1 mutant is a mutant in which lysine at position 165 of the wild-type nonspecific peroxygenase is replaced with leucine (M is the amino acid corresponding to ATG and 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;

[0091] The M2 mutant is obtained by replacing the 210th methionine of the wild-type non-specific 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.

[0092] Example 3 Construction of a non-specific peroxygenase mutant library

[0093] 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 aforementioned sites. Primers were designed using snapgene software (see Table 1, synthesized by Shanghai Bioengineering Co., Ltd.), followed by PCR-directed mutagenesis. 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 25 μL. The PCR reaction conditions were as follows: preheating at 98°C for 3 min; 28 cycles of denaturation at 98°C for 10 s, annealing at 62°C for 30 s, and extension at 72°C for 90 s; and extension at 72°C for 5 min.

[0094] Table 1. Primer sequences

[0095]

[0096]

[0097] The PCR product was detected by 1% agarose gel electrophoresis and then recovered using the Shanghai Biotechnology Nucleic Acid Purification Kit. After the concentration was determined, ligation was performed.

[0098] The seamless cloning procedure is as follows: Mix the linearized vector and target fragment at a molar ratio of 2:6-9, add water to make up to 10 μL, then add 10 μL of HB-infusion. Ligate at 50°C for 30-40 minutes. Then, transform into competent E. coli C43 (DE3) cells, plate on LB plates (containing 50 μg / mL kanamycin), and incubate at 37°C overnight. The next day, positive transformants are screened using universal primers T7-F (taatacgactcactatagg) and T7-R (gctagttattgctcagcgg). Recombinant plasmids are then extracted and sent to Shanghai Bioengineering Co., Ltd. for sequencing verification.

[0099] Using the recombinant plasmid pET28a-UPO as a template, PCR was performed using primer pairs 165L-F / UPO-R and UPO-F / 165L-R to obtain target fragments A and B, respectively. PCR was performed using primer pairs 28a-F and 28a-R to obtain linearized vector A. Target fragments A, B, and linearized vector A were mixed at a molar ratio of 3:3:2 and seamlessly cloned according to the above steps to obtain pET28a-M1.

[0100] Using the recombinant plasmid pET28a-UPO as a template, PCR was performed using primer pairs M210L-F / UPO-R and UPO-F / M210L-R to obtain target fragments C and D, respectively. PCR was performed using 28a-F and 28a-R to obtain linearized vector A. Target fragments C, D, and linearized vector A were mixed at a molar ratio of 3:3:2 and seamlessly cloned according to the above steps to obtain pET28a-M2.

[0101] 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 generate target fragments E, F, and G, respectively. PCR was performed using 28a-F and 28a-R to generate 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 seamlessly cloned according to the above steps to generate pET28a-M3.

[0102] 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, respectively, to generate target fragments H, I, and J. PCR was performed using 28a-F and 28a-R to generate 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 seamlessly cloned according to the above steps to generate pET28a-M4.

[0103] 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, respectively, to generate target fragments K, L, and M. PCR was performed using 28a-F and 28a-R to generate 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 seamlessly cloned according to the above steps to generate pET28a-M5.

[0104] 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 generate target fragments N, O, and P, respectively. PCR was performed using 28a-F and 28a-R to generate linearized vector A. Target fragments N, O, and P were mixed with linearized vector A at a molar ratio of 3:3:3:2 and seamlessly cloned according to the above steps to generate pET28a-M6.

[0105] 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 generate target fragments Q, R, and S, respectively. PCR was performed using 28a-F and 28a-R to generate linearized vector A. Target fragments Q, R, and S were mixed with linearized vector A at a molar ratio of 3:3:3:2 and seamlessly cloned according to the above steps to generate pET28a-M7.

[0106] 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, respectively, to generate target fragments T and U. PCR was performed using 28a-F and 28a-R to generate linearized vector A. Target fragments T and U were mixed with linearized vector A in a molar ratio of 3:3:2 and seamlessly cloned according to the above steps to generate pET28a-M8.

[0107] 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 fragments V and W were mixed with linearized vector A in a molar ratio of 3:3:2 and seamlessly cloned according to the above steps to obtain pET28a-M9.

[0108] 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 fragments X and Y were mixed with linearized vector A in a molar ratio of 3:3:2 and seamlessly cloned according to the above steps to obtain pET28a-M10.

[0109] 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 generate target fragments B2, B3, and B4, respectively. PCR was performed using primers 28a-F and 28a-R to generate 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 seamlessly cloned according to the above steps to generate pET28a-M11.

[0110] 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 generate target fragments C2, C3, and C4, respectively. PCR was performed using primers 28a-F and 28a-R to generate 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 seamlessly cloned according to the above steps to generate pET28a-M12.

[0111] 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 generate target fragments D2, D3, and D4, respectively. PCR was performed using primers 28a-F and 28a-R to generate 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 seamlessly cloned according to the above steps to generate pET28a-M13.

[0112] Example 4 Catalytic efficiency and selectivity detection of wild-type and mutant non-specific peroxygenase

[0113] The wild type and mutant proteins were purified as in Example 1 ( Figure 3 ), followed by catalytic efficiency and selectivity testing.

[0114] The reaction system contains 50 mM buffer at pH 5.0-9.0 (specifically, sodium hydrogen phosphate-citrate buffer at pH 5-6, sodium 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), substrates (ethylbenzene, styrene, tetralin, p-bromoethylbenzene, o-bromoethylbenzene, m-bromoethylbenzene, p-chlorostyrene, m-chlorostyrene, o-chlorostyrene, indane, 3,4-dihydrobenzopyran, 6-bromobenzopyran) are added to a final concentration of 1 mM, acetonitrile at a final concentration of 10% v / v, 8 mg / L pure enzyme, and 1 mM H2O2, and the system is made up to 1 mL with water to obtain a reaction solution.

[0115] Equal concentrations of wild-type and mutant pure enzymes were reacted in a metal bath at 30°C for 2 hours (rxn. Time = 2 h) with 1 mM hydrogen peroxide (1 mM substrate, pH 7, 50 mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer) and 1 mM hydrogen peroxide. The reaction was terminated by adding ethyl acetate in a 1:1 volume ratio. The sample was vortexed for 30 seconds and then centrifuged at 12,000 rpm for 1 minute. The upper organic phase was collected, dried over anhydrous sodium sulfate, and analyzed for the content of the corresponding chiral alcohol using gas chromatography (chiral capillary column CP-Chirasil-Dex-CB). The assay conditions were: nitrogen carrier gas at a rate of 1.0 mL / min, 100°C maintained for 2 minutes, increased to 180°C at a rate of 10°C / min and held for 5 minutes, then increased to 200°C at a rate of 2°C / min and held for 3 minutes. TON refers to the amount of product divided by the enzyme concentration. When the enzyme concentration and reaction time are the same, it reflects the amount of product generated, that is, the size of the catalytic activity. For example, when ethylbenzene is used as the substrate, the TON of M7 (TON is 173) is 5.2 times that of the wild type (TON is 33). The ee value reflects the configuration preference in the product. The larger the ee value, the purer the product. ee(R) or ee(S) is mostly acceptable, because the product is a chiral alcohol, and its R or S configuration can be used as a chiral building block. For example, when using para-bromoethylbenzene as the substrate, M5 not only has a significant improvement in activity, but also a significant improvement in ee value compared to the wild type. As for M3, while the activity is improved, the selectivity is reversed compared to the wild type, which is also very beneficial. Analysis of the gas phase detection results shows that the activity of the mutants M1-M10 is 1-78.3 times that of the wild type, the selectivity is improved or the chirality is reversed compared to the wild type, and the ee value can be as high as 99% ( Figure 4 However, not every mutation can improve or reverse selectivity while maintaining activity. For example, with ethylbenzene as the substrate, mutants M11 and M12 exhibited 63% and 84% of the wild-type activity, respectively. With styrene as the substrate, mutant M13 exhibited only 44% of the wild-type activity. However, our mutants, M1-M10, achieve both improved and reversed selectivity while maintaining activity equal to or greater than that of the wild-type.

[0116] Example 5 Application of non-specific peroxygenase mutants in the production of chiral alcohols

[0117] The mutant enzyme obtained in Example 3 was used as a catalyst. The reaction system contained 50 mM buffer at pH 5.0-9.0 (specifically, sodium hydrogen phosphate-citrate buffer at pH 5-6, sodium 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 1 and Formula 2 were substrates) and 5%-30% acetonitrile were added, and chiral alcohol production was carried out at 30°C.

[0118] Preferably, 6-bromobenzopyran is used as the substrate (final concentration 10 mM), the final concentration of the mutant M3 enzyme is 8 mg / L, the final concentration of acetonitrile is 5%, the initial concentration of hydrogen peroxide in the reaction system is 2 mM, pH = 5 (disodium hydrogen phosphate-citrate buffer, final concentration 50 mM); 1-3 mM is added every 2 hours (after regular sampling and detection, it is added according to the product generation rate to maintain a relatively constant value), the reaction is carried out at 30°C for 10 hours, and finally an equal volume of ethyl acetate is added to terminate the reaction. The content of the remaining substrate and the product (6-bromochrome-4-ol (R)) is detected by gas chromatography (chiral capillary column CP-Chirasil-Dex-CB). According to the analysis of the gas phase detection results, the final conversion rate can reach 60% ( Figure 5 ).

[0119] The above description is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and modifications without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A non-specific peroxygenase mutant, characterized in that: The present invention relates to a mutant obtained by mutating at least one of amino acids 64, 87, 88, 154, 157, 158, 161, 165 and 210 in a non-specific peroxygenase having an amino acid sequence as shown in SEQ ID NO.1; the mutant has higher catalytic performance than the non-specific peroxygenase having an amino acid sequence as shown in SEQ ID NO.

1.

2. The nonspecific peroxygenase mutant according to claim 1, characterized in that: The non-specific peroxygenase mutant is any of the following mutants: (1) The 165th lysine in the nonspecific peroxygenase of the amino acid sequence shown in SEQ ID NO. 1 was replaced with leucine to obtain mutant M1; (2) The methionine at position 210 in the nonspecific peroxygenase shown in the amino acid sequence of SEQ ID NO. 1 was replaced with leucine to obtain mutant M2; (3) In mutant M1, leucine at position 64 was replaced by glutamine, glutamine at position 154 was replaced by leucine, isoleucine at position 157 was replaced by lysine, and threonine at position 158 was replaced by leucine to obtain mutant M3; (4) In mutant M1, leucine at position 64 was replaced with glutamine, glutamine at position 154 was replaced with leucine, and threonine at position 158 was replaced with leucine to obtain mutant M4; (5) In mutant M2, phenylalanine at position 88 was replaced with alanine, glutamine at position 154 was replaced with leucine, isoleucine at position 157 was replaced with leucine, threonine at position 158 was replaced with leucine, and glycine at position 161 was replaced with serine to obtain mutant M5; (6) In mutant M2, phenylalanine at position 88 was replaced with alanine, isoleucine at position 157 was replaced with leucine, threonine at position 158 was replaced with leucine, and glycine at position 161 was replaced with serine to obtain mutant M6; (7) In mutant M2, leucine at position 87 was replaced by valine, phenylalanine at position 88 was replaced by isoleucine, glutamine at position 154 was replaced by threonine, and threonine at position 158 was replaced by leucine to obtain mutant M7; (8) In mutant M2, glutamine at position 154 was replaced with lysine, isoleucine at position 157 was replaced with asparagine, threonine at position 158 was replaced with isoleucine, and lysine at position 165 was replaced with phenylalanine to obtain mutant M8; (9) The 165th lysine in the nonspecific peroxygenase of the amino acid sequence shown in SEQ ID NO. 1 was replaced with alanine to obtain mutant M9; (10) The 165th lysine in the nonspecific peroxygenase shown in the amino acid sequence of SEQ ID NO. 1 was replaced with glycine to obtain mutant M10.

3. A nucleic acid molecule, characterized in that: The present invention is a nucleic acid encoding the non-specific peroxygenase mutant according to claim 1 or 2.

4. The nucleic acid molecule according to claim 3, characterized in that: The nucleotide sequence of the nucleic acid encoding the variant M1 is shown in SEQ ID NO.3; The nucleotide sequence of the nucleic acid encoding the variant M2 is shown in SEQ ID NO.

5.

5. A recombinant expression vector, characterized in that: Containing the nucleic acid molecule according to claim 3 or 4.

6. An expression cell, characterized in that: Contains the recombinant expression vector according to claim 5.

7. Use of the nonspecific peroxygenase mutant according to claim 1 or 2 in catalyzing the synthesis of chiral compounds.

8. The use according to claim 7, characterized in that The method comprises the following steps: preparing a reaction system: mixing a substrate, the nonspecific peroxygenase mutant, an organic solvent for promoting dissolution, hydrogen peroxide, and a buffer solution to obtain a reaction system; reacting the obtained reaction system at 25 to 35° C., supplementing hydrogen peroxide during the reaction according to the product generation rate, to obtain a chiral compound; The substrate is an aliphatic hydrocarbon, a cycloalkane or an aromatic hydrocarbon compound; The pH of the reaction system is 3-9.

9. The use according to claim 8, characterized in that: The substrate is a compound as shown in Formula I or Formula II: Wherein, R1 is an alkyl group, an alkenyl group or an 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 is disodium hydrogen phosphate-citric acid buffer, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, Tris-HCl buffer or glycine-sodium hydroxide buffer.

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

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