Mutant based on cytochrome P450BM3 and application of mutant in chloroaniline biodegradation

Abstract

The invention relates to a mutant based on cytochrome P450BM3 and application of the mutant in chloroaniline biodegradation, and belongs to the field of enzymology. The mutant is obtained by mutating threonine at the 268 site of an amino acid sequence shown in SEQ ID NO.1 into aspartic acid or glutamic acid, mutating alanine at the 328 site into phenylalanine, and mutating alanine at the 328 site into phenylalanine. According to the present invention, the amino acid at the 87 site is mutated into alanine, and the valine at the 78 site is mutated into alanine, such that a variety of mutants such as T268D / A328F, T268E / A328F, T268D / A328F / F87A, T268E / A328F / F87A, T268D / A328F / F87A / V78A and T268E / A328F / F87A / V78A are obtained; according to the present invention, the degradation rate of the mutant on 4-chloroaniline, 2-chloroaniline, 2, 4-dichloroaniline, 3, 4-dichloroaniline and 3, 5-dichloroaniline is achieved; the method has important significance and application value for promoting rapid degradation of chloroaniline pollutants in a water environment and maintaining ecological balance.

Description

Technical Field

[0001] The invention belongs to the field of enzymology, and specifically relates to a mutant based on cytochrome P450BM3 and application thereof in biodegradation of chloroaniline. Background Art

[0002] Chloroaniline is an important organic pollutant, commonly used as an intermediate in the synthesis of dyes, drugs, pesticides, plastics and preservatives. Chloroaniline mainly includes 4-chloroaniline, 2-chloroaniline, 2,4-dichloroaniline, 3,4-dichloroaniline and 3,5-dichloroaniline. They enter the environment through various pathways, including wastewater discharge, atmospheric deposition and herbicide metabolism (diuron, linuron). The accumulation, persistence and acute toxicity (teratogenic, carcinogenic and mutagenic effects) of chloroaniline in the environment pose a significant risk to mammals, aquatic organisms and ecosystems. Therefore, it is urgent to develop effective and environmentally friendly methods to degrade chloroaniline.

[0003] The treatment or removal of chloroaniline is of vital importance. At present, many methods have been studied for the removal of chloroaniline in water environments, including physical and chemical methods such as adsorption, electrochemical oxidation, photochemistry and ozone oxidation. However, these physical and chemical methods all have certain limitations. The efficiency of physical adsorption methods is relatively low, and electrochemical oxidation, photochemistry and ozone oxidation usually produce by-products. In addition, they are costly and energy-intensive, and are not suitable for large-scale industrial applications. Therefore, it is particularly important to explore a more efficient and environmentally friendly method for degrading chloroaniline pollutants. Microbial-mediated bioremediation has the potential to efficiently degrade chloroaniline, so it is a promising alternative. However, challenges such as long culture cycles and slow reaction rates still exist. The enzymatic degradation of chloroaniline has mild conditions and high efficiency, and is particularly promising in future applications.

[0004] Cytochrome P450 is a heme-containing monooxygenase that is essential for drug metabolism, xenobiotic detoxification, and steroid biosynthesis. However, most natural P450s require the cofactor NAD(P)H and a complex electron transfer system to activate molecular oxygen, which greatly limits their applications. Low-cost hydrogen peroxide is an ideal alternative to the expensive cofactor NAD(P)H and can be directly used to unleash the catalytic potential of P450. However, the hydrogen peroxide shunt pathway is usually inefficient or even non-existent in driving P450 catalysis. Except for a few natural P450s, most natural P450s lack the structural features required to activate hydrogen peroxide (general acid-base catalysts). Given the great 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 P450BM3, which integrates anchoring and acid-base catalysis within the active site via bifunctional small molecules. Despite the many advantages of bifunctional small molecules, their synthesis remains complex and costly. In addition, myoglobin and P450 can be converted into peroxidases or peroxygenases by introducing an acid-base residue through site-directed mutagenesis. However, ensuring that the acid-base residue is oriented in the correct direction so that it can effectively function as a direct acid-base catalyst remains a major challenge. Summary of the invention

[0005] The purpose of the present invention is to provide a mutant based on cytochrome P450BM3 and its application. The engineered P450BM3 mutant shows extraordinary ability in degrading chloroaniline without expensive bifunctional small molecules, providing new ideas and possibilities for the degradation of chloroaniline pollutants.

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

[0007] A mutant based on cytochrome P450BM3, wherein the threonine at position 268 of the amino acid sequence shown in SEQ ID NO.1 is mutated to aspartic acid or glutamic acid, the alanine at position 328 is mutated to phenylalanine, the phenylalanine at position 87 is mutated to alanine and the valine at position 78 is mutated to alanine, and multiple mutants including T268D / A328F, T268E / A328F, T268D / A328F / F87A, T268E / A328F / F87A, T268D / A328F / F87A / V78A and T268E / A328F / F87A / V78A are obtained.

[0008] The nucleotide sequence of the WT P450BM3 is (SEQ ID NO.1):

[0009]

[0010] The present invention also provides an expression vector of the mutant, wherein the expression vector contains the gene of the mutant.

[0011] The present invention also provides a preparation for treating chloroaniline pollutants, wherein the preparation contains one or more of the mutants.

[0012] The present invention also provides use of the mutant in degrading chloroaniline pollutants, wherein the chloroaniline pollutants are 4-chloroaniline, 2-chloroaniline, 2,4-dichloroaniline, 3,4-dichloroaniline or / and 3,5-dichloroaniline, and the mutant is one or more of T268D / A328F, T268E / A328F, T268D / A328F / F87A, T268E / A328F / F87A, T268D / A328F / F87A / V78A and T268E / A328F / F87A / V78A.

[0013] Specifically, the application method is to utilize the obtained mutant, use H2O2 as an oxidant, and use the P450BM3 peroxygenase system to biodegrade 4-chloroaniline, 2-chloroaniline, 2,4-dichloroaniline, 3,4-dichloroaniline and / or 3,5-dichloroaniline.

[0014] The beneficial effects of the present invention compared with the prior art are as follows:

[0015] The degradation performance of WT P450BM3 and its mutants on five chloroaniline pollutants was tested by high performance liquid chromatography. Within 10 minutes, the degradation rates of 4-chloroaniline, 2-chloroaniline and 2,4-dichloroaniline by the T268D / A328F / F87A mutant were 99.16%, 98.62% and 98.38%, respectively; within 15 minutes, the degradation rates of 3,4-dichloroaniline and 3,5-dichloroaniline by the T268D / A328F / F87A mutant were 98.85% and 99.18%, respectively, while the degradation rate of 3,4-dichloroaniline by the T268D / A328F / F87A / V78A mutant was even higher, at 98.99%.

[0016] The catalytic efficiency of P450BM3 and its series of mutants on chloroaniline was determined. The results showed that the T268D / A328F / F87A / V78A mutant exhibited excellent catalytic efficiency on 4-chloroaniline, 2,4-dichloroaniline, 3,4-dichloroaniline and 3,5-dichloroaniline. cat / K m The values ​​were 106.75mM -1 s -1 、80.39mM -1 s -1 、78.05mM-1 s -1 and 98.38mM -1 s -1 ; The T268D / A328F / F87A mutant has excellent catalytic activity towards 2-chloroaniline, k cat / K m The value is 7.02mM -1 s -1 .

[0017] The degradation reaction system of the mutants for chloroaniline pollutants 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 economically feasible solution for the pollution control of chloroaniline pollutants in aquatic environments, which is of great significance for protecting ecosystems and human health. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The SDS-PAGE electrophoresis diagram of WT P450BM3 and a series of mutant proteins;

[0019] Figure 2 is the degradation rate of 4-chloroaniline by WT P450BM3 and a series of mutants;

[0020] Figure 3 is the degradation rate of 2-chloroaniline by WT P450BM3 and a series of mutants;

[0021] Figure 4 is the degradation rate of 2,4-dichloroaniline by WT P450BM3 and a series of mutants;

[0022] Figure 5 is the degradation rate of 3,4-dichloroaniline by WT P450BM3 and a series of mutants;

[0023] Figure 6 The degradation rates of 3,5-dichloroaniline by WT P450BM3 and its series of mutants. DETAILED DESCRIPTION

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] Example 1P450BM3 site-directed mutagenesis:

[0026] (1) Design of mutation sites: Based on previous research foundation and theoretical analysis, amino acid sites 268, 328, 87, and 78 were identified as sites that may improve the catalytic activity of P450BM3 towards chloroaniline pollutants. Table 1 lists the primers used for each site.

[0027] Table 1

[0028]

[0029]

[0030] (2) Polymerase chain reaction (PCR) site-directed mutagenesis: The total system is a 25 μL reaction solution, which contains 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. Add the above components to a sterile centrifuge tube, put it into a PCR instrument, and perform amplification reaction according to the preset program.

[0031] (3) Plasmid ligation reaction: Perform the ligation reaction of the open-circular plasmid according to a specific reaction system. The total reaction system is 10 μL, which contains 1 μL of PCP product, 5 μL of reaction buffer, 1 μL of ligation enzyme mixture (including kinase, ligase and DpnI) and 5 μL of sterile water. Add the above components to a sterile centrifuge tube in sequence and react at room temperature for 5 minutes.

[0032] (4) Plasmid transformation and bacterial liquid acquisition: Add 10 μL of KLD product to 100 μL of DH5α competent cells, mix well, place in an ice bath for 30 minutes, then heat shock in a 42°C water bath for 30 seconds, and quickly transfer to ice and let stand for 5 minutes. Then, add 1 mL of LB liquid culture medium without antibiotics and culture at 37°C and 180 rpm for 1 hour. The cultured bacterial liquid was centrifuged at 12000 rpm for 1 minute, the supernatant was discarded, the remaining bacterial liquid was mixed well and spread on an LB solid plate containing 50 μg / mL kanamycin, and cultured at 37°C for 12 hours. Pick a single colony from the plate, inoculate it in 5 mL of LB liquid culture medium containing kanamycin, and culture it at 37°C and 180 rpm for 12 hours to obtain E. coli DH5α competent cells containing the P450BM3 plasmid.

[0033] (5) Determination of bacterial liquid gene sequence and preservation of strains: 1 mL of the bacterial liquid obtained by the above culture was selected for sequencing, and the sequencing results were analyzed using the sequence alignment analysis software ApE to screen out the bacterial liquid with successful site-directed mutation. 1000 μL of the bacterial liquid with successful site-directed mutation was taken, mixed with 500 μL of 70% sterile glycerol, and placed in a -80°C ultra-low temperature refrigerator for strain preservation.

[0034] Example 2 P450BM3 expression:

[0035] (1) Plasmid extraction: A plasmid rapid extraction kit was used to extract the pET-28a(+)-BM3 recombinant plasmid from the E. coli DH5α competent cells that showed successful mutation after sequence comparison. The operation steps were the same as the conventional plasmid extraction method.

[0036] (2) Plasmid transformation and bacterial strain preservation: Take 10 μL of the extracted recombinant plasmid and add 100 μL of E. coli BL21 (DE3) competent cells. After ice bathing for 30 minutes, heat shock in a 42°C water bath for 60 seconds, and transfer to ice bath for 5 minutes. Add 1 mL of LB liquid culture medium without antibiotics and culture at 37°C and 180 rpm for 1 hour. Centrifuge the bacterial solution at 12000 rpm for 1 minute, discard 900 μL of the supernatant, mix the remaining bacterial solution and spread it on an LB solid plate containing 50 μg / mL kanamycin, and culture at 37°C for 12 hours. Inoculate the single colony on the plate into 5 mL of LB liquid culture medium containing kanamycin, and culture at 37°C and 180 rpm for 12 hours to obtain competent cells containing the P450BM3 recombinant plasmid. Take 1000 μL of the bacterial solution and mix it with 500 μL of 70% sterile glycerol and store it at -80°C.

[0037] (3) Induced expression: The preserved E. coli BL21 (DE3) strain was inoculated into LB solid medium for activation by plate streak method and cultured at 37°C for 12h. A single colony was picked from the plate and inoculated into 5mL LB liquid medium (5mL) containing 50μg / mL kanamycin and cultured at 37°C and 180rpm for 12h. 500μL of the bacterial solution (1% inoculation amount) was transferred to 50mL LB liquid medium (50mL) containing 50μg / mL kanamycin and cultured at 37°C and 180rpm for 12h. Another 5mL of the bacterial solution (1% inoculation amount) was transferred to 500mL LB liquid medium (500mL) containing 50μg / mL kanamycin and cultured at 37°C and 200rpm until the absorbance at 600nm wavelength reached about 0.8. Finally, 5-Ala and FeCl3 with a final concentration of 0.5 mM were added to the bacterial solution in sequence, and after culturing at 30°C and 200 rpm for 40 min, 1 mM IPTG was added to continue induction for 20 h.

[0038] Example 3P450BM3 protein purification:

[0039] (1) Pretreatment before protein purification: The bacterial solution after induced expression in Example 2 was centrifuged at 4°C and 8000 rpm for 10 min, and the supernatant was discarded to obtain the bacterial precipitate. After the bacterial precipitate was dissolved, it was fully resuspended. Repeat the above centrifugation operation and resuspend the bacterial solution again. Then, the bacterial solution was subjected to ultrasonic disruption for 15 min (5 s ultrasonication, 15 s cooling) to fully release the intracellular protein. The disrupted bacterial solution was centrifuged at 4°C and 10000 rpm for 90 min, the precipitate was discarded, the supernatant was collected, and the crude protein was obtained after filtering through a 0.22 μm filter membrane.

[0040] (2) Protein purification: Since the P450BM3 gene has a His tag at the end, His Trap was used to TM HP affinity chromatography column and AKTA explorer 10S protein rapid purification instrument were used. Dual wavelength detection uses 280nm protein absorption wavelength and 418nm P450 characteristic absorption wavelength. Pre-treatment first, rinse the instrument and pump with ultrapure water, A solution (100mM KPi, 500mM NaCl, pH 7.4), B solution (100mMKPi, 500mM NaCl, 500mM imidazole, pH 7.4). When loading, balance with 100% A solution, flow rate 5mL / min, load at 3mL / min after the baseline is stable, impurities flow through, and the target protein binds. After the baseline is stable again, 96% A solution and 4% B solution are used to elute the impurities, and then 100mL of 94% A solution and 6% B solution are used to rinse. After the baseline is stable again, adjust the concentration of B solution to 40% to elute the target protein, and collect when the characteristic peak appears at 418nm. At this time, the target protein is replaced by high concentration imidazole. Then adjust the B solution to 100% for flushing, flush the system and pump with ultrapure water and 20% ethanol. Remove imidazole by dialysis, first with pure water and then with dialysis solution, each time for 2 hours. Finally, ultrafiltration concentration is used to remove salt, the filter membrane is 30kDa, and glycerol is added and stored at -80℃.

[0041] Example 4 P450BM3 protein purity detection:

[0042] (1) Pretreatment: The protein purified in Example 3 was diluted to 2 μM, and then evenly mixed with SDS-PAGE protein loading buffer (5×) at a volume ratio of 4:1, boiled for 10 min to completely denature and depolymerize the protein, and then centrifuged at 10,000 rpm for 1 min.

[0043] (2) Sample loading: Insert the precast gel into the electrophoresis tank and add electrophoresis solution. Take 5 μL of the supernatant after centrifugation and add it to each lane along with the protein marker.

[0044] (3) Electrophoresis: Set the initial voltage of electrophoresis to 80 V. When the bands run in a straight line, adjust the voltage to 120 V until the bands run to the bottom of the gel and stop electrophoresis.

[0045] (4) Staining: After electrophoresis, remove the gel and stain it with Coomassie Brilliant Blue for 2 h.

[0046] (5) Decolorization: After being fully stained, the gel is placed in a decolorizing solution (the volume ratio of pure water: ethanol: acetic acid is 7:2:1) for 2 hours, and then placed in pure water for decolorization until the desired effect is achieved.

[0047] (6) Observation results: Observe and analyze the destained gel in a gel imager, compare it with the protein maker to determine the purity of the target protein, and take photos to record the experimental results. Figure 1 shown.

[0048] Example 5 P450BM3 protein characterization and protein concentration determination:

[0049] (1) Characterization of P450BM3 protein: The purified P450BM3 protein of Example 3 was characterized by UV-visible spectrophotometry. The wild type and a series of mutants were diluted to 5 μM, 10 mg of Na2S2O4 was added to 3 mL of the protein dilution, CO was passed through the solution for 3 min until saturation, and the absorbance in the 350-700 nm band before and after reduction was measured, and the shift in the characteristic absorption peak position was compared to achieve characterization.

[0050] (2) Determination of P450BM3 protein concentration: Based on the heme binding experiment, the heme cofactor concentration in the purified wild-type P450BM3 and a series of mutants in Example 3 was measured to indirectly obtain the protein concentration. First, 1.75 mL of pyridine was mixed with 0.75 mL of 1 mol / L NaOH, centrifuged at 8500 rpm for 1 min at room temperature, 0.25 mL of supernatant was mixed with protein diluent at a ratio of 3:1 to 1 mL, 2.5-3 mg of Na2S2O4 was added and mixed, and the ultraviolet spectrum of 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 protein absorbance was used as the protein absorbance to calculate the P450BM3 protein concentration.

[0051] Example 6 Study on the degradation rate and kinetics of 4-chloroaniline by P450BM3 mutants:

[0052] (1) Degradation rate determination: WTP450BM3 and its mutants were added to the reaction system containing 4-chloroaniline (substrate concentration 100 μM). After the reaction, the mixture was filtered through a 0.22 μm filter membrane and then filtered using a Waters Sunfire TM The analysis was performed by high performance liquid chromatography using a C18 column (4.6 mm × 150 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 240 nm. The degradation results are shown in Figure 2Under the optimal conditions (30°C, pH 8.5, 25 mM H2O2), the degradation rate of the T268D / A328F / F87A mutant reached 99.16%.

[0053] (2) Kinetic analysis: The total volume of the reaction system was 1 mL, containing 4-chloroaniline at different concentration gradients and 0.5 μM P450BM3, all dissolved in 50 mM KPi at pH 8.5. After incubation in the metal bath for 5 min, 25 mM H2O2 was added to start the reaction. The concentration of 4-chloroaniline before and after the reaction was determined using a high performance liquid chromatograph. According to the Michaelis-Menten equation v / [protein] = k cat [substrate] / (K m +[substrate]) to calculate the kinetic parameter k cat , K m , as shown in Table 2. The catalytic efficiency k of the T268D / A328F mutant for 4-chloroaniline was obtained. cat / K m 18.40mM -1 s -1 , while the k cat / K m 106.75mM -1 s -1 , which is about 5.8 times that of the T268D / A328F mutant;

[0054] Table 2 Kinetic parameters of 4-chloroaniline catalyzed by P450BM3 and its mutants

[0055] P450BM3 <![CDATA[K cat (s -1 )]]> <![CDATA[K m (μM)]]> <![CDATA[k cat / K m (mM -1 s -1 )]]> WT nd nd nd T268D / A328F 1.43±0.032 77.73±4.10 18.40 T268E / A328F 1.14±0.013 50.63±1.60 22.52 T268D / A328F / F87A 1.60±0.0090 22.09±0.51 72.43 T268E / A328F / F87A 3.82±0.086 70.96±3.88 53.83 T268D / A328F / F87A / V78A 3.94±0.11 36.91±3.28 106.75 T268E / A328F / F87A / V78A 3.85±0.11 63.13±4.77 60.99

[0056] Example 7 Degradation rate and kinetics of 2-chloroaniline by P450BM3 mutant:

[0057] (1) Degradation rate determination: WTP450BM3 and its mutants were added to the reaction system containing 2-chloroaniline (substrate concentration 100 μM). After the reaction, the mixture was filtered through a 0.22 μm filter membrane and then filtered using a Waters Sunfire TM The analysis was performed by high performance liquid chromatography using a C18 column (4.6 mm × 150 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 240 nm. The degradation results are shown in Figure 3Under the optimal conditions (20°C, pH 8.5, 25 mM H2O2), the degradation rate of the T268D / A328F / F87A mutant reached 98.62%.

[0058] (2) Kinetic analysis: The total volume of the reaction system was 1 mL, containing 2-chloroaniline at different concentration gradients and 0.5 μM P450BM3, all dissolved in 50 mM KPi at pH 8.5. After incubation in the metal bath for 5 min, 25 mM H2O2 was added to start the reaction. The concentration of 2-chloroaniline before and after the reaction was determined using a high performance liquid chromatograph. According to the Michaelis-Menten equation v / [protein] = k cat [substrate] / (K m +[substrate]) to calculate the kinetic parameter k cat , K m , see Table 3. The catalytic efficiency k of the T268D / A328F mutant for 2-chloroaniline was obtained. cat / K m 1.37mM -1 s -1 , while the k cat / K m 7.02mM -1 s -1 , which is approximately 5.1 times that of the T268D / A328F mutant;

[0059] Table 3 Kinetic parameters of 2-chloroaniline catalyzed by P450BM3 and its mutants

[0060] P450BM3 <![CDATA[k cat (s -1 )]]> <![CDATA[K m (mM)]]> <![CDATA[k cat / K m (M -1 s -1 )]]> WT nd nd nd T268D / A328F 0.73±0.029 534.06±39.94 1.37 T268E / A328F 1.44±0.051 422.47±31.31 3.41 T268D / A328F / F87A 4.17±0.15 594.17±39.46 7.02 T268E / A328F / F87A 3.54±0.21 520.64±59.64 6.80 T268D / A328F / F87A / V78A 3.43±0.059 635.04±19.47 5.40 T268E / A328F / F87A / V78A 2.17±0.083 381.71±31.60 5.68

[0061] Example 8

[0062] Degradation rate and kinetics of 2,4-dichloroaniline by P450BM3 mutant:

[0063] (1) Degradation rate determination: WTP450BM3 and its mutants were added to the reaction system containing 2,4-dichloroaniline (substrate concentration 100 μM). After the reaction, the mixture was filtered through a 0.22 μm filter membrane and then filtered using a Waters Sunfire TM The analysis was performed by high performance liquid chromatography using a C18 column (4.6 mm × 150 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 240 nm. The degradation results are shown in Figure 4Under the optimal conditions (25°C, pH 8.5, 25 mM H2O2), the degradation rate of the T268D / A328F / F87A mutant reached 98.38%.

[0064] (2) Kinetic analysis: The total volume of the reaction system was 1 mL, containing 2,4-dichloroaniline at different concentration gradients and 0.5 μM P450BM3, all dissolved in 50 mM KPi at pH 8.5. After incubation in the metal bath for 5 min, 25 mM H2O2 was added to start the reaction. The concentration of 2,4-dichloroaniline before and after the reaction was determined using a high performance liquid chromatograph. According to the Michaelis-Menten equation v / [protein] = k cat [substrate] / (K m +[substrate]) to calculate the kinetic parameter k cat , K m , see Table 4. The catalytic efficiency k of the T268D / A328F mutant for 2,4-dichloroaniline was obtained. cat / K m 40.65mM -1 s -1 , while the k cat / K m 80.39mM -1 s -1 , which is approximately 2.0 times that of the T268D / A328F mutant;

[0065] Table 4 Kinetic parameters of 2,4-dichloroaniline catalyzed by P450BM3 and its mutants

[0066] P450BM3 <![CDATA[k cat (s -1 )]]> <![CDATA[K m (mM)]]> <![CDATA[k cat / K m (M -1 s -1 )]]> WT nd nd nd T268D / A328F 7.91±0.34 194.61±13.84 40.65 T268E / A328F 2.84±0.062 67.88±3.63 41.84 T268D / A328F / F87A 2.67±0.026 35.80±1.14 74.58 T268E / A328F / F87A 5.27±0.10 96.36±3.95 54.69 T268D / A328F / F87A / V78A 11.04±0.15 137.33±3.54 80.39 T268E / A328F / F87A / V78A 6.13±0.12 85.74±3.84 71.50

[0067] Example 9 Study on the degradation rate and kinetics of 3,4-dichloroaniline by P450BM3 mutant:

[0068] (1) Degradation rate determination: WTP450BM3 and its mutants were added to the reaction system containing 3,4-dichloroaniline (substrate concentration 100 μM). After the reaction, the mixture was filtered through a 0.22 μm filter membrane and then filtered using a Waters Sunfire TM The analysis was performed by high performance liquid chromatography using a C18 column (4.6 mm × 150 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 240 nm. The degradation results are shown in Figure 5Under the optimal conditions (30°C, pH 8.5, 25 mM H2O2), the degradation rate of the T268D / A328F / F87A mutant reached 98.85%, and the degradation rate of the T268D / A328F / F87A / V78A mutant reached 98.99%.

[0069] (2) Kinetic analysis: The total volume of the reaction system was 1 mL, containing 3,4-dichloroaniline at different concentration gradients and 0.5 μM P450BM3, all dissolved in 50 mM KPi at pH 8.5. After incubation in the metal bath for 5 min, 25 mM H2O2 was added to start the reaction. The concentration of 3,4-dichloroaniline before and after the reaction was determined using a high performance liquid chromatograph. According to the Michaelis-Menten equation v / [protein] = k cat [substrate] / (K m +[substrate]) to calculate the kinetic parameter k cat , K m , see Table 5. The catalytic efficiency k of the T268D / A328F mutant for 3,4-dichloroaniline was obtained. cat / K m 58.60mM -1 s -1 , while the k cat / K m 78.05mM -1 s -1 , which is approximately 1.3 times that of the T268D / A328F mutant;

[0070] Table 5 Kinetic parameters of 3,4-dichloroaniline catalyzed by P450BM3 and its mutants

[0071] P450BM3 <![CDATA[k cat (s -1 )]]> <![CDATA[K m (mM)]]> <![CDATA[k cat / K m (M -1 s -1 )]]> WT nd nd nd T268D / A328F 5.50±0.18 93.86±6.79 58.60 T268E / A328F 1.75±0.026 49.62±2.12 35.27 T268D / A328F / F87A 2.28±0.040 45.18±2.37 50.46 T268E / A328F / F87A 2.02±0.042 62.38±3.33 32.38 T268D / A328F / F87A / V78A 19.98±0.99 255.98±19.11 78.05 T268E / A328F / F87A / V78A 8.71±0.30 116.60±7.91 74.70

[0072] Example 10 Study on the degradation rate and kinetics of 3,5-dichloroaniline by P450BM3 mutant:

[0073] (1) Degradation rate determination: WTP450BM3 and its mutants were added to the reaction system containing 3,5-dichloroaniline (substrate concentration 100 μM). After the reaction, the mixture was filtered through a 0.22 μm filter membrane and then filtered using a Waters Sunfire TM The analysis was performed by high performance liquid chromatography using a C18 column (4.6 mm × 150 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 240 nm. The degradation results are shown in Figure 6Under the optimal conditions (30°C, pH 7.0, 25 mM H2O2), the degradation rate of the T268D / A328F / F87A mutant reached 99.18%.

[0074] (2) Kinetic analysis: The total volume of the reaction system was 1 mL, containing 3,5-dichloroaniline at different concentration gradients and 0.5 μM P450BM3, all dissolved in 50 mM KPi at pH 8.5. After incubation in the metal bath for 5 min, 25 mM H2O2 was added to start the reaction. The concentration of 3,5-dichloroaniline before and after the reaction was determined using a high performance liquid chromatograph. According to the Michaelis-Menten equation v / [protein] = k cat [substrate] / (K m +[substrate]) to calculate the kinetic parameter k cat , K m , see Table 6. The catalytic efficiency k of the T268D / A328F mutant for 3,5-dichloroaniline was obtained. cat / K m 41.50mM -1 s -1 , while the k cat / K m 98.38mM -1 s -1 , which is approximately 2.4 times that of the T268D / A328F mutant;

[0075] Table 6 Kinetic parameters of 3,5-dichloroaniline catalyzed by P450BM3 and its mutants

[0076] P450BM3 <![CDATA[k cat (s -1 )]]> <![CDATA[K m (mM)]]> <![CDATA[k cat / K m (M -1 s -1 )]]> WT nd nd nd T268D / A328F 6.15±0.13 148.19±5.70 41.50 T268E / A328F 3.75±0.11 125.60±6.89 29.86 T268D / A328F / F87A 2.35±0.029 31.87±1.38 73.74 T268E / A328F / F87A 3.66±0.037 120.33±2.38 30.42 T268D / A328F / F87A / V78A 3.94±0.086 40.05±2.73 98.38 T268E / A328F / F87A / V78A 5.33±0.064 67.29±1.99 79.21

[0077] In summary, the P450BM3 mutant provided by the present invention exhibits significantly improved catalytic performance for chloroaniline pollutants, providing a potential biotechnological approach for the effective degradation of chloroaniline pollutants in the environment. At the same time, the embodiments of the present invention are detailed and repeatable, providing an important reference for research and application in related fields.

Claims

1. A mutant based on cytochrome P450BM3, characterized in that: The mutant is a mutant in which the threonine at position 268 of the amino acid sequence shown in SEQ ID NO.1 is mutated to aspartic acid or glutamic acid, the alanine at position 328 is mutated to phenylalanine, the phenylalanine at position 87 is mutated to alanine and the valine at position 78 is mutated to alanine, and multiple mutants including T268D / A328F, T268E / A328F, T268D / A328F / F87A, T268E / A328F / F87A, T268D / A328F / F87A / V78A and T268E / A328F / F87A / V78A are obtained.

2. An expression vector based on a mutant of cytochrome P450BM3, characterized in that: The expression vector contains the gene of the mutant according to claim 1.

3. A preparation for treating chloroaniline pollutants, characterized in that: The preparation contains one or more mutants described in claim 1.

4. Application of mutants of cytochrome P450BM3 in the degradation of chloroaniline pollutants, characterized in that: The chloroaniline pollutants are 4-chloroaniline, 2-chloroaniline, 2,4-dichloroaniline, 3,4-dichloroaniline or / and 3,5-dichloroaniline, and the mutants are one or more of T268D / A328F, T268E / A328F, T268D / A328F / F87A, T268E / A328F / F87A, T268D / A328F / F87A / V78A and T268E / A328F / F87A / V78A.

5. The use according to claim 4, characterized in that: The application method is to utilize the mutant, take H2O2 as an oxidant, and use the P450BM3 peroxygenase system to biodegrade 4-chloroaniline, 2-chloroaniline, 2,4-dichloroaniline, 3,4-dichloroaniline and / or 3,5-dichloroaniline.

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