A mutant for catalytic synthesis of phenazine and phenoxazine ketone compounds and its application

Abstract

The present invention relates to a mutant for catalytic synthesis of phenazine and phenoxazine ketone compounds and its application, belonging to the field of bioenzyme engineering technology, wherein the mutant is a cytochrome P450BM3 oxidase in which one or more of the following amino acid sites are mutated: the 87th phenylalanine is mutated to alanine or glycine, the 78th valine is mutated to alanine, and the 264th threonine is changed to alanine, and the specific mutants are F87A, F87G, F87A / V78A / A264G or F87G / V78A / A264G. The present invention also provides an expression vector of any one of the mutants and a genetic engineering bacterium containing the expression vector, and the mutant catalyzes the self-oxidative coupling of aromatic amine substrates including 2,5-diaminobenzenesulfonic acid, o-phenylenediamine or o-aminophenol to generate phenazine and phenoxazine ketone compounds through H2O2, and the yield of the mutant catalyzing o-aminophenol to generate phenoxazine ketone is as high as 99%.

Description

Technical Field

[0001] The invention belongs to the technical field of bioenzyme engineering, and specifically relates to a mutant for catalyzing the synthesis of phenazine and phenoxazine ketone compounds and application thereof. Background Art

[0002] Natural phenazines and their derivatives are a class of nitrogen-containing heterocyclic compounds that are ubiquitous in nature. They are mainly distributed in the secondary metabolites of marine and soil microorganisms and have a variety of important biological activities such as antibacterial, antiviral and antitumor. To date, more than 150 natural phenazine products from organisms such as Pseudomonas, Actinomycetes and Streptomyces have been identified. Inspired by the active skeleton of natural phenazines, it is of great research value to discover and explore phenazine compounds with novel structures and clear functions. At present, more than 6,000 compounds containing phenazine structures have been synthesized by biological, chemical and other methods, and have shown excellent performance.

[0003] Phenoxazinone (PXO) is widely found in nature and is often used as the parent structure of antimicrobial agents, antibiotics, antitumor drugs, pesticides, and dyes. It is also an important component of optoelectronic materials and biosensors. The most studied phenoxazinone is 2-aminophenoxazinone (APX), which is the key component of the famous anticancer natural peptide antibiotic actinomycin D and plays an inhibitory role in the process of DNA-directed synthesis of RNA. In addition, APX also exists in other bacterial and fungal secondary metabolites and has a variety of application values.

[0004] Artificial chemical synthesis of phenazines and phenoxazinones can be traced back to the mid-19th century. Due to the excellent stability, good dyeing properties and excellent biological activity of phenazines and phenoxazinones, researchers began to look for convenient and efficient artificial synthesis methods, mainly focusing on chemical reagents, electrochemistry, photocatalysis and metal complexes, and achieved certain results. However, traditional chemical synthesis methods have many disadvantages such as long reaction time, large-scale use of chemical reagents, and heavy metal pollution, while the biological preparation of phenazines and phenoxazinones is still in the development stage. Therefore, it is urgent to find biocatalysts with excellent catalytic performance to achieve green and efficient synthesis of phenazines and phenoxazinones.

[0005] P450BM3 is known as a "universal biocatalytic oxidant" due to its wide substrate adaptability and diverse catalytic functions, and has great potential for application in biocatalysis and transformation, synthetic chemistry, and synthetic biology. However, its dependence on expensive cofactors NAD(P)H and redox chaperone proteins limits its practical catalytic application in vitro. Although there is a H2O2 shunt pathway in the P450BM3 catalytic cycle, its efficiency in driving H2O2 is low. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a mutant for catalytic synthesis of phenazine and phenoxazinone compounds and its application. The present invention further optimizes the DFSM-P450BM3 system through enzyme engineering and chemical modification strategies to provide an economical and efficient green synthesis method for phenazine and phenoxazinone compounds.

[0007] The present invention is achieved through the following technical solutions:

[0008] A mutant catalyzing the synthesis of phenazine and phenoxazine ketone compounds, wherein one or more of the following amino acid sites of cytochrome P450BM3 oxidase are mutated: phenylalanine at position 87 is mutated to alanine or glycine, valine at position 78 is mutated to alanine, and threonine at position 264 is changed to alanine. The specific mutants are F87A, F87G, F87A / V78A / A264G or F87G / V78A / A264G.

[0009] Specifically, a mutant catalyzing the synthesis of phenazine and phenoxazinone compounds, wherein the phenylalanine at position 87 of the amino acid sequence shown in SEQ ID NO.1 is replaced with alanine or glycine; the obtained mutants are named F87A or F87G respectively.

[0010] Furthermore, a mutant catalyzing the synthesis of phenazine and phenoxazine ketone compounds, based on the mutation at position 87, simultaneously replaces the valine at position 78 of the amino acid sequence shown in SEQ ID NO.1 with alanine; the mutants are named F87A / V78A or F87G / V78A respectively.

[0011] Furthermore, based on the mutations at positions 87 and 78, the alanine at position 264 of the amino acid sequence shown in SEQ ID NO.2 was replaced with glycine; the mutants were named F87A / V78A / A264G or F87G / V78A / A264G, respectively.

[0012] An expression vector comprising any one of the mutants, wherein the mutant is one of F87A, F87G, F87A / V78A / A264G or F87G / V78A / A264G.

[0013] A genetically engineered bacterium, which contains the expression vector.

[0014] An application of a mutant, wherein the mutant is used in catalyzing the synthesis of biological phenazine and phenoxazine ketone compounds, and the mutant is at least one of F87A, F87G, F87A / V78A / A264G or F87G / V78A / A264G.

[0015] As a more specific embodiment, the catalytic system in the application is a bifunctional small molecule compound (DFSM), any one of the mutants, H2O2 and a substrate; wherein the bifunctional small molecule compound is one of the N-(ω-imidazolyl, fatty acyl)-L-phenylalanine derivatives Im-C7-Phe / Im-C6-Phe / Im-C5-Phe.

[0016] As a further application, the substrate of the catalytic reaction is one of 2,5-diaminobenzenesulfonic acid, o-phenylenediamine, and o-aminophenol. The specific catalytic process is to use a bifunctional small molecule N-(ω-imidazole, fatty acyl)-L-phenylalanine derivative to induce the mutant, and realize the self-oxidative coupling of aromatic amine substrates including 2,5-diaminobenzenesulfonic acid, o-phenylenediamine or o-aminophenol to generate phenazine and phenoxazine ketone compounds through H2O2 catalysis. The mutant is a single, double or triple mutant containing the mutations at the 87th, 78th and 264th positions, wherein the phenylalanine at the 87th position is mutated to glycine or alanine, the valine at the 78th position is mutated to alanine, and the alanine at the 264th position is changed to glycine, that is, at least one of F87A, F87G, F87A / V78A / A264G or F87G / V78A / A264G.

[0017] The oxidative coupling system is to sequentially add 1 mM substrate, 1 μM wild-type P450BM3 and a series of mutants, and 0.5 mM bifunctional small molecules to 1 mL of potassium phosphate buffer (100 mM, pH 6.0), incubate at 30° C. for 5 min, then add 20 mM H2O2 and react for 15 min.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention combines the dual regulatory capabilities of protein engineering technology with the synergistic strategy of bifunctional small molecules to achieve green and efficient conversion of phenazine and phenoxazinone compounds, optimize the P450BM3 catalytic system and expand the substrate range, providing new ideas and methods for further research in the field of biocatalysis.

[0020] The invention uses random mutation high-throughput screening to obtain superior mutants that catalyze the biosynthesis of phenazine and phenoxazine ketone through directed evolution of P450BM3, constructs a "small and precise" mutant library through semi-rational design of superior mutation sites, improves screening efficiency, obtains high-yield indigo superior mutant enzymes under a P450 peroxygenase system coordinated by a bifunctional small molecule, and obtains the highest catalytic efficiency of F87G / V78A for 2,5-diaminobenzenesulfonic acid; the yield of F87A / V78A / A264G catalyzing the synthesis of phenazine compounds from 2,5-diaminobenzenesulfonic acid is as high as 99%; F87G has the highest catalytic efficiency for o-phenylenediamine (79.13 mM-1s-1), and the yield of catalyzing o-phenylenediamine to generate phenazine compounds is the highest (84%); F87G has the highest catalytic efficiency for o-aminophenol (276.92 mM-1s-1), and the yield of F87G catalyzing o-phenylenediamine to generate phenazine compounds is the highest (84%). -1 s -1 ), catalyzing o-aminophenol to produce phenoxazinone with a yield of up to 98%. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 HPLC analysis chart of the bifunctional small molecule provided in the embodiment of the present invention cooperating with the typical mutant of P450BM3 to catalyze 2,5-diaminobenzenesulfonic acid using H2O2;

[0022] Figure 2 HPLC analysis chart of the bifunctional small molecule provided in the embodiment of the present invention cooperating with a typical mutant of P450BM3 to catalyze o-phenylenediamine using H2O2;

[0023] Figure 3 HPLC analysis chart of the bifunctional small molecule provided in the embodiment of the present invention cooperating with the typical mutant of P450BM3 to catalyze o-aminophenol using H2O2. DETAILED DESCRIPTION

[0024] The principles and features of the present invention are further described below. The examples listed are only used to explain the present invention and are not used to limit the scope of protection of the present invention. In addition, the experimental methods used in the following examples are conventional methods in the art unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0025] The present invention firstly constructs a DFSM-P450BM3 peroxygenase system by semi-rationally designing amino acid mutations through protein engineering and chemical modification strategies. The system uses green and economical H2O2 as an oxidant to catalyze three substrates of 2,5-diaminobenzenesulfonic acid, o-phenylenediamine and o-aminophenol to generate phenazine and phenoxazinone compounds. The system is simple in composition and can achieve efficient biocatalysis without relying on expensive cofactors NAD(P)H and electron transfer chaperone proteins, providing a green and efficient alternative method for the synthesis of phenazine and phenoxazinone compounds.

[0026] Meanwhile, the bifunctional small molecule compounds of the following examples can be purchased commercially or synthesized according to the prior art.

[0027] The following methods were used for the construction of engineering bacteria, induced expression and purification of enzymes, and determination of catalytic activity in the following examples, as follows:

[0028] 1. Construction of mutants

[0029] The process of obtaining the mutation is as follows: using the oxidase part of the cytochrome P450-BM3 monooxygenase from Bacillus megaterium as a template, using the mutation primer containing the 87 mutation site to perform PCR mutation, sequencing to obtain the F87 single mutant; then using the single mutant gene as a template, PCR amplification of the F87-V78 combination double mutant is performed in the same way; then using the double mutant gene as a template, PCR amplification of the F87-V78-A264 combination triple mutant is performed in the same way, and the catalytic effect of synthesizing phenazine and phenoxazine ketone compounds by this P450BM3-H2O2 system is greatly improved, and at the same time, efficient regulation of biosynthesis can be achieved by combining different amino acids and organically combining with different bifunctional small molecules. Then, multiple mutants containing the 87 mutation site and one or more other sites around it are obtained by combined mutation, and introduced into the oxidative coupling body, which greatly improves the efficiency of this system in catalyzing the synthesis of phenazine and phenoxazine ketone compounds.

[0030] The wild-type cytochrome P450BM3 oxidase gene (the gene sequence is shown in SEQ ID NO.1) was subjected to site-directed mutagenesis using PCR mutagenesis technology. Using the recombinant plasmid pET-28a(+)-BMP connected to the P450BM3 oxidase part (BMP) gene as a template, a pair of forward and reverse primers containing the mutation site were designed, and PCR was performed to obtain the recombinant gene.

[0031] The PCR amplification procedure is:

[0032]

[0033] The amplified PCR product was detected by 0.7% agarose gel electrophoresis, and a band with the same length as the P450BM3 gene was observed, which determined that the gene encoding the target mutant had been amplified. The PCR product was transformed into E. coli DH5ɑ competent cells after KLD connection, and evenly coated on a solid LB plate containing 50μl / mg kanamycin after adding culture medium for 1h. After overnight culture at 37℃, a single clone was selected and cultured in 5mL LB liquid culture medium containing 50μg / mL kanamycin, and then sent to a sequencing company for sequencing to obtain the correct mutant.

[0034] 2. Induced expression and purification of mutants

[0035] Pick a single colony with successful mutation, randomly pick a single colony into 5mL LB medium containing Kana, activate it for a period of time, transfer 500μL of bacterial solution into 50mL LB liquid medium (Kana), activate it for a period of time, transfer 5mL of bacterial solution into 500mL LB liquid medium (Kana), when the OD value is 0.6-0.8, add 0.5mM FeCl3, 5'-ALA, culture at 37℃, 180rpm for 30min, then add 1mM IPTG, induce at 30℃, 180rpm for 20h.

[0036] The expressed bacterial solution was centrifuged at 8000 rpm and 4°C for 10 min, the supernatant was discarded, and the bacterial cells were resuspended and washed twice with Binding buffer (100 mM K2HPO4-KH2PO4, 500 mM NaCl, pH 7.4) to obtain resting cells. The resting cells were resuspended with one-tenth of the bacterial solution volume of Binding buffer, mixed and then ultrasonically disrupted, and the disrupted product was centrifuged at 10000 rpm and 4°C for 90 min to remove cell debris, and the crude enzyme solution of the mutant was obtained.

[0037] The crude enzyme solution was purified by Ni column affinity chromatography. The crude enzyme solution was filtered through a 0.22 μm filter membrane. The pretreated series of P450BM3 crude proteins were further purified using the AKTAexplorer 10S protein rapid purification instrument, and dual wavelength detection was performed using the 280nm protein absorption wavelength and the 418nm P450 protein characteristic absorption wavelength. Specific steps: First, balance the His Trap with 100% loading solution TM HP affinity chromatography column, the flow rate is 5mL / min, remove the impurities that may remain in the column, start loading after the baseline is stable, the loading flow rate is 3mL / min; at this time, most of the impurities will directly pass through the chromatography column to show a penetration peak, and the target protein P450BM3 and a small amount of impurities are bound to the column due to ion interaction; when the baseline is stable again, the impurities are eluted with 96% loading solution and 4% eluent (100mM K2HPO4-KH2PO4, 500mM NaCl, 500mM imidazole, pH 7.4), and after the baseline is stable, the impurities are washed again with 100mL (20 column volumes) of 94% loading solution and 6% eluent; after the baseline is stable again, the elution of the target protein begins, and the eluent concentration ratio is adjusted to 40%. When the characteristic absorption peak appears at 418nm, the target protein is collected from the outlet pipeline; at this time, P450BM3 no longer binds to His Trap TMThe HP affinity chromatography column undergoes specific binding and is displaced by high concentration of imidazole; the concentration ratio of the eluent is adjusted to 100%, and the protein in the column is washed with high concentration of imidazole. After the baseline is stable, the system and pump are washed with ultrapure water and 20% ethanol respectively; the high concentration of imidazole in the target protein is removed by dialysis, first dialyzing once with pure water 100 times the volume of the target protein, and then dialyzing the target protein twice with dialysate, each time for 2 hours; the target protein is placed in an ultrafiltration concentration cup for concentration and desalination, and the filter membrane size is 30kDa; an equal volume of glycerol is added to the concentrated protein, and stored at -80°C.

[0038] 3. Test of catalytic performance of DFSM-P450BM3 system:

[0039] Reaction kinetics test: The reaction system is 1 mL. Different concentrations of substrate (0.0125, 0.025, 0.05, 0.075, 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1.0, 1.5 and 2.0 mM), 1 μM wild-type P450BM3 and a series of mutants, and 0.5 mM Im-C6-Phe are added to 100 mM potassium phosphate buffer at pH 6.0. The reaction is incubated at 30°C for 5 min, and 20 mM H2O2 is added to start the reaction. The initial reaction velocity is calculated by measuring the change in absorbance at a wavelength of 460 nm within 15 min and the molar absorptivity of the product. According to the Michaelis-Menten equation v / [protein]=k cat [substrate] / (K m +[substrate]) to obtain the kinetic parameter k cat , K m .

[0040] 4. Detection of reaction products: To the reaction system of 1 mL potassium phosphate buffer (100 mM, pH 6.0), add 1 mM 2,5-diaminobenzenesulfonic acid, 1 μM wild-type P450BM3 and a series of mutants, and 0.5 mM bifunctional small molecules in sequence. After incubation at 30°C for 5 min, add 20 mM H2O2 and react for 15 min.

[0041] 5. HPLC detection conditions: The oxidation products were qualitatively and quantitatively detected by HPLC using Waters sunfire TM C18 column 5.0μm (4.6mm×150mm chromatographic column), isocratic elution, flow rate 1mL / min, column temperature 40℃, injection volume 20μL. The specific detection conditions of the three substrates and their corresponding products are as follows:

[0042] (1) 2,5-Diaminobenzenesulfonic acid: The mobile phase was acetonitrile and buffer (1 g / L tetrabutylammonium bromide and 2 g / L NaH2PO4) (volume ratio of 20:80), and the detection wavelengths were 245 nm and 460 nm.

[0043] (2) o-phenylenediamine: the mobile phase was acetonitrile and water (volume ratio was 20:80), and the detection wavelengths were 288 nm and 419 nm.

[0044] (3) o-Aminophenol: The mobile phase was acetonitrile and water (volume ratio 40:60), and the detection wavelengths were 286 nm and 433 nm.

[0045] Example 1 Preparation of F87 single mutant

[0046] Using the recombinant plasmid pET-28a(+)-BMP connected to the P450BM3 oxidase part (BMP) gene as a template, and the forward and reverse primers containing different mutation sites (see Table 1) as primer pairs, PCR was performed to obtain the recombinant genes of F87 single mutants F87A and F87G. The amplification system and PCR conditions are shown in the general method.

[0047] Table 1

[0048]

[0049] Example 2 Preparation of F87-V78 double mutation:

[0050] Using the recombinant plasmid pET-28a(+)-F87A connected with the P450BMP-F87 single mutation as a template, and the forward and reverse primers containing different mutation sites as primer pairs (see Table 2), PCR was performed to obtain the recombinant genes of different F87-V78 double mutants. The amplification system and PCR conditions are shown in the general method.

[0051] Table 2

[0052]

[0053] Example 3 Preparation of F87-V78-A264 triple mutation

[0054] Using the recombinant plasmid pET-28a(+)-F87A connected with the P450BMP-F87-V78 double mutation as a template, and using forward and reverse primers containing different mutation sites as primer pairs (see Table 3), PCR was performed to obtain the recombinant genes of different F87-V78-A264 triple mutants. The amplification system and PCR conditions are shown in the general method.

[0055] Table 3

[0056]

[0057] Example 4 P450BM3 catalyzes the production of phenazines and phenoxazinones from aromatic amine substrates

[0058] In this example, three aromatic amines, 2,5-diaminobenzenesulfonic acid (2,5-DABSA), o-phenylenediamine (OPD) and o-aminophenol (OAP), were used as substrates to synthesize phenazines and phenoxazine ketone compounds under the catalysis of the P450BM3 peroxygenase system, and the catalytic performance of the two catalytic systems, the DFSM-P450BM3 system and the P450BM3 system with artificially introduced acidic and basic amino acids, were tested.

[0059] The catalytic activity of the mutants prepared in Example 1-3 to synthesize phenazine and phenoxazine ketone compounds from 2,5-diaminobenzenesulfonic acid (2,5-DABSA), o-phenylenediamine (OPD) and o-aminophenol (OAP) was measured. The reaction conditions were as described in General Method 3 (Testing of Catalytic Performance of DFSM-P450BM3 System). All mutants were not added with small molecules as reaction controls; the specific activities are shown in the following table:

[0060] 1) 2,5-Diaminobenzenesulfonic acid (2,5-DABSA)

[0061] Table 4

[0062]

[0063] From the data in Table 4, it can be seen that under the cooperation of Im-C6-Phe, the catalytic efficiency of single mutants F87A and F87G increased significantly to 76.47 mM -1 s -1 and 125.21mM -1 s -1 , which are 2.6 and 4.2 times the catalytic efficiency of wild-type P450BM3, respectively. In order to further improve the catalytic ability of F87A and F87G, the valine at position 78 located in the substrate channel was replaced with alanine, which is smaller in size, to obtain the catalytic efficiency of the double mutants F87A / V78A and F87G / V78A. cat / K m 129.85mM respectively -1 s -1 (1.7 times that of F87A) and 175.69mM -1 s -1 The double mutants F87A / V78A and F87G / V78A were used as parents to detect the effect of the 264th amino acid, which is closer to the heme active center, on the catalytic performance of 2,5-diaminobenzenesulfonic acid oxidative coupling. The obtained triple mutants F87A / V78A / A264G and F87G / V78A / A264G increased kcat The value increased to 20.76s -1 (1.3 times that of F87A / V78A) and 19.16s -1 (1.9 times that of F87G / V78A).

[0064] 2) o-phenylenediamine (OPD)

[0065] Table 5

[0066]

[0067] In the presence of Im-C6-Phe, the catalytic efficiency of wild-type P450BM3 was low, and k cat The value is 0.050s -1 , catalytic efficiency k cat / K m 0.29mM -1 s -1 The catalytic efficiency of single mutants F87A and F87G was significantly improved compared with that of wild-type P450BM3. cat The value is increased to 0.88s -1 , about 17.6 times that of wild-type P450BM3; K m The value decreased to 0.023mM, the affinity between the enzyme and the substrate increased, and the catalytic efficiency k cat / K m Increased to 38.26mM -1 s -1 , which is about 131.9 times that of wild-type P450BM3; F87G further increases k cat The value is increased to 1.82s -1 , which is about 36.4 times that of wild-type P450BM3. cat / K m The value increased to 79.13mM -1 s -1 , which is approximately 272.9 times that of wild-type P450BM3.

[0068] 3) o-Aminophenol (OAP)

[0069] Table 6

[0070]

[0071] Under the synergistic effect of Im-C6-Phe, the catalytic efficiency of wild-type P450BM3 towards o-aminophenol was low, k cat The value is only 0.15s -1 , catalytic efficiency k cat / K m Only 11.54mM -1 s-1 The catalytic efficiency of the single mutants F87A and F87G obtained by mutating position 87 to alanine and glycine was significantly improved compared with the wild-type P450BM3. cat The value is increased to 1.66s -1 , which is about 11.1 times that of wild-type P450BM3, increasing the catalytic efficiency to 50.30 mM -1 s -1 , which is about 4.4 times that of wild-type P450BM3. Among all mutants in the DFSM-P450BM3 system, F87G exhibits the highest catalytic efficiency, k cat The value is 7.20s -1 , about 48.0 times that of wild-type P450BM3; k cat / K m The value is 276.92mM -1 s -1 , which is approximately 24.0 times that of wild-type P450BM3.

[0072] Example 5 Comparison of DFSM Control Capabilities

[0073] This example compares the control ability of DFSMs with different chain lengths, taking the catalysis of o-aminophenol as an example. The control ability of DFSM with a carbon chain length of 5 carbon atoms (Im-C5-Phe) and DFSM with a carbon chain length of 7 carbon atoms (Im-C7-Phe) were selected and compared with Im-C6-Phe to determine the most suitable DFSM structure.

[0074] Table 7

[0075]

[0076] The above results show that the bifunctional small molecule Im-C6-Phe has a much higher ability to regulate P450BM3 than Im-C5-Phe, and Im-C5-Phe is better than Im-C7-Phe. DFSM with different carbon chain lengths can affect the positioning of the imidazole group as an acid-base catalyst in the heme active cavity, thereby affecting the catalytic performance of the DFSM-P450BM3 peroxygenase system.

[0077] Example 6 Yield of Oxidative Coupling Reaction of Aromatic Amine Substrates Catalyzed by DFSM-P450BM3 System The reaction of 2,5-diaminobenzenesulfonic acid, o-phenylenediamine and o-aminophenol catalyzed by DFSM-P450BM3 system was detected by HPLC. The specific method is described in General Methods 4 (4, Detection of Reaction Products) and 5 (5, HPLC Detection Conditions).

[0078] In the oxidation reaction of 2,5-diaminobenzenesulfonic acid, the yield of the wild-type P450BM3 catalyzed by Im-C6-Phe was only 10% within 15 minutes, while the single mutants F87A and F87G increased the yield to 66% and 92%, respectively. The F87A / V78A / A264G mutant further increased the yield to 99%. The specific HPLC chart is shown in Figure 2. Figure 1 shown.

[0079] In the oxidation reaction of o-phenylenediamine, the yield of the F87G mutant catalyzed by the synergistic effect of Im-C6-Phe was as high as 84% ​​within 15 minutes, which was 74% higher than that of the wild-type P450BM3. The yield of the F87G mutant in the oxidation of o-aminophenol was as high as 99% within 10 minutes. The specific HPLC graph is shown in Figure 2. Figure 2 and Figure 3 shown.

[0080] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

[0081] Bacillus megaterium P450BM3 (SEQ ID NO.1)

[0082]

Claims

1. An application of a mutant for catalytic synthesis of phenazine and phenoxazine ketone compounds, characterized in that: The application is to use the mutant to catalyze the synthesis of biological phenazine and phenoxazine ketone compounds, the mutant is at least one of F87A, F87G, F87A / V78A / A264G or F87G / V78A / A264G, the mutant is a mutation based on the amino acid sequence encoded by the gene of the nucleotide sequence shown in SEQ ID NO.1, and the substrate of the catalytic reaction is one of 2,5-diaminobenzenesulfonic acid, o-phenylenediamine, and o-aminophenol; The catalytic system in the application is a bifunctional small molecule compound, any one of the mutants, H2O2 and a substrate; wherein the bifunctional small molecule compound is one of the N-(ω-imidazole, fatty acyl)-L-phenylalanine derivatives Im-C7-Phe / Im-C6-Phe / Im-C5-Phe.

2. The use according to claim 1, characterized in that: The catalytic reaction was performed by sequentially adding 1 mM substrate, 1 μM mutant, and 0.5 mM bifunctional small molecule to 1 mL, 100 mM, pH 6.0 potassium phosphate buffer, incubating at 30 °C for 5 min, then adding 20 mM H2O2 and reacting for 15 min.

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