Mutant based on p450bm3 and its application in catalyzing bromophenol pollutants

Abstract

The application relates to a mutant based on P450BM3 and application of the mutant in catalyzing bromophenol pollutants, and belongs to the field of enzymology. The application provides a new mutant T268D / A328F / F87G / L272S. Wild-type P450BM3 has no degradation function on bromophenol pollutants. The mutant T268D, T268D / A328F, T268D / A328F / F87A, T268D / A328F / F87G, T268D / A328F / F87A / V78A, T268D / A328F / F87G / V78A and T268D / A328F / F87G / L272S at a series of sites based on the wild-type P450BM3 have degradation functions on bromophenol pollutants.

Description

Technical Field

[0001] This invention belongs to the field of enzymology, specifically relating to a P450BM3-based mutant and its application in catalyzing bromophenol contaminants. Background Technology

[0002] Bromophenols are significant organic pollutants, commonly used as intermediates in the synthesis of flame retardants, dyes, pharmaceuticals, pesticides, and preservatives. The main bromophenols include 4-bromophenol, 2,4-dibromophenol, and 2,4,6-tribromophenol. They enter the environment through various pathways, including wastewater discharge, atmospheric deposition, and pesticide metabolism (bromofenoxam). The cumulative, persistent, and highly toxic (teratogenic, carcinogenic, and mutagenic) nature of bromophenols in the environment poses a significant risk to mammals, aquatic organisms, and ecosystems. Therefore, the development of effective and environmentally friendly methods for degrading bromophenols is urgently needed.

[0003] The treatment or removal of bromophenol is crucial. Currently, numerous methods have been researched for the removal of bromophenol from aquatic environments, including physicochemical methods such as adsorption, electrochemical oxidation, photochemical oxidation, and ozone oxidation. However, these physicochemical methods all have certain limitations. Physical adsorption methods are relatively inefficient, while electrochemical oxidation, photochemical oxidation, and ozone oxidation often produce byproducts and are costly and energy-intensive, making them unsuitable for large-scale industrial applications. Therefore, exploring a more efficient and environmentally friendly method for degrading bromophenol pollutants is particularly important. Microbial-mediated bioremediation has the potential to efficiently degrade bromophenol and is thus a promising alternative. However, challenges such as long cultivation cycles and slow reaction rates remain. Enzymatic degradation of bromophenol, on the other hand, offers milder conditions and higher efficiency, making it particularly promising for future applications.

[0004] Cytochrome P450 is a heme-containing monooxygenase crucial for drug metabolism, detoxification of exogenous compounds, and steroid biosynthesis. However, most natural P450s require the cofactor NAD(P)H and a complex electron transport system to activate molecular oxygen, which significantly limits their applications. Low-cost hydrogen peroxide is an ideal alternative to the expensive cofactor NAD(P)H and can be used directly to unlock the catalytic potential of P450s. However, the hydrogen peroxide split pathway is generally inefficient or even absent in driving P450 catalysis. With the exception of a few natural P450s, most lack the structural properties required to activate hydrogen peroxide (generally acid-base catalysts). Given the enormous potential of peroxidases as practical biocatalysts, efforts have been made to develop hydrogen peroxide-driven P450 systems. Recently, an artificial P450-H2O2 system was constructed using P450 BM3, which integrates anchoring and acid-base catalysis within the active site via a bifunctional small molecule. Despite the many advantages of bifunctional small molecules, their synthesis remains complex and costly. Furthermore, myoglobin and P450 can be converted into peroxidases or peroxygenases by introducing an acid-base residue through directed mutagenesis. However, ensuring the correct orientation of the acid-base residue to effectively function as a direct acid-base catalyst remains a significant challenge.

[0005] By inserting aspartic acid near the active site of P450BM3, the aim was to improve its catalytic efficiency by directly integrating the acid-base catalyst into the active site of P450BM3. The engineered P450BM3 mutant demonstrated remarkable ability to degrade bromophenol without the need for expensive bifunctional small molecules, providing new ideas and possibilities for the degradation of bromophenol pollutants. Summary of the Invention

[0006] To address the shortcomings of existing technologies in treating bromophenol pollutants in aquatic environments, this invention aims to provide a highly efficient biodegradation solution based on cytochrome P450BM3.

[0007] This invention is achieved through the following technical solution:

[0008] This invention develops a mutant based on cytochrome P450BM3. The mutant undergoes mutations at the following sites based on the amino acid sequence shown in SEQ ID NO.1: threonine at position 268 is mutated to aspartic acid, alanine at position 328 is mutated to phenylalanine, phenylalanine at position 87 is mutated to alanine or glycine, valine at position 78 is mutated to alanine, and leucine at position 272 is mutated to serine, resulting in the mutant T268D / A328F / F87G / L272S.

[0009] This invention also provides the application of the cytochrome P450BM3-based mutant in the degradation of bromophenol pollutants, wherein the mutant is T268D, T268D / A328F, T268D / A328F / F87A, T268D / A328F / F87G, T268D / A328F / F87A / V78A, T268D / A328F / F87G / V78A, or T268D / A328F / F87G / L272S; wherein T268D is formed by mutating threonine at position 268 to aspartic acid based on the amino acid sequence of SEQ ID NO.1;

[0010] The T268D / A328F is based on the amino acid sequence of SEQ ID NO.1, where threonine at position 268 is mutated to aspartic acid and alanine at position 328 is mutated to phenylalanine.

[0011] The T268D / A328F / F87A is based on the amino acid sequence of SEQ ID NO.1, where threonine at position 268 is mutated to aspartic acid, alanine at position 328 is mutated to phenylalanine, and phenylalanine at position 87 is mutated to alanine or glycine.

[0012] The T268D / A328F / F87A / V78A has the following amino acid sequence based on SEQ ID NO.1: threonine at position 268 is mutated to aspartic acid, alanine at position 328 is mutated to phenylalanine, phenylalanine at position 87 is mutated to alanine, and valine at position 78 is mutated to alanine.

[0013] In the T268D / A328F / F87G / V78A sequence, threonine at position 268 is mutated to aspartic acid, alanine at position 328 is mutated to phenylalanine, phenylalanine at position 87 is mutated to glycine, and valine at position 78 is mutated to alanine.

[0014] Furthermore, the method of the application involves using H2O2 as an oxidant and employing a P450BM3 peroxygenase system to biodegrade bromophenol, 2,4-dibromophenol, and 2,4,6-tribromophenol bromophenol pollutants.

[0015] The nucleotide sequence of WT P450BM3 is (SEQ ID NO.1):

[0016]

[0017] The degradation performance of WT P450BM3 and its mutants on three bromophenol pollutants was tested using high performance liquid chromatography. Wild-type P450BM3 does not degrade bromophenol. However, a series of mutants based on wild-type P450BM3, namely T268D, T268D / A328F, T268D / A328F / F87A, T268D / A328F / F87G, T268D / A328F / F87A / V78A, T268D / A328F / F87G / V78A, and T268D / A328F / F87G / L272S, all exhibit degradation capabilities for bromophenol. In particular, the T268D / A328F / F87G mutant showed degradation rates of 100% for 4-bromophenol and 99.88% for 2,4-dibromophenol within 2 minutes; and within 5 minutes, the T268D / A328F / F87G mutant showed degradation rates of 2,4-dibromophenol. The degradation rate of 6-tribromophenol was 99.28%.

[0018] The catalytic efficiency of P450BM3 and its series of mutants for bromophenol was determined. The results showed that the T268D / A328F / F87G mutant exhibited excellent catalytic efficiency for 4-bromophenol, 2,4-dibromophenol, and 2,4,6-tribromophenol. k cat / K m The values ​​were 166.95 mM. -1 s -1 203.68 mM -1 s -1 and 85.69 mM -1 s -1 .

[0019] The beneficial effects of this invention compared to the prior art are as follows:

[0020] (1) The present invention successfully developed a novel biocatalyst based on cytochrome P450BM3 and its various mutants, providing a new and efficient pathway for the degradation of bromophenol pollutants.

[0021] (2) This invention clarifies the highly efficient catalytic effect of each mutant on different bromophenol pollutants, providing a scientific basis for selection and optimization in practical applications.

[0022] (3) The expression vector containing mutants and the genetically engineered bacteria constructed in this invention provide a foundation for large-scale production and application, which is conducive to the promotion and popularization of the technology.

[0023] (4) The degradation reaction system of the present invention is simple and the conditions are mild, which reduces the difficulty and cost of operation and has good practical application feasibility. It provides an innovative, efficient and economical solution for the pollution control of bromophenol pollutants in the aquatic environment, which is of great significance for protecting the ecosystem and human health. Attached Figure Description

[0024] Figure 1 SDS-PAGE electrophoresis images of WT P450BM3 and a series of mutant proteins;

[0025] Figure 2 The degradation rate of 4-bromophenol by WT P450BM3 and its series of mutants;

[0026] Figure 3 The degradation rate of 2,4-dibromophenol by WT P450BM3 and its series of mutants;

[0027] Figure 4 The degradation rate of 2,4,6-tribromophenol by WT P450BM3 and its series of mutants;

[0028] Figure 5A The protein characterization diagram of WTP450BM3;

[0029] Figure 5B The protein characterization diagram for T268D;

[0030] Figure 5C Protein characterization diagram of T268D / A328F;

[0031] Figure 5D Protein characterization diagram of T268D / A328F / F87A;

[0032] Figure 5E Protein characterization diagram of T268D / A328F / F87G;

[0033] Figure 5F Protein characterization diagrams for T268D / A328F / F87A / V78A;

[0034] Figure 5G Protein characterization diagrams for T268D / A328F / F87G / V78A;

[0035] Figure 5H The protein characterization diagram is for T268D / A328F / F87G / L272S. Detailed Implementation

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] Example 1: P450BM3 site-directed mutagenesis:

[0038] (1) Design of mutation sites: Based on previous research and theoretical analysis, amino acid sites 268, 328, 87, 78 and 272 that may improve the catalytic activity of P450BM3 against bromophenol pollutants were identified. Table 1 lists the primers used for each site.

[0039] Table 1

[0040] ;

[0041] .

[0042] (2) Polymerase chain reaction (PCR) site-directed mutagenesis: The total reaction volume was 25 μL, which included 12.5 μL of Q5 hot-start ultra-fidelity 2×Master Mix, 1.25 μL of 10 μM forward primer, 1.25 μL of 10 μM reverse primer, 1 μL of template DNA, and 9 μL of sterile water. The above components were added to a sterile centrifuge tube, placed in a PCR instrument, and the amplification reaction was performed according to the preset program.

[0043] (3) KLD ligation reaction: KLD ligation reaction of open-ring plasmid was carried out according to a specific reaction system. The reaction system was 10 μL. After the reaction was completed, it was placed at room temperature for 5 min.

[0044] (4) Plasmid transformation and bacterial culture acquisition: 10 μL of KLD product was added to 100 μL of DH5α competent cells, mixed well, and then incubated on ice for 30 min, followed by heat shock at 42 ℃ for 30 s, and then quickly transferred to ice and allowed to stand for 5 min. Then, 1 mL of antibiotic-free LB liquid medium was added, and the culture was carried out at 37 ℃ and 180 rpm for 1 h. The cultured bacterial culture was centrifuged at 12000 rpm for 1 min, 900 μL of supernatant was discarded, and the remaining bacterial culture was mixed well and spread on LB solid plates containing 50 μg / mL kanamycin, and cultured at 37 ℃ for 12 h. Single colonies were picked from the plates and inoculated into 5 mL of LB liquid medium containing kanamycin, and cultured at 37 ℃ and 180 rpm for 12 h to obtain E. coli DH5α competent cells containing P450BM3 plasmid.

[0045] (5) Gene sequencing of bacterial culture and preservation of bacterial strain: 1 mL of the bacterial culture obtained above was sequenced, and the sequencing results were analyzed using ApE sequence alignment analysis software to screen out bacterial cultures that successfully underwent site-directed mutagenesis. 1000 μL of bacterial culture that successfully underwent site-directed mutagenesis was mixed with 500 μL of 70% sterile glycerol and placed in an ultra-low temperature freezer at -80℃ for bacterial culture preservation.

[0046] Example 2 Expression of P450BM3 series mutants

[0047] (1) Plasmid extraction: The pET-28a(+)-BM3 recombinant plasmid in E. coli DH5α competent cells in Example 1, which showed successful mutation after sequence comparison, was extracted using a rapid plasmid extraction kit. The operation steps were the same as those of conventional plasmid extraction methods.

[0048] (2) Plasmid transformation and strain preservation: 10 μL of the extracted recombinant plasmid was added to 100 μL of E. coli BL21(DE3) competent cells, incubated on ice for 30 min, then heat-shocked in a water bath at 42 ℃ for 60 s, and then transferred to ice for 5 min. 1 mL of antibiotic-free LB liquid medium was added, and the cells were cultured at 37 ℃ and 180 rpm for 1 h. The bacterial culture was centrifuged at 12000 rpm for 1 min, 900 μL of the supernatant was discarded, and the remaining bacterial culture was spread on LB agar plates containing 50 μg / mL kanamycin and cultured at 37 ℃ for 12 h. Single colonies on the plates were inoculated into 5 mL of LB liquid medium containing kanamycin and cultured at 37 ℃ and 180 rpm for 12 h to obtain competent cells containing the P450BM3 recombinant plasmid. 1000 μL of the bacterial culture was mixed with 500 μL of 70% sterile glycerol and stored at -80 ℃.

[0049] (3) Induced expression: The preserved samples were expressed using the streak plating method. E. coliBL21(DE3) strain was activated by inoculation on LB solid medium and cultured at 37°C for 12 h. A single colony was picked from a plate and inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C and 180 rpm for 12 h. 500 μL of this bacterial culture (1% inoculum) was transferred to 50 mL of LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C and 180 rpm for 12 h. Another 5 mL of this bacterial culture (1% inoculum) was transferred to 500 mL of LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C and 200 rpm until the absorbance at 600 nm reached approximately 0.8. Finally, 0.5 mM 5'-Ala and FeCl3 were added sequentially to the bacterial culture, and the culture was incubated at 30°C and 200 rpm for 40 min. Then, 1 mM IPTG was added for further induction for 20 h.

[0050] Example 3: Purification of P450BM3 series mutant proteins

[0051] (1) Pretreatment before protein purification: The induced bacterial culture was centrifuged at 4℃ and 8000 rpm for 10 min, and the supernatant was discarded to obtain the bacterial cell pellet. The bacterial cell pellet was dissolved and resuspended thoroughly. The above centrifugation operation was repeated, and the bacterial culture was resuspended again. Then, the bacterial culture was sonicated for 15 min (5 s sonication, 15 s cooling) to fully release the intracellular protein. The disrupted bacterial culture was centrifuged at 4℃ and 10000 rpm for 90 min, the pellet was discarded, the supernatant was collected, and the crude protein was obtained after filtration through a 0.22 μm filter membrane.

[0052] (2) Protein purification: Given that the P450BM3 gene has a His tag at its end, His Trap™ HP affinity chromatography column and AKTA explorer 10S protein rapid purification instrument were used. Dual wavelength detection was performed using the 280 nm protein absorption wavelength and the 418 nm P450 characteristic absorption wavelength. Pretreatment was performed by rinsing the instrument and pump with ultrapure water, solution A (100 mM KPi, 500 mM NaCl, pH 7.4), and solution B (100 mM KPi, 500 mM NaCl, 500 mM imidazole, pH 7.4). When loading the sample, equilibrate with 100% solution A at a flow rate of 5 mL / min. After the baseline stabilizes, load the sample at 3 mL / min, allowing impurities to flow through and the target protein to bind. After the baseline stabilizes again, elute impurities with 96% solution A and 4% solution B, and then rinse with 100 mL of 94% solution A and 6% solution B. After the baseline stabilized again, the concentration of solution B was adjusted to 40% to elute the target protein. The protein was collected when a characteristic peak appeared at 418 nm, at which point it had been replaced by a high concentration of imidazole. Then, solution B was adjusted to 100% for rinsing, followed by rinsing the system and pump with ultrapure water and 20% ethanol. Imidazole was removed by dialysis, first with pure water then with dialysate, for 2 hours each time. Finally, ultrafiltration was performed to concentrate and remove salts, using a 30 kDa filter membrane. Glycerol was added, and the solution was stored at -80 °C.

[0053] Example 4: Purity Detection of P450BM3 Series Mutant Proteins

[0054] (1) Pretreatment: Dilute the purified protein to 2 μM, then mix it with protein loading buffer (5×) at a volume ratio of 4:1, boil for 10 min to completely denature and depolymerize the protein, and then centrifuge at 10000 rpm for 1 min.

[0055] (2) Sample loading: Insert the pre-cast gel into the electrophoresis tank and add the electrophoresis buffer. Take 5 μL of the supernatant after centrifugation and add it to each lane along with the protein marker.

[0056] (3) Electrophoresis: Set the initial voltage for electrophoresis to 80 V. When the bands run in a straight line, adjust the voltage to 120 V until the bands reach the bottom of the gel and stop electrophoresis.

[0057] (4) Staining: After electrophoresis, remove the gel and stain it with Coomassie brilliant blue staining agent for 2 h.

[0058] (5) Decolorization: After the gel is fully stained, it is placed in a decolorization solution (pure water: ethanol: acetic acid volume ratio of 7:2:1) for 2 hours, and then placed in pure water for decolorization until the desired effect is achieved.

[0059] (6) Observation results: The decolorized gel was observed and analyzed in a gel imaging system. The purity of the target protein was determined by comparing it with the protein marker, and the experimental results were recorded by taking pictures. Figure 1As shown.

[0060] Example 5: Characterization and concentration determination of P450BM3 protein

[0061] (1) Characterization of P450BM3 protein: P450BM3 protein was characterized using a UV-Vis spectrophotometer. Wild-type and a series of mutants were diluted to 5 μM. 10 mg Na2S2O4 was added to 3 mL of protein dilution buffer, and the mixture was sealed and purged with CO2 for 3 min until saturation. The absorbance in the 350-700 nm band before and after reduction was measured. Characterization was achieved by comparing the shift in the position of the characteristic absorption peak. The results are shown in […]. Figures 5A-5H .

[0062] (2) Determination of P450BM3 protein concentration: The concentration of heme cofactor in wild-type and a series of mutants was determined based on the heme binding experiment to indirectly obtain the protein concentration. First, 1.75 mL of pyridine was mixed with 0.75 mL of 1 mol / L NaOH and centrifuged at 8500 rpm for 1 min at room temperature. 0.25 mL of the supernatant was taken and mixed with the protein dilution solution at a ratio of 3:1 to 1 mL. 2.5-3 mg of Na2S2O4 was added and mixed well. The UV spectrum in the 350-700 nm band was recorded. The blank group only replaced the protein solution. The average absorbance at 390 nm and 450 nm was used as the benchmark, and the difference between the absorbance at 418 nm and the average absorbance at 418 nm was the protein absorbance. The concentration of P450BM3 protein was calculated. The specific results are shown in Table 2.

[0063] Table 2

[0064] .

[0065] Example 6: Degradation rate and kinetics of 4-bromophenol by the P450BM3 mutant

[0066] (1) Degradation rate determination: WTP450BM3 and its mutant were added to the reaction system containing 4-bromophenol (substrate concentration 100 μM). After the reaction, the mixture was filtered through a 0.22 μm filter membrane and analyzed using high-performance liquid chromatography (HPLC) with a WatersSunfire™ C18 column (4.6 mm × 250 mm, 5 μm). The column temperature was set at 30℃, the injection volume was 20 μL, the mobile phase was water and acetonitrile (v:v = 60:40), the flow rate was 1 mL / min, and the detection wavelength was 280 nm. The degradation results are as follows: Figure 2 Under optimal conditions (30°C, pH 7.0, 25 mM H2O2), the degradation rate of the T268D / A328F / F87G mutant reached 100%.

[0067] (2) Kinetic analysis: The total volume of the reaction system was 1 mL, containing 4-bromophenol at different concentration gradients and 1.0 μM 450BM3, all dissolved in 50 mM KPi at pH 7.0. After incubation in a metal bath for 5 min, 25 mM H2O2 was added to initiate the reaction. The concentration of 4-bromophenol before and after the reaction was determined using high performance liquid chromatography. According to the Michaelis-Menten equation, v / [protein] = k cat [substrate] / (K m The dynamic parameters are calculated using [substrate]). k cat K m The catalytic efficiency of the T268D mutant for 4-bromophenol was determined. k cat / K m It is 7.67 mM -1 s -1 The kcat / Km of the T268D / A328F / F87G mutant is 166.95 mM. -1 s -1 It is approximately 21.76 times that of the T268D mutant;

[0068] Table 3 Kinetic parameters of 4-bromophenol catalyzed by P450BM3 and its mutants

[0069] .

[0070] Example 7: Degradation rate and kinetics of 2,4-dibromophenol by the P450BM3 mutant.

[0071] (1) Degradation rate determination: WTP450BM3 and its mutant were added to the reaction system containing 2,4-dibromophenol (substrate concentration 100 μM). After the reaction, the mixture was filtered through a 0.22 μm filter membrane and analyzed using high-performance liquid chromatography (HPLC) with a WatersSunfire™ C18 column (4.6 mm × 250 mm, 5 μm). The column temperature was set at 30 °C, the injection volume was 20 μL, the mobile phase was water and acetonitrile (v:v = 50:50), the flow rate was 1 mL / min, and the detection wavelength was 280 nm. The degradation results are as follows: Figure 3 Under optimal conditions (25 °C, pH 7.0, 25 mM H2O2), the degradation rate of the T268D / A328F / F87G mutant reached 99.88%.

[0072] (2) Kinetic analysis: The total volume of the reaction system was 1 mL, containing 2,4-dibromophenol and 1.0 μM P450BM3 at different concentration gradients, all dissolved in 50 mM KPi at pH 7.0. After incubation in a metal bath for 5 min, 25 mM H2O2 was added to initiate the reaction. The concentration of 2,4-dibromophenol before and after the reaction was determined using high performance liquid chromatography. According to the Michaelis-Menten equation, v / [protein] = k cat [substrate] / (K m The dynamic parameters are calculated using [substrate]). k cat K m The catalytic efficiency of the T268D mutant for 2,4-dibromophenol was determined. k cat / K m It is 9.91 mM -1 s -1 The T268D / A328F / F87G mutant k cat / K m 203.68 mM -1 s -1 It is approximately 20.55 times that of the T268D mutant;

[0073] Table 4 Kinetic parameters of 2,4-dibromophenol catalyzed by P450BM3 and its mutants

[0074] .

[0075] Example 8: Degradation rate and kinetics of 2,4,6-tribromophenol by the P450BM3 mutant.

[0076] (1) Degradation rate determination: WTP450BM3 and its mutant were added to the reaction system containing 2,4,6-tribromophenol (substrate concentration 100 μM). After the reaction, the mixture was filtered through a 0.22 μm filter membrane and analyzed by high performance liquid chromatography (HPLC) with a WatersSunfire™ C18 column (4.6 mm × 250 mm, 5 μm). The column temperature was set at 30 °C, the injection volume was 20 μL, the mobile phase was water and acetonitrile (v:v = 40:60), the flow rate was 1 mL / min, and the detection wavelength was 270 nm. The degradation results are as follows: Figure 4 Under optimal conditions (30 °C, pH 7.0, 25 mM H2O2), the degradation rate of the T268D / A328F / F87G mutant reached 99.28%.

[0077] (2) Kinetic analysis: The total volume of the reaction system was 1 mL, containing 2,4,6-tribromophenol at different concentration gradients and 1.0 μM P450BM3, all dissolved in 50 mM KPi at pH 7.0. After incubation in a metal bath for 5 min, 25 mM H2O2 was added to start the reaction. The concentration of 2,4,6-tribromophenol before and after the reaction was determined using high performance liquid chromatography. The kinetic parameters kcat and Km were calculated according to the Michaelis-Menten equation v / [protein] = kcat[substrate] / (Km+[substrate]). The catalytic efficiency kcat / Km of the T268D mutant for 2,4,6-tribromophenol was found to be 4.01 mM. -1 s -1 The kcat / Km of the T268D / A328F / F87G mutant is 85.69 mM. -1 s -1 It is approximately 21.36 times that of the T268D mutant;

[0078] Table 5 Kinetic parameters of 2,4,6-tribromophenol catalyzed by P450BM3 and its mutants

[0079] .

[0080] In summary, the P450BM3 mutant provided by this invention exhibits significantly enhanced catalytic performance against bromophenol pollutants, offering a promising biotechnological pathway for the effective degradation of bromophenol pollutants in the environment. Furthermore, the detailed and reproducible embodiments of this invention provide important reference for research and applications in related fields.

Claims

1. A mutant based on P450BM3, characterized in that, The mutant is based on the amino acid encoded by the nucleotide sequence shown in SEQ ID NO. 1, and is mutated at the following positions: threonine at position 268 is mutated to aspartic acid, alanine at position 328 is mutated to phenylalanine, phenylalanine at position 87 is mutated to glycine, and leucine at position 272 is mutated to serine, to obtain a mutant of T268D / A328F / F87G / L272S.

2. Use of the mutant of P450BM3 according to claim 1 in degrading bromophenol pollutants.

3. Use according to claim 2, characterized in that, The method of use is to biodegrade 4-bromophenol, 2, 4-dibromophenol and 2, 4, 6-tribromophenol bromophenol pollutants using the P450BM3 peroxygenase system with H2O2 as the oxidant.

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