Non-specific peroxygenase mutants and use thereof

Abstract

The application discloses a non-specific peroxygenase mutant and application thereof. The application uses a multiple computer-aided strategy to rationally design Collariella virescens UPO of a resolved protein structure, and successfully obtains a UPO mutant library with rich enantioselectivity to various substrates and improved activity through function verification, thereby laying a solid foundation 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 application has high catalytic activity in the selective oxidation of carbon-hydrogen bonds of various substrates, and the selectivity is significantly improved or reversed, the obtained reaction product has high optical purity, the reaction efficiency is improved, the separation process is simplified, the cost is reduced, and the non-specific peroxygenase mutant has a good industrial application prospect.

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] Current 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 obtained an AaeUPO mutant PaDa with improved expression and activity through directed evolution (Appl Environ Microbiol. 2014, 80, 11, 3496-3507); then, using this mutant as a starting point, a variant pada-1_A77L with improved catalytic activity for fatty acids was obtained through rational design (Angew. Chem. Int. Ed. 2023, e202217372); recently, the research group obtained an AaeUPO_PaDa mutant with significantly improved enantiomeric selectivity and activity through combinatorial mutation (J. Am. Chem. Soc. 2023, 145, 6, 3443-3453). The team of Zhang Wuyuan improved the ability of AaeUPO to catalyze sterols through semi-rational design (ACS Catal. 2025, 15, 1952-1960). However, the catalytic selectivity and activity of the currently reported UPOs are still very limited and far from meeting the requirements of industrial production. Therefore, improving the activity and / or selectivity of UPOs has important application value for establishing an efficient synthesis system of chiral alcohols. SUMMARY

[0004] In view of the problems existing in the prior art non-specific peroxygenase, the primary object of the present application is to provide a non-specific peroxygenase mutant.

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

[0006] The object of the present application is achieved by the following technical solutions:

[0007] A non-specific peroxygenase mutant, referred to as CviUPO mutant, is a mutant obtained by mutating at least one of the 64th, 87th, 88th, 154th, 157th, 158th, 161st, 165th, and 210th amino acids in the non-specific peroxygenase with the amino acid sequence shown in SEQ ID NO. 1; and the mutant has higher catalytic performance than the non-specific peroxygenase with the amino acid sequence 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 application of the above-mentioned non-specific peroxygenase mutant in catalyzing synthesis of chiral compounds; preferably comprising the following steps: preparing a reaction system: mixing a substrate, the above-mentioned non-specific peroxygenase mutant, an organic solvent for solubilization, hydrogen peroxide and a buffer to obtain a reaction system; reacting the obtained reaction system at 25-35°C, and supplementing hydrogen peroxide according to the product generation rate during the reaction to obtain a chiral compound.

[0038] The substrate is a fatty 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, n = 1; R is hydrogen or bromine, X is oxygen, 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, 6-bromobenzopyran.

[0048] The concentration of the substrate in the reaction system is preferably 1-10 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 5-30% by volume; more preferably 5-10% by volume.

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

[0053] The concentration of the buffer in the reaction system is preferably 40-60 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; most preferably 7.

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

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

[0057] The time of the reaction is preferably 1-10 h; more preferably 2-10 h.

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

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

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

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

[0062] When the substrate is styrene, the chiral compound is a chiral epoxide, specifically epoxystyrene.

[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] When the substrate is 3,4-dihydrobenzopyran, the chiral compound is a chiral alcohol, specifically 4-diacetol.

[0072] The present application has the following advantages and effects relative to the prior art:

[0073] The applicant uses multiple computer-aided strategies to rationally design Collariella virescens UPO (CviUPO) based on the resolved protein structure, and successfully obtains a library of UPO mutants with rich enantioselectivity and improved activity for a variety of substrates through functional verification, thereby laying a solid foundation for the research and development of green and efficient chiral compound production methods.

[0074] Compared with the prior art, the non-specific peroxygenase mutant provided by the present application exhibits high catalytic activity, significantly improved or reversed selectivity in the selective oxidation of carbon-hydrogen bonds of various substrates, and high optical purity of the reaction product, thereby improving the reaction efficiency, simplifying the separation process, reducing the cost, and having good industrial application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0075] Figure 1 It is a schematic diagram of recombinant expression plasmid pET28a-UPO.

[0076] Figure 2 It is an SDS-PAGE protein electrophoresis diagram of wild-type non-specific peroxygenase; wherein: lane M is a protein marker; lane 1 is a protein loading flow-through liquid; lane 2 is a 40 mM imidazole eluent; lane 3 is a 90 mM imidazole eluent; lane 4 is a 125 mM imidazole eluent; lane 5 is a 175 mM imidazole eluent; lane 6 is a 200 mM imidazole eluent; and lane 7 is a 250 mM imidazole eluent.

[0077] Figure 3SDS-PAGE electrophoretogram of non-specific peroxygenase mutant pure enzyme; wherein: lane M is protein Marker; lanes 1-6 in a are mutants M1-M6; lanes 1-4 in b are mutants M7-M10.

[0078] Figure 4 Figure for substrate scope of non-specific peroxygenase wild type and performance excellent mutant catalyzed asymmetric oxidation.

[0079] Figure 5 Figure for mutant non-specific peroxygenase production of chiral alcohol under optimal conditions. DETAILED DESCRIPTION

[0080] The technical solutions in the embodiments of the present application will be described clearly and completely below in combination with specific related embodiments. However, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

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

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

[0083] The wild type non-specific peroxygenase gene (derived from Collariella virescens, nucleotide sequence as shown in SEQ ID NO. 6) was cloned into pET28a through EcoR I and Sac I enzyme cutting sites to obtain the recombinant plasmid pET28a-UPO as shown in Figure 1 The recombinant plasmid pET28a-UPO was transformed into Escherichia coli C43 (DE3) (Ouobaobi, Changsha) to obtain the recombinant bacteria.

[0084] The 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. Then, the bacteria were transferred into 200 mL of ZYM-5052 medium containing 50 mg / L kanamycin and cultured at 16°C and 180 rpm for 72 hours. The bacteria were collected by centrifugation, and the supernatant was discarded. 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 ultrasonic treatment, and centrifuged at 12,000 rpm for 30 min. The supernatant was collected and injected into a nickel column. Phosphate buffer containing 40, 90, 125, 250, and 500 mM imidazole was used to elute the column in sequence, and the eluate at a concentration of 250 mM imidazole was collected. The eluate was desalted and concentrated by ultrafiltration until the remaining volume was about 1 mL. The purified protein was analyzed by SDS-PAGE, and the results are shown in FIG. 8. The protein concentration was quantified using a protein concentration test kit from Shanghai Bioengineering Co., Ltd. Figure 2

[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 additionally contains 20 g / L agar powder.

[0086] ZYM-5052 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% a-lactose, 200 μM hematin chloride. Except for glycerol, glucose, and a-lactose, the medium was sterilized at 121°C for 20 min, and the sterile liquid was obtained by filtration.

[0087] The solvent of the medium was deionized water.

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

[0089] Using the wild-type non-specific peroxygenase amino acid sequence or crystal structure (PDB ID 7zcl) as input, M1 and M2 were first obtained by rational design of site-directed mutation, and then the 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 mutation calculation to obtain combinatorial mutants with M1 and M2 as the parent, respectively.

[0090] ​The M1 mutant is a mutant in which the wild-type nonspecific peroxygenase has a lysine at position 165 replaced by a leucine (M is the amino acid corresponding to ATG, which is not counted in the numbering, the same below), the amino acid sequence of which is shown in SEQ ID NO. 2, and the encoding nucleotide sequence is shown in SEQ ID NO. 3;

[0091] The M2 mutant is a mutant in which the wild-type nonspecific peroxygenase has a methionine at position 210 replaced by a leucine, the amino acid sequence of which is shown in SEQ ID NO. 4, and the encoding nucleotide sequence is shown in SEQ ID NO. 5.

[0092] Example 3 Construction of a library of nonspecific peroxygenase mutants

[0093] The pET28a recombinant plasmid containing the wild-type UPO gene (i.e. pET28a-UPO) was used as a template for site-directed mutagenesis of the above-mentioned sites. Primers were designed using snapgene software (synthesized by Shanghai Biotechnology Co., Ltd.), and then PCR site-directed mutagenesis was performed. The PCR reaction system was as follows: 1 μL of template, 12.5 μL of DNA polymerase (Takara), 1 μL of F primer, 1 μL of R primer, and ddH2O to make up to 25 μL. The PCR reaction conditions were as follows: 98 °C preheating for 3 min; 98 °C denaturation for 10 s, 62 °C annealing for 30 s, 72 °C extension for 90 s, 28 cycles; 72 °C extension for 5 min.

[0094] Table 1. Primer sequences

[0095]

[0096]

[0097] The PCR products were detected by 1% agarose gel electrophoresis, then recovered using a Shanghai Biotechnology nucleic acid purification kit, and after determining the concentration, were ligated.

[0098] The steps of seamless cloning were as follows: the linearized vector and the target fragment were mixed at a molar ratio of 2:6-9, water was added to make up to 10 μL, 10 μL of HB-infusion was then added, and ligation was performed at 50 °C for 30-40 min, followed by transformation into E. coli C43(DE3) competent cells, which were then plated on LB plates containing 50 μg / mL of kanamycin and incubated at 37 °C overnight. The next day, positive transformants were screened using universal primers T7-F (taatacgactcactatagg) and T7-R (gctagttattgctcagcgg), and the recombinant plasmids were extracted and sent to Shanghai Biotechnology Co., Ltd. for sequencing verification.

[0099] The recombinant plasmid pET28a-UPO was used as a template, and the primer pair 165L-F / UPO-R and UPO-F / 165L-R was used to obtain the target fragment A and the target fragment B by PCR, and 28a-F and 28a-R were used to obtain the linearized carrier A. The target fragment A, the target fragment B and the linearized carrier A were mixed in a molar ratio of 3:3:2, and the seamless cloning was performed according to the above steps to obtain pET28a-M1.

[0100] The recombinant plasmid pET28a-UPO was used as a template, and the primer pair M210L-F / UPO-R and UPO-F / M210L-R was used to obtain the target fragment C and the target fragment D by PCR, and 28a-F and 28a-R were used to obtain the linearized carrier A. The target fragment C, the target fragment D and the linearized carrier A were mixed in a molar ratio of 3:3:2, and the seamless cloning was performed according to the above steps to obtain pET28a-M2.

[0101] The recombinant plasmid pET28a-M1 was used as a template, and the primer pair L64Q-F / Q154L_I157K_T158L_165L-R, UPO-F / L64Q-R and Q154L_I157K_T158L_165L-F / UPO-R was used to obtain the target fragment E, F and G by PCR, and 28a-F and 28a-R were used to obtain the linearized carrier A. The target fragment E, the target fragment F, the target fragment G and the linearized carrier A were mixed in a molar ratio of 3:3:3:2, and the seamless cloning was performed according to the above steps to obtain pET28a-M3.

[0102] The recombinant plasmid pET28a-M1 was used as a template, and the primer pair L64Q-F / Q154L_T158L_165L-R, UPO-F / L64Q-R and Q154L_T158L_165L-F / UPO-R was used to obtain the target fragment H, I and J by PCR, and 28a-F and 28a-R were used to obtain the linearized carrier A. The target fragment H, the target fragment I, the target fragment J and the linearized carrier A were mixed in a molar ratio of 3:3:3:2, and the seamless cloning was performed according to the above steps to obtain pET28a-M4.

[0103] PCR was performed using primers F88A-F / Q154L_I157L_T158L_G161S-R, UPO-F / F88A-R, Q154L_I157L_T158L_G161S-F / UPO-R to obtain target fragments K, L and M, respectively, with pET28a-M2 as the template; and 28a-F and 28a-R to obtain linearized vector A. The target fragment K, the target fragment L, the target fragment M and the linearized vector A were mixed in a molar ratio of 3:3:3:2, and seamless cloning was performed according to the above procedure to obtain pET28a-M5.

[0104] PCR was performed using primers F88A-F / I157L_T158L_G161S-R, UPO-F / F88A-R, I157L_T158L_G161S-F / UPO-R to obtain target fragments N, O and P, respectively, with pET28a-M2 as the template; and 28a-F and 28a-R to obtain linearized vector A. The target fragment N, the target fragment O, the target fragment P and the linearized vector A were mixed in a molar ratio of 3:3:3:2, and seamless cloning was performed according to the above procedure to obtain pET28a-M6.

[0105] PCR was performed using primers L87V_F88I-F / Q154T_T158L-R, UPO-F / L87V_F88I-R, Q154T_T158L-F / UPO-R to obtain target fragments Q, R and S, respectively, with pET28a-M2 as the template; and 28a-F and 28a-R to obtain linearized vector A. The target fragment Q, the target fragment R, the target fragment S and the linearized vector A were mixed in a molar ratio of 3:3:3:2, and seamless cloning was performed according to the above procedure to obtain pET28a-M7.

[0106] PCR was performed using primers Q154K_I157N_T158I_165F-F / UPO-R, UPO-F / Q154K_I157N_T158I_165F-R to obtain target fragments T and U, respectively, with pET28a-M2 as the template; and 28a-F and 28a-R to obtain linearized vector A. The target fragment T, the target fragment U and the linearized vector A were mixed in a molar ratio of 3:3:2, and seamless cloning was performed according to the above procedure to obtain pET28a-M8.

[0107] With pET28a-M1 as the template, the primers 165A-F / UPO-R, UPO-F / 165A-R are used respectively to obtain the target fragments V and W by PCR; the primers 28a-F and 28a-R are used to obtain the linearized carrier A by PCR. The target fragment V, the target fragment W and the linearized carrier A are mixed in a molar ratio of 3:3:2, and the seamless cloning is performed according to the above steps to obtain pET28a-M9.

[0108] With pET28a-M1 as the template, the primers 165G-F / UPO-R, UPO-F / 165G-R are used respectively to obtain the target fragments X and Y by PCR; the primers 28a-F and 28a-R are used to obtain the linearized carrier A by PCR. The target fragment X, the target fragment Y and the linearized carrier A are mixed in a molar ratio of 3:3:2, and the seamless cloning is performed according to the above steps to obtain pET28a-M10.

[0109] With pET28a-M1 as the template, the primers UPO-F / L64M-R, L64M-F / Q154L_I157E_T158L_165M-R, Q154L_I157E_T158L_165M-F / UPO-R are used respectively to obtain the target fragments B2, B3 and B4 by PCR; the primers 28a-F and 28a-R are used to obtain the linearized carrier A by PCR. The target fragment B2, the target fragment B3, the target fragment B4 and the linearized carrier A are mixed in a molar ratio of 3:3:3:2, and the seamless cloning is performed according to the above steps to obtain pET28a-M11.

[0110] With pET28a-M1 as the template, the primers UPO-F / T60A-R, T60A-F / Q154T_I157N_T158F_165M-R, Q154T_I157N_T158F_165M-F / UPO-R are used respectively to obtain the target fragments C2, C3 and C4 by PCR; the primers 28a-F and 28a-R are used to obtain the linearized carrier A by PCR. The target fragment C2, the target fragment C3, the target fragment C4 and the linearized carrier A are mixed in a molar ratio of 3:3:3:2, and the seamless cloning is performed according to the above steps to obtain pET28a-M12.

[0111] With pET28a-M2 as the template, the primers UPO-F / L87V-R, L87V-F / Q154K_I157K_T158I-R, Q154K_I157K_T158I-F / UPO-R are used respectively to obtain the target fragments D2, D3 and D4 by PCR; the primers 28a-F and 28a-R are used to obtain the linearized carrier A by PCR. The target fragment D2, the target fragment D3, the target fragment D4 and the linearized carrier A are mixed in a molar ratio of 3:3:3:2, and the seamless cloning is performed according to the above steps to obtain pET28a-M13.

[0112] Example 4: Detection of catalytic efficiency and selectivity of wild-type and mutant nonspecific peroxyases

[0113] Wild-type and mutant proteins were purified according to Example 1. Figure 3 ), and then catalytic efficiency and selectivity tests were conducted.

[0114] 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 pure enzyme, and 1 mM H2O2. Water was then added to bring the total volume to 1 mL to obtain the reaction solution.

[0115] The reaction was carried out in a metal bath at 30°C for 2 hours (rxn. Time = 2h) with 1 mM substrate (final concentration), 10% acetonitrile, 1 mM hydrogen peroxide, 50 mM Na2HPO4-NaH2PO4 buffer, pH = 7. After the reaction, the reaction was terminated by adding ethyl acetate in a volume ratio of 1:1. The sample was vortexed for 30 seconds and then centrifuged at 12000 rpm for 1 minute. The upper organic phase was collected and dried with anhydrous sodium sulfate. The content of the corresponding chiral alcohol was detected by gas chromatography (chiral capillary column CP-Chirasil-Dex-CB). The detection conditions were as follows: the carrier gas was nitrogen, the rate was 1.0 mL / min, 100°C was maintained for 2 min, the temperature was increased to 180°C at a rate of 10°C / min and maintained for 5 min, and then the temperature was increased to 200°C at a rate of 2°C / min and maintained for 3 min. TON refers to the amount of product divided by the enzyme concentration, so under the same enzyme concentration and reaction time, it reflects the amount of product generated, that is, the catalytic activity. For example, when ethylbenzene is used as the substrate, the TON of M7 (TON = 173) is 5.2 times that of the wild type (TON = 33). The ee value reflects the preference of the product configuration, and the larger the ee value, the purer the product. The ee(R) or ee(S) can be used because the product is a chiral alcohol, and both R and S configurations can be used as chiral building blocks. For example, when p-bromoethylbenzene is used as the substrate, M5 not only has a significant increase in activity, but also has a large increase in ee value compared with the wild type. M3 has an increase in activity and a reversal in selectivity compared with the wild type, which is also very advantageous. According to the gas chromatography results, 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 with the wild type, and the ee value can be as high as 99%. Figure 4 ) However, not all mutations can improve or reverse the selectivity while maintaining the activity. For example, when ethylbenzene is used as the substrate, the activities of mutant M11 and mutant M12 are 63% and 84% of the wild type, respectively. When styrene is used as the substrate, the activity of mutant M13 is only 44% of the wild type. The mutants M1-M10 provided by us can improve or reverse the selectivity while ensuring that the activity is greater than or equal to that of the wild type.

[0116] Example 5 Application of Mutants of Non-specific Peroxygenase in Chiral Alcohol Production

[0117] The mutant enzyme obtained in Example 3 was used as catalyst, and the reaction system contained 50 mM buffer solution with pH 5.0-9.0 (specifically, sodium phosphate dibasic-citric acid buffer solution with pH 5-6, sodium phosphate dibasic-sodium phosphate monobasic buffer solution with pH 6-7, Tris-HCl buffer solution with pH 7-8, and glycine-sodium hydroxide buffer solution with pH 8-9), 10 mM substrate (formula 1 and formula 2) was added, 5%-30% acetonitrile was added, and chiral alcohol production was carried out at 30°C.

[0118] Preferably, 6-bromobenzopyran was used as substrate (final concentration 10 mM), the final concentration of 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, pH=5 (sodium phosphate dibasic-citric acid buffer solution, final concentration 50 mM); 1-3 mM was added every 2 hours (after timed sampling detection, according to the product generation rate, so as to keep a relatively constant value), the reaction was carried out at 30°C for 10 h, and finally an equal volume of ethyl acetate was added to terminate the reaction. Gas chromatography (chiral capillary column CP-Chirasil-Dex-CB) was used to detect the content of residual substrate and product (6-bromochroman-4-ol (R)). It can be known from the analysis of gas chromatography detection results that the final conversion rate can reach 60% ( Figure 5 ).

[0119] The above only describes the preferred embodiments of the present application, and it should be noted that, for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A non-specific peroxygenase mutant, characterized in that: The nonspecific peroxidase mutant is mutant M1, mutant M3 or mutant M4. The mutant M1 is to replace the lysine at the 165th amino acid in the nonspecific peroxidase with leucine, and the amino acid sequence is shown in SEQ ID NO.

1. The mutant M3 is to replace the leucine at the 64th amino acid in the mutant M1 with glutamine, replace the glutamine at the 154th amino acid with leucine, replace the isoleucine at the 157th amino acid with lysine, and replace the threonine at the 158th amino acid with leucine. The mutant M4 is to replace the leucine at the 64th amino acid in the mutant M1 with glutamine, replace the glutamine at the 154th amino acid with leucine, and replace the threonine at the 158th amino acid with leucine.

2. A nucleic acid molecule, characterized in that: The nucleic acid encoding the nonspecific peroxidase mutant of claim 1.

3. The nucleic acid molecule of claim 2, wherein: The nucleotide sequence of the nucleic acid encoding the mutant M1 is shown in SEQ ID NO.

3.

4. A recombinant expression vector, characterized by: The nucleic acid molecule of claim 2 or 3.

5. An expression cell, characterized by: The recombinant expression vector of claim 4.

6. The nonspecific peroxidase mutant of claim 1 for use in catalyzing synthesis of chiral compounds.

7. Use according to claim 6, characterized in that The method comprises the following steps: preparing a reaction system by mixing a substrate, the nonspecific peroxidase mutant, an organic solvent for solubilization, hydrogen peroxide and a buffer to obtain the reaction system; and reacting the obtained reaction system at 25-35°C, and supplementing hydrogen peroxide during the reaction according to the product generation rate to obtain a chiral compound. The substrate is a fatty hydrocarbon, a cycloalkane or an aromatic hydrocarbon compound. The pH of the reaction system is 3-9.

8. The use of claim 7, wherein: The substrate is a compound shown in formula I or formula II: I II; 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 is disodium hydrogen phosphate-citric acid buffer, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, Tris-HCl buffer or glycine-sodium hydroxide buffer.

9. The use of claim 7, wherein: The concentration of the substrate in the reaction system is 1-10 mmol / L; The concentration of the nonspecific peroxidase 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 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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