P450 BM3 protein mutant and its application

Abstract

The present invention relates to the field of biotechnology, and in particular to P450BM3 protein mutants and applications thereof. The present invention discloses two P450BM3 protein mutants, wherein the P450BM3-mut1 amino acid sequence has 13 mutation sites, specifically K70A / L72G / G86A / L87G / F88A / W91A / E94A / W97A / I154A / F262A / F332A / I358A / R399A. The P450BM3-mut2 amino acid sequence has 15 mutation sites, specifically K70A / L72G / G86A / L87G / F88A / W91A / E94A / W97A / I154A / F332A / R399A / F108S / L234S / F262S / I358S. The P450BM3 protein mutant can bind to large-framework porphyrins and has good application prospects in enzyme catalysis.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a P450 BM3 protein mutant and an application thereof. Background Art

[0002] The directed evolution technology of enzyme molecules is to artificially create special evolutionary conditions, simulate the natural evolution mechanism, randomly mutate genes in vitro, and finally obtain the expected evolved enzymes with certain characteristics from one or more artificial mutant enzyme libraries through certain screening or selection methods. Compared with natural evolution. The directed evolution process of enzyme molecules is completely under artificial control, so that the enzyme molecules move towards the specific goals people expect. In recent years, with the application of technologies such as error-prone PCR and DNA shuffling, under the condition of efficient detection and screening system for target gene expression, although the structure and other characteristics of enzyme molecules are not clear, the directed strategy of enzyme molecules can obtain new enzymes with expected characteristics and realize the artificial rapid evolution of enzyme molecules. Many studies have shown that under the current situation that the understanding of enzyme molecules is still immature, it is possible to produce new enzymes in recombinant organisms by changing the amino acid sequence of enzymes through appropriate modification at the DNA level, and then changing the performance of enzymes, and obtain evolved enzymes with higher activity, better stability and catalytic performance than natural enzymes to meet the needs of research and application.

[0003] The P450 enzyme system is a class of metabolic enzymes widely distributed in different organisms such as animals, plants and microorganisms. It is named because its main component, the P450 protein, has a characteristic light absorption peak at 450nm after binding to CO. Studies have shown that it can catalyze thousands of chemical reactions, and the main reactions involved are alkyl hydroxylation, alkyl epoxidation, hydroxyl oxidation. Dealkylation at ammonia, oxygen, and sulfur sites, hydroxylation and oxidation at ammonia sites, oxidation at sulfur sites, oxidative deamination, dehydrogenation and dehalogenation, oxidative CC bond cleavage, and some other reduction catalytic reactions. Some monographs even point out that it may be the most catalytically diverse biological catalyst in nature. Due to the properties and functions of this enzyme system and its role in the process of life activities, the P450 enzyme system has received great attention from researchers. The importance of the role of the P450 enzyme system has become a noteworthy field in contemporary biological research. Foreign research work is mainly distributed in the role of this enzyme system in biological systems and the large-scale production of chiral drugs using the catalytic effect of this enzyme system. In China. Although there are a large number of review and research reports on this enzyme system. However, the research work is limited to the role of this enzyme system in biological organisms. There is no report on the catalytic effect of this enzyme system in the preparation of chiral drugs. Some scholars pointed out that the main reason is that the appropriate amount of enzymes cannot be obtained in China.

[0004] In the P450 enzyme system, P450BM-3 is an enzyme with fatty acid hydroxylation activity discovered from Bacillus megaterium and is now used as an important model of mammalian microsomal P450 monooxygenase.

[0005] P450BM-3 is a soluble monooxygenase with a molecular weight of 117,641Da. Unlike other P450 enzymes that require additional proteins for catalysis, P450BM-3 has the monooxygenase activity to rapidly catalyze saturated fatty acids with a chain length of C12 to C18 in the presence of NADPH and O2.

[0006] Cytochrome P450 BM3 is a prokaryotic cytochrome P450 enzyme, in which heme (ferroprotoporphyrin) plays an important role in the function of P450 BM3 protein. In recent years, researchers have used a variety of methods to replace the cofactors of heme-containing proteins to generate artificial metalloporphyrin proteins for catalyzing high-value reactions. Currently, it is possible to replace the iron protoporphyrin of hemoglobin with other metal protoporphyrins or metalloporphyrin frameworks of similar size, but it is impossible to efficiently replace the large porphyrin framework, which limits the development and application of artificial metalloporphyrin proteins.

[0007] So far, no P450 BM3 protein mutants that can bind large-framed porphyrins have been found. Summary of the invention

[0008] In view of this, the present invention provides a P450 BM3 protein mutant and its application. The present invention provides two P450BM3 protein mutants, namely P450 BM3-mut1 and P450 BM3-mut2. The present invention finds through experiments that the two P450BM3 protein mutants can bind to large-framework porphyrins.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0010] The present invention provides a P450 BM3 protein mutant, which includes P450 BM3-mut1 and / or P450BM3-mut2;

[0011] The P450 BM3-mut1 includes any one or more mutations of the following sites:

[0012] K70A; and / or

[0013] L72G; and / or

[0014] G86A; and / or

[0015] L87G; and / or

[0016] F88A; and / or

[0017] W91A; and / or

[0018] E94A; and / or

[0019] W97A; and / or

[0020] I154A; and / or

[0021] F262A; and / or

[0022] F332A; and / or

[0023] I358A; and / or

[0024] R399A;

[0025] The P450 BM3-mut2 includes any one or more of the following site mutations:

[0026] K70A; and / or

[0027] L72G; and / or

[0028] G86A; and / or

[0029] L87G; and / or

[0030] F88A; and / or

[0031] W91A; and / or

[0032] E94A; and / or

[0033] W97A; and / or

[0034] I154A; and / or

[0035] F332A; and / or

[0036] R399A; and / or

[0037] F108S; and / or

[0038] L234S; and / or

[0039] F262S; and / or

[0040] I358S.

[0041] The present invention also provides a nucleic acid molecule encoding the P450 BM3 protein mutant.

[0042] The nucleic acid molecule encoding the P450 BM3-mut1 has:

[0043] (I), the nucleotide sequence shown in SEQ ID NO: 3; or

[0044] (II) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I) but is different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0045] (III) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and having the same or similar function as the nucleotide sequence shown in (I) or (II); or

[0046] (IV), a nucleotide sequence having at least 90% sequence homology with the nucleotide sequence of (I), (II) or (III); and / or

[0047] The nucleic acid molecule encoding the P450 BM3-mut2 has:

[0048] (I), the nucleotide sequence shown in SEQ ID NO:4; or

[0049] (II) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I) but is different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0050] (III) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and having the same or similar function as the nucleotide sequence shown in (I) or (II); or

[0051] (IV) A nucleotide sequence having at least 90% sequence homology with the nucleotide sequence of (I), (II) or (III).

[0052] The present invention also provides a recombinant vector, which comprises the nucleic acid molecule and a backbone vector.

[0053] The present invention also provides a host, which comprises the recombinant vector.

[0054] The present invention also provides a primer set, wherein the primer set has:

[0055] (I) an upstream primer having a nucleotide sequence as shown in SEQ ID No: 2n-1; and

[0056] A downstream primer of the nucleotide sequence shown in SEQ ID No: 2n; or

[0057] (II) having a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (I), but differing from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0058] (III) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and having the same or similar function as the nucleotide sequence shown in (I) or (II); or

[0059] (IV), a nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III);

[0060] The n includes any integer between 3 and 11.

[0061] The present invention also provides the use of any of the following items in the preparation of P450 BM3 protein mutants:

[0062] (I), the nucleic acid molecule; and / or

[0063] (II), the recombinant vector; and / or

[0064] (III), the host; and / or

[0065] (IV), the primer set.

[0066] The present invention also provides a method for preparing the P450 BM3 protein mutant, which comprises preparing the P450 BM3 protein mutant based on any of the following items:

[0067] (I), the nucleic acid molecule; and / or

[0068] (II), the recombinant vector; and / or

[0069] (III), the host; and / or

[0070] (IV), the primer set.

[0071] The present invention also provides the use of any of the following items in the preparation of metalloporphyrin proteins and / or artificial enzymes:

[0072] (I), the P450 BM3 protein mutant; and / or

[0073] (II), the nucleic acid molecule; and / or

[0074] (III), the recombinant vector; and / or

[0075] (IV), the host; and / or

[0076] (V), the primer set; and / or

[0077] (VI) P450 BM3 protein mutant obtained by the preparation method.

[0078] The metalloporphyrin protein and / or artificial enzyme comprises the P450 BM3 protein mutant and metalloporphyrin; the metalloporphyrin comprises tetraphenylporphyrin manganese (III) chloride and / or 5,10,15,20-tetrakis (pentafluorophenyl) porphyrin manganese (III) chloride.

[0079] The present invention also provides a metalloporphyrin protein and / or an artificial enzyme, wherein the metalloporphyrin protein and / or the artificial enzyme comprises any of the following items and a metalloporphyrin:

[0080] (I), the P450 BM3 protein mutant; and / or

[0081] (II), the nucleic acid molecule; and / or

[0082] (III), the recombinant vector; and / or

[0083] (IV), the host; and / or

[0084] (V), the primer set; and / or

[0085] (VI) P450 BM3 protein mutant obtained by the preparation method.

[0086] The metalloporphyrin protein and / or artificial enzyme of the present invention includes but is not limited to structures produced by adding, reducing or replacing branches, ions, etc., polymers, isomers, chelates and other forms of the structures, and mixtures of any of the above structures or forms.

[0087] The present invention also provides the use of the metalloporphyrin protein and / or artificial enzyme in enzyme catalysis.

[0088] The present invention includes but is not limited to achieving the following beneficial effects:

[0089] The P450 BM3 protein mutant P450 BM3-mut1 or P450 BM3-mut2 prepared by the invention loses the ability to bind to heme (ferroprotoporphyrin), can bind to large-frame porphyrin, and has good application prospects in enzyme catalysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0091] Figure 1 Spectral characterization diagram showing that the P450 BM3-mut1 protein mutant can bind to tetraphenylporphyrin manganese chloride (III) and the P450BM3-mut1 protein mutant cannot bind to heme;

[0092] Figure 2 Spectral characterization graphs showing the binding of the P450 BM3-mut2 protein mutant to 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin manganese (III) chloride and the inability of the P450 BM3-mut2 protein mutant to bind heme. DETAILED DESCRIPTION

[0093] The present invention discloses a P450 BM3 protein mutant and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0094] The present invention provides two P450 BM3 protein mutants, whose amino acid sequences are shown in Table 1 and whose nucleic acid sequences are shown in Table 2:

[0095] Table 1 Amino acid sequences of P450 BM3 protein mutants

[0096]

[0097]

[0098] Table 2 Nucleotide sequences of P450 BM3 protein mutants

[0099]

[0100]

[0101]

[0102]

[0103] Unless otherwise specified, the P450 BM3 protein mutant provided by the present invention and the raw materials and reagents used in its application can be purchased from the market.

[0104] The present invention will be further described below in conjunction with embodiments:

[0105] Preparation Example 1 Construction of P450 BM3 protein mutant expression strain and bacterial cell preparation

[0106] The present invention synthesized the P450 BM3 gene, and used protein visualization software to analyze mutation sites. 12 mutation sites were selected for P450 BM3-mut1, and 14 mutation sites were selected for P450 BM3-mut2. The wild-type P450 BM3 gene optimized by codons of Escherichia coli was used as a template, and mutations were introduced using site-directed mutagenesis. P450 BM3-mut2 used the P450 BM3-mut1 gene as a template, and mutations were introduced using site-directed mutagenesis.

[0107] 1. Construction of P450 BM3-mut1 protein mutant expression plasmid

[0108] (1) Primer design of P450 BM3-mut1 is shown in Table 3:

[0109] Table 3 P450 BM3-mut1 primer design

[0110]

[0111] For PCR amplification, the primer combinations are as follows:

[0112] Fragment 1 (232 bp in length): BM3-1-for and BM3-1-rev;

[0113] Fragment 2 (length 357 bp): BM3-2-for and BM3-2-rev;

[0114] Fragment 3 (256 bp in length): BM3-3-for and BM3-3-rev;

[0115] Fragment 4 (98 bp in length): BM3-4-for and BM3-4-rev;

[0116] Fragment 5 (149 bp in length): BM3-5-for and BM3-5-rev;

[0117] Fragment 6 (length 7528 bp): pET28-BM3-for and pET28-BM3-rev.

[0118] PCR amplification conditions are as follows:

[0119] Fragment 1, fragment 2, fragment 3, fragment 4, fragment 5: first pre-denaturation at 95°C for 10 min, then 98°C for 30 s, 60°C for 15 s, 72°C for 15 s, for a total of 25 cycles; finally extension at 72°C for 5 min.

[0120] Fragment 6: pre-denaturation at 95°C for 10 min, then 98°C for 30 s, 58°C for 15 s, and 72°C for 4 min 30 s, for a total of 25 cycles; finally, extension at 72°C for 5 min.

[0121] After the PCR was completed, 10 U of DpnⅠ was added and the template was digested at 37°C for 2 h.

[0122] The PCR products were recovered by gel extraction to obtain fragments of corresponding sizes. The concentration of each fragment was measured and each fragment was added according to the molar ratio of vector PET28a: fragment = 1:10 during Gibson Assembly. The reaction was carried out at 50°C for 1 hour.

[0123] (2) After Gibson Assembly, the cells were transferred into DH5а medium, placed on ice for 30 min, heat-shocked at 42°C for 1 min, placed on ice again for 3 min, added with 900 μL of resuscitation solution, resuscitated at 37°C for 1 h, and plated onto LB plates containing kanamycin and cultured at 37°C overnight.

[0124] (3) Pick a single clone from the plate and culture overnight to extract the plasmid, and then sequence and verify it to obtain the correct P450BM3-mut1 protein mutant expression plasmid.

[0125] 2. Construction of P450 BM3-mut2 protein mutant expression plasmid

[0126] (1) Primer design of P450 BM3-mut2 is shown in Table 4:

[0127] Table 4 P450 BM3-mut2 primer design

[0128]

[0129] For PCR amplification, the primer combinations are as follows:

[0130] Fragment 1 (length 8449 bp): BM3-F108S-for and BM3-F108S-rev;

[0131] Fragment 2 (length 8449 bp): BM3-F262S-L234S-for and BM3-F262S-L234S-rev;

[0132] Fragment 3 (length 8449 bp): BM3-I358S-for and BM3-I358S-rev.

[0133] PCR amplification conditions are as follows:

[0134] A: Fragment 1: pre-denaturation at 95°C for 10 min, then 98°C for 30 s, 60°C for 15 s, and 72°C for 4 min 30 s, for a total of 25 cycles; finally, extension at 72°C for 5 min.

[0135] B: After the PCR of fragment 1 was completed, 10U DpnⅠ was added and the template was digested at 37℃ for 2h.

[0136] C: Gel recovery of the above PCR products to obtain fragments of corresponding sizes. Measure the concentration of the fragments, transfer them into DH5а, place them on ice for 30 minutes, heat shock them at 42℃ for 1 minute, place them on ice again for 3 minutes, add 900μL of recovery solution, recover at 37℃ for 1 hour, and spread them on LB plates containing kanamycin and culture them at 37℃ overnight.

[0137] D: Pick a single clone from the plate and extract the plasmid from the overnight culture. Perform sequencing verification to obtain a mutant containing the mutation site of fragment 1.

[0138] E: Continue to amplify the correct plasmid obtained in D using the fragment 2 primer and repeat steps A to D.

[0139] F: Continue to amplify the correct plasmid obtained in E with the fragment 3 primer and repeat steps B to D to obtain the correct P450 BM3-mut2 protein mutant expression plasmid.

[0140] 3. Preparation of E. coli expressing P450 BM3 protein mutant

[0141] (1) The P450 BM3-mut1 protein mutant expression plasmid or the P450 BM3-mut2 protein mutant expression plasmid was transformed into the expression host Escherichia coli BL21 (DE3).

[0142] (2) The above strain was inoculated into 4 mL LB medium (50 μg / mL kanamycin) and cultured overnight.

[0143] (3) Transfer to 1L LB medium (50μg / mL kanamycin) and grow to OD 600 =0.6-0.8, add IPTG at a final concentration of 0.5 mmol / L, induce expression overnight at 18°C ​​and 220 rpm to obtain bacteria expressing P450BM3-mut1 protein mutant or bacteria expressing P450BM3-mut2 protein mutant. Preparation Example 2 Construction of P450 BM3 protein mutant containing large framework porphyrin

[0144] Collect the bacteria expressing the P450 BM3-mut1 protein mutant or the P450 BM3-mut2 protein mutant induced overnight obtained in Preparation Example 1, resuspend the bacteria with 30 mL of pH = 7.5 cell suspension, add 1 mL of tetraphenylporphyrin manganese (III) chloride to 30 mL of the P450 BM3-mut1 protein mutant cell suspension or add 1 mL of 5,10,15,20-tetrakis (pentafluorophenyl) porphyrin manganese (III) chloride porphyrin solution to 30 mL of the P450 BM3-mut2 protein mutant cell suspension (the macro-framework porphyrin is dissolved in DMF at a concentration of 5 mg / mL), and then use an ultrasonic disruptor (Biosafer900-92) for ultrasonic disruption, with a 6-horn, 45% power, 3s on, 6s off, and disruption for 30 min. After the disruption is completed, place it at 4°C and incubate for 30 min. After that, the supernatant was collected by high-speed centrifugation at 12000rpm for 60min, incubated with nickel gel, and then the impurities were eluted with Tris-HCl buffer containing low concentration of imidazole (40mmol / L imidazole), and then the mutant protein bound to the large framework porphyrin was collected with Tris-HCl buffer containing 500mmol / L imidazole. Tris-HCl buffer: 50mmol / L Tris, 200mmol / L sodium chloride, 1mmol / L tris (2-carboxyethyl) phosphine (TCEP), 10% (v / v) glycerol, pH 7.5-8.0.

[0145] The eluted mutant protein bound to the large framework porphyrin was dialyzed into a buffer solution of 100 mmol / L KPi, pH 7.5-8.0.

[0146] Preparation Example 3 Construction of a P450 BM3 protein mutant containing a large framework porphyrin

[0147] Collect the bacteria expressing the P450 BM3-mut1 protein mutant or the bacteria expressing the P450 BM3-mut2 protein mutant obtained in Preparation Example 1 overnight, resuspend the bacteria with 30 mL of pH = 7.5 cell suspension, and use a high-pressure homogenizer to break it up at 850 bar for 5 minutes. After the breaking, add 1 mL of tetraphenylporphyrin manganese (III) chloride to 30 mL of the P450 BM3-mut1 protein mutant breaking solution or add 1 mL of 5,10,15,20-tetrakis (pentafluorophenyl) porphyrin manganese (III) chloride porphyrin solution to 30 mL of the P450 BM3-mut2 protein mutant breaking solution (the large framework porphyrin is dissolved in DMF at a concentration of 5 mg / mL), and incubate at 4°C for 30 minutes. After that, the supernatant was collected by high-speed centrifugation at 12000rpm for 60min, incubated with nickel gel, and then the impurities were eluted with Tris-HCl buffer containing low concentration of imidazole (40mmol / L imidazole), and then the mutant protein bound to the large framework porphyrin was collected with Tris-HCl buffer containing 500mmol / L imidazole. Tris-HCl buffer: 50mmol / L Tris, 200mmol / L sodium chloride, 1mmol / L tris (2-carboxyethyl) phosphine (TCEP), 10% (v / v) glycerol, pH 7.5-8.0.

[0148] The eluted mutant protein bound to the large framework porphyrin was dialyzed into a buffer solution of 100 mmol / L KPi, pH 7.5-8.0.

[0149] Preparation Example 4 Construction of a P450 BM3 protein mutant containing a large framework porphyrin

[0150] Collect the bacteria expressing the P450 BM3-mut1 protein mutant or the bacteria expressing the P450 BM3-mut2 protein mutant obtained in Preparation Example 1 overnight, resuspend the bacteria with 30 mL of pH = 7.5 cell suspension, and crush them with a high-pressure homogenizer at 850 bar for 5 minutes. After crushing, high-speed centrifugation at 12000 rpm for 60 minutes was performed to collect the supernatant, and after incubation with nickel colloid, the impurities were eluted with a Tris-HCl buffer containing a low concentration of imidazole (40 mmol / L imidazole), and then the mutant protein was collected with 30 mL of a Tris-HCl buffer containing 500 mmol / L imidazole. Add 1 mL of tetraphenylporphyrin manganese (III) chloride to 30 mL of P450 BM3-mut1 protein mutant eluate or add 1 mL of 5,10,15,20-tetrakis (pentafluorophenyl) porphyrin manganese (III) chloride to 30 mL of P450 BM3-mut2 protein mutant eluate (the large framework porphyrin is dissolved in DMF at a concentration of 5 mg / mL), incubate at 4°C for 30 min, and repeat the above purification steps. Tris-HCl buffer: 50 mmol / L Tris, 200 mmol / L sodium chloride, 1 mmol / L tris (2-carboxyethyl) phosphine (TCEP), 10% (v / v) glycerol, pH 7.5-8.0.

[0151] The eluted mutant protein bound to the large framework was dialyzed into a buffer solution of 100 mmol / L KPi, pH 7.5-8.0.

[0152] Spectroscopic determination of mutant proteins binding to large framework porphyrins

[0153] 100 μL of the mutant protein bound to the large-framework porphyrin obtained in Preparation 2, the mutant protein bound to the large-framework porphyrin obtained in Preparation Example 3, and the mutant protein bound to the large-framework porphyrin obtained in Preparation Example 4 were taken for spectral measurement (the protein amount was about 500 μg). The measurement was performed using an enzyme marker with a measurement range of 300 to 500 nm and a step size of 1 nm.

[0154] The experimental results are as follows Figure 1 , Figure 2 As shown in Table 5:

[0155] Table 5 Spectral characterization results

[0156]

[0157]

[0158]

[0159]

[0160]

[0161] The experiment of the present invention proves that, when spectroscopically measured by an enzyme marker, myoglobin has the highest absorption at 450nm when it binds to heme, while the P450 BM3-mut1 and P450 BM3-mut2 mutants have no absorption at 450nm, and therefore lose the ability to bind to heme. When tetraphenylporphyrin manganese (III) chloride is added to P450 BM3-mut1 or 5,10,15,20-tetrakis (pentafluorophenyl) porphyrin manganese (III) chloride is added to the P450 BM3-mut2 mutant, the mutants have corresponding absorption peaks at specific positions (Table 5).

[0162] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A P450 BM3 protein mutant, characterized in that: It consists of P450 BM3-mut1 and / or P450 BM3-mut2; The P450 BM3-mut1 includes the following multiple site mutations: K70A; L72G; G86A; L87G; F88A; W91A; E94A; W97A; I154A; F262A; F332A; I358A; and R399A; The amino acid sequence of the P450 BM3-mut1 is shown in SEQ ID NO: 1; The P450 BM3-mut2 includes the following multiple site mutations: K70A; L72G; G86A; L87G; F88A; W91A; E94A; W97A; I154A; F332A; R399A; F108S; L234S; F262S; and I358S; The amino acid sequence of the P450 BM3-mut2 is shown in SEQ ID NO:

2.

2. A nucleic acid molecule encoding the P450 BM3 protein mutant according to claim 1, characterized in that: The nucleic acid molecule encoding the P450 BM3-mut1 is: (I), the nucleotide sequence shown in SEQ ID NO: 3; or (II) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I) but is different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or The nucleic acid molecule encoding the P450 BM3-mut2 is: (I), the nucleotide sequence shown in SEQ ID NO: 4; or (II) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I) but is different from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code.

3. A recombinant vector, characterized in that: It comprises the nucleic acid molecule and the backbone vector as claimed in claim 2.

4. A host, characterized in that It comprises the recombinant vector as claimed in claim 3.

5. A primer set, characterized in that: The primer set consists of: The primers consist of the nucleotide sequences shown in SEQ ID No: 5 to 16; or The primers consist of the nucleotide sequences shown in SEQ ID No: 17-22.

6. Use of any of the following in the preparation of P450 BM3 protein mutants: (I), the nucleic acid molecule according to claim 2; and / or (II), the recombinant vector according to claim 3; and / or (III), the host according to claim 4; and / or (IV) The primer set according to claim 5.

7. The method for preparing a P450 BM3 protein mutant according to claim 1, characterized in that: The method comprises preparing a P450 BM3 protein mutant based on any of the following items: (I), the nucleic acid molecule according to claim 2; and / or (II), the recombinant vector according to claim 3; and / or (III), the host according to claim 4; and / or (IV) The primer set according to claim 5.

8. Use of any of the following in the preparation of metalloporphyrin protein: (I) The P450 BM3 protein mutant according to claim 1; and / or (II), the nucleic acid molecule according to claim 2; and / or (III), the recombinant vector according to claim 3; and / or (IV), the host according to claim 4; and / or (V) The primer set according to claim 5; and / or (VI) A P450 BM3 protein mutant prepared by the preparation method according to claim 7; When P450 BM3-mut1 was used, the metalloporphyrin added was tetraphenylporphyrin manganese(III) chloride; When P450 BM3-mut2 was used, the metalloporphyrin added was 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin manganese(III) chloride porphyrin.

9. A metalloporphyrin protein, characterized in that The metalloporphyrin protein and / or artificial enzyme includes any of the following items and metalloporphyrin: (I) The P450 BM3 protein mutant according to claim 1; and / or (II) a P450 BM3 protein mutant prepared by the preparation method according to claim 7; When P450 BM3-mut1 is used, the metalloporphyrin added is tetraphenylporphyrin manganese(III) chloride; When P450 BM3-mut2 is used, the metalloporphyrin added is 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin manganese(III) chloride porphyrin.

10. Use of the metalloporphyrin protein according to claim 9 in enzyme catalysis.

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