A mutant based on cytochrome P450BM3, a preparation and application thereof in degrading non-steroidal anti-inflammatory drugs

Abstract

The present invention relates to a mutant based on cytochrome P450BM3, a preparation and an application thereof for degrading non-steroidal anti-inflammatory drugs, belonging to the field of bioengineering technology, wherein the mutant is F87G, T268D, F87G / T268D, F87A / T268D, F87V / T268D, F87I / T268D, and the amino acid sequence of cytochrome P450BM3 is shown in SEQ ID NO. 1. The present invention also provides an expression vector or a recombinant cell comprising the mutant gene, and a biological preparation comprising the mutant, the expression vector or the recombinant cell, wherein the biological preparation can degrade non-steroidal anti-inflammatory drugs including diclofenac, meloxicam, phenylbutazone and acetaminophen.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a mutant based on cytochrome P450BM3, a preparation and application thereof in degrading non-steroidal anti-inflammatory drugs. Background Art

[0002] NSAIDs are the first choice for the treatment of common diseases such as pain and inflammation due to their low price and wide range of uses. They are widely used in the human and veterinary fields. Common NSAIDs mainly include diclofenac, acetaminophen, meloxicam, phenylbutazone, etc., and about 30 million people use such drugs every day around the world. According to statistics, NSAIDs are one of the most frequently detected drugs in wastewater, surface water, groundwater and even drinking water, and the detected concentrations are mainly in the range of ng / L-μg / L. In 2013, the EU Water Framework Directive included diclofenac in the EU micropollutant priority substance observation list based on factors such as toxicity, persistence and incidence. Due to factors such as frequent and large-scale use, excretion by humans and animals, limitations of sewage treatment technology, and unreasonable disposal of discarded drugs, NSAIDs and their metabolites that are not fully absorbed and utilized eventually enter the water environment through various pathways, and can be enriched in organisms through the action of the food chain, causing serious consequences for the ecosystem and human health.

[0003] The treatment or removal of NSAIDs is of vital importance. At present, many methods have been studied for the removal of NSAIDs in water environments, such as physical adsorption, photocatalysis, electrochemical oxidation, and sonocatalysis. However, these technologies all have certain limitations. The efficiency of physical adsorption methods is relatively low, and advanced oxidation processes are not only costly but also have adverse effects on the environment. Therefore, it is particularly important to explore a more efficient and environmentally friendly method for the degradation of NSAIDs. In the scope of biodegradation, it mainly includes microbial strain degradation and enzyme-catalyzed degradation. However, microbial strains have the problems of long culture cycle and slow reaction rate, which has formed a significant limitation in the actual wastewater treatment process. In contrast, enzyme conversion, as a mild, rapid, green and environmentally friendly biodegradation pathway with high catalytic efficiency, has shown great potential in the remediation of various environmental pollutants. Therefore, finding a suitable enzyme to catalyze the degradation of NSAIDs may become a very promising remediation strategy.

[0004] Over the past 20 years, the use of chemical and biological approaches to create artificial metalloenzymes that surpass the capabilities of natural metalloenzymes has become a major focus in bioinorganic chemistry and chemical biology. Cytochrome P450BM3 is an iron-heme-containing monooxygenase that shows considerable promise in green biocatalysis. However, its dependence on expensive cofactors such as NAD(P)H limits its practical applications. To address this issue, we developed an artificial P450BM3 catalase system by dynamically installing a bifunctional small molecule (DFSM) into the protein. DFSM uses an acyl amino acid as an anchor to bind to the enzyme, and the basic imidazole group at the other end acts as a general acid-base catalyst to synergistically activate H2O2. In addition, the enzyme's substrate range was expanded by site-directed mutagenesis around its active site.

[0005] As a substance with potential biocatalytic activity, cytochrome P450BM3 provides new ideas and possibilities for the degradation of non-steroidal anti-inflammatory drugs. However, before the present invention, the research on the development of efficient biocatalysts for non-steroidal anti-inflammatory drugs based on cytochrome P450BM3 was still in the preliminary stage, and a mature and effective technical solution had not yet been formed. Summary of the invention

[0006] In view of the shortcomings of the prior art in treating non-steroidal anti-inflammatory drugs in aquatic environments, the present invention aims to provide an efficient biodegradation solution based on cytochrome P450BM3.

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

[0008] A mutant based on cytochrome P450BM3, wherein the mutant is F87G, T268D, F87G / T268D, F87A / T268D, F87V / T268D, and F87I / T268D, wherein the amino acid sequence of cytochrome P450BM3 is as shown in SEQ ID NO.1, wherein F87G, F87A, F87V, and F87I respectively represent that the phenylalanine at the 87th position of the amino acid sequence of cytochrome P450BM3 is mutated to glycine, alanine, valine, and isoleucine, and T268D represents that the threonine at the 268th position of the amino acid sequence of cytochrome P450BM3 is mutated to aspartic acid.

[0009] The amino acid sequence of WT P450BM3 is (SEQ ID NO.1):

[0010] MTIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDKNLSQALKFVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYFLVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLKHFDFEDHTNYELDIKETLTLKPEGFVVKAKSKKIPLGGIPSPSTEQSAKKVRKKAEN。

[0011] The nucleotide sequence of the WT P450BM3 is (SEQ ID NO.2):

[0012]

[0013] The present invention also provides a method for constructing an expression vector or a recombinant cell containing the mutant gene.

[0014] The present invention also provides a biological preparation, which comprises the mutant, expression vector or recombinant cell.

[0015] The present invention also provides an application of the mutant, expression vector or recombinant cell, wherein the application method is to utilize the obtained mutant, expression vector or recombinant cell, use H2O2 as an oxidant, and under the action of the P450BM3 catalase system promoted by DFSM, biodegrade non-steroidal anti-inflammatory drugs including diclofenac, meloxicam, phenylbutazone and acetaminophen.

[0016] The degradation performance of WT P450BM3 and its mutants on four non-steroidal anti-inflammatory drugs was tested by high performance liquid chromatography. Within 10 minutes, the degradation rates of diclofenac by WT P450BM3 and T268D mutant were 61.62% and 63.26%, respectively, while the degradation rates of diclofenac by P450BM3 mutants F87G, F87G / T268D, F87A / T268D, F87V / T268D and F87I / T268D were all over 95%, among which the degradation rate of F87I / T268D mutant was as high as 99.22%. At the same time, the degradation rates of meloxicam and phenylbutazone by F87I / T268D mutant were 98.86% and 90.51% respectively within 5 minutes, and the degradation of acetaminophen by F87G mutant was 99.08% within 2 minutes.

[0017] The catalytic efficiency of P450BM3 and its series of mutants was determined. The results showed that the F87I / T268D P450BM3 mutant exhibited excellent catalytic efficiency for diclofenac, meloxicam and phenylbutazone. k cat / K m The values ​​are 3625.00M -1 s -1 、5000.00M -1 s -1 and 3882.35M -1 s -1 ; The F87G P450BM3 mutant has excellent catalytic activity towards acetaminophen, k cat / K m The value is 3740.74M -1 s -1 .

[0018] The beneficial effects of the present invention are mainly reflected in the following aspects:

[0019] 1) A new biocatalyst based on cytochrome P450BM3 and its various mutants were successfully developed, providing a new and efficient way for the degradation of non-steroidal anti-inflammatory drugs. (2) The efficient catalytic effect of each mutant on different non-steroidal anti-inflammatory drugs was clarified, providing a scientific basis for selection and optimization in practical applications. (3) The constructed expression vectors and genetically engineered bacteria containing mutants provide a basis for large-scale production and application, which is conducive to the promotion and popularization of technology. (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. (5) The present invention provides an innovative, efficient and economically feasible solution for the pollution control of non-steroidal anti-inflammatory drugs in aquatic environments, which is of great significance for protecting ecosystems and human health. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The SDS-PAGE electrophoresis diagram of WT P450BM3 and a series of mutant proteins;

[0021] Figure 2 is the degradation rate of diclofenac by WT P450BM3 and a series of mutants;

[0022] Figure 3 is the degradation rate of meloxicam by WT P450BM3 and a series of mutants;

[0023] Figure 4 is the degradation rate of phenylbutazone by WT P450BM3 and a series of mutants;

[0024] Figure 5 The degradation rates of acetaminophen by WT P450BM3 and its series of mutants are shown in Table 2 . DETAILED DESCRIPTION

[0025] 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.

[0026] The present invention develops a mutant based on cytochrome P450BM3, wherein the phenylalanine at position 87 of the amino acid sequence shown in SEQ ID NO.1 is mutated to glycine, and the threonine at position 268 is mutated to aspartic acid to obtain two single mutants, and on this basis, the phenylalanine at position 87 is further mutated to alanine, valine and isoleucine, thereby obtaining multiple mutants including F87G, T268D, F87G / T268D, F87A / T268D, F87V / T268D and F87I / T268D.

[0027] The components and final concentrations of the LB liquid culture medium involved are: 1% tryptone, 0.5% yeast extract, 1% sodium chloride (% is the mass ratio). Autoclave at 121°C for 20 min, and add kanamycin (50 μg / mL) after cooling.

[0028] The composition and final concentration of LB solid plate medium are: 1% tryptone, 0.5% yeast extract, 1% sodium chloride, 2% agar (% is mass ratio). Autoclave at 121°C for 20 min, add kanamycin (50 μg / mL) after the medium cools down slightly, shake well, pour into the plate, and place at 4°C for later use.

[0029] The specific implementation is as follows:

[0030] Example 1 P450BM3 site-directed mutagenesis:

[0031] (1) Design of mutation sites: Based on previous research foundation and theoretical analysis, amino acid sites 87 and 268 that may improve the catalytic activity of P450BM3 for non-steroidal anti-inflammatory drugs were determined. Table 1 lists the primers used for each site (SEQ ID NO.3-SEQ ID NO.9);

[0032] Table 1 Primer sequences of P450BM3 mutants

[0033] .

[0034] (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, place it in a PCR instrument, and perform the amplification reaction according to the preset program.

[0035] (3) KLD (kinase-ligase-dpni) enzyme ligation reaction: The KLD enzyme ligation reaction of the open plasmid was carried out according to a specific reaction system. The reaction system was 10 μL and was left at room temperature for 5 minutes after the reaction was completed.

[0036] (4) Plasmid transformation and bacterial liquid acquisition: Add 10 μL of KLD product to 100 μL of DH5α competent cells, mix well, and 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 13000 rpm for 1 minute, and 900 μL of the supernatant was discarded. The remaining bacterial liquid was mixed 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 a plasmid containing P450BM3. E. coli DH5α competent cells.

[0037] (5) Determination of bacterial liquid gene sequence and preservation of strains: 1 mL of competent cell liquid obtained from the above culture was selected for sequencing, and the sequencing results were analyzed using sequence alignment analysis software ApE to screen out bacterial liquid with successful site-directed mutation. 750 μL of bacterial liquid with successful site-directed mutation was taken and mixed with 250 μL of 70% sterile glycerol and placed in a -80°C ultra-low temperature refrigerator for strain preservation.

[0038] Example 2 P450BM3 expression:

[0039] (1) Plasmid extraction: Use a plasmid rapid extraction kit to extract the plasmids that have successfully mutated after sequence comparison. E. coli The pET-28a(+)-BM3 recombinant plasmid was extracted from DH5α competent cells, and the operation steps were the same as the conventional plasmid extraction method.

[0040] (2) Plasmid transformation and strain preservation: Take 10 μL of the extracted recombinant plasmid and add 100 μL E. coli BL21 (DE3) competent cells, ice bath for 30 minutes, heat shock in 42℃ water bath for 60s, transfer to ice bath for 5 minutes. Add 1mL LB liquid medium without antibiotics, and culture at 37℃, 180rpm for 1h. Centrifuge the bacterial solution at 12000rpm for 1min, discard 900μL of supernatant, mix the remaining bacterial solution and spread it on LB solid plate containing 50μg / mL kanamycin, and culture at 37℃ for 12h. Inoculate the single colony on the plate into 5mL LB liquid medium containing kanamycin, culture at 37℃, 180rpm for 12h, and obtain competent cells containing P450BM3 recombinant plasmid. Take 750μL of bacterial solution and mix it with 250μL of 70% sterile glycerol, and store at -80℃.

[0041] (3) Induced expression: The stored E. coliThe BL21 (DE3) strain was inoculated in LB solid medium for activation and cultured at 37°C for 12 hours. A single colony was picked from the plate and inoculated into 5 mL of LB liquid medium containing kanamycin, and cultured at 37°C and 180 rpm for 12 hours. 500 μL of the bacterial solution (1% inoculation amount) was transferred to 50 mL of LB liquid medium containing kanamycin and cultured at 37°C and 180 rpm for 12 hours. Another 5 mL of bacterial solution (1% inoculation amount) was transferred to 500 mL of LB liquid medium containing kanamycin, and cultured at 37°C and 200 rpm until the absorbance at 600 nm reached about 0.8. Finally, 5¢-Ala and FeCl3 were added to the bacterial solution in sequence with a final concentration of 0.5 mM. After culturing at 30°C and 200 rpm for 40 minutes, 1 mM of isopropyl-β-D-thiogalactoside was added.

[0042] (IPTG for short) and continue induction for 20h.

[0043] Example 3 P450BM3 mutant protein purification:

[0044] (1) Pretreatment before protein purification: Centrifuge the induced bacterial solution at 4°C and 8000 rpm for 10 min, discard the supernatant and obtain the bacterial precipitate. After the bacterial precipitate is dissolved, resuspend it fully. Repeat the above centrifugation operation and resuspend the bacterial solution again. Then, ultrasonically disrupt the bacterial solution for 15 min (5s ultrasonication, 15s cooling) to fully release the intracellular protein. Centrifuge the disrupted bacterial solution at 4°C and 10000 rpm for 90 min, discard the precipitate, collect the supernatant, and filter it through a 0.22μm filter membrane to obtain crude protein.

[0045] (2) Protein purification: Since the P450BM3 gene has a His tag at the end, His Trap TMHP affinity chromatography column and AKTA explorer 10S protein rapid purification instrument were used. Dual wavelength detection used 280nm protein absorption wavelength and 418nm P450 characteristic absorption wavelength. Pre-treatment was performed first, and the instrument and pump were rinsed with ultrapure water, A solution (100mM K2HPO4-KH2PO4, 500mM NaCl, pH7.4), and B solution (100mM K2HPO4-KH2PO4, 500mM NaCl, 500mM imidazole, pH7.4). When loading, 100% A solution was used for balance, the flow rate was 5mL / min, and the sample was loaded at 3mL / min after the baseline was stable, the impurities were passed through, and the target protein was bound. After the baseline was stabilized again, the impurities were eluted with 96% A solution and 4% B solution, and then rinsed with 100mL of 94% A solution and 6% B solution. After the baseline is stable again, adjust the concentration of liquid B to 40% to elute the target protein. Collect when the characteristic peak appears at 418nm. At this time, the target protein is replaced by high-concentration imidazole. Then adjust liquid B to 100% for flushing, and flush the system and pump with ultrapure water and 20% ethanol. Dialyze to remove imidazole, first with pure water and then with dialysate, 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℃.

[0046] Example 4 P450BM3 mutant protein purity detection:

[0047] (1) Pretreatment: The purified protein was diluted to 5 μM, then mixed evenly with protein loading buffer (5×) at a volume ratio of 4:1, boiled for 10 min to completely denature and disaggregate the protein, and then centrifuged at 10,000 rpm for 1 min.

[0048] (2) 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.

[0049] (3) Electrophoresis: Set the initial voltage of electrophoresis to 80 V. When the band reaches the middle position, adjust the voltage to 120 V until the band reaches the bottom of the gel and stop electrophoresis.

[0050] (4) Staining: After electrophoresis, remove the gel and stain it with Coomassie Brilliant Blue for 2 h.

[0051] (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.

[0052] (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, such as Figure 1 shown.

[0053] Example 5 Characterization of P450BM3 mutant protein and determination of protein concentration:

[0054] (1) Characterization of P450BM3 mutant protein: P450BM3 protein was characterized using a UV-visible spectrophotometer. The wild type and a series of mutants were diluted to 5 μM, 10 mg of Na2S2O4 was added to 3 mL of protein dilution, CO was passed through a closed chamber 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.

[0055] (2) Determination of protein concentration of P450BM3 mutants: The heme cofactor concentration in the wild type and a series of mutants was measured 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, centrifuged at 6000 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, 3 mg of Na2S2O4 was added and mixed, and the UV 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 to calculate the P450BM3 protein concentration.

[0056] Example 6 Study on the degradation rate and kinetics of diclofenac by P450BM3 mutants:

[0057] (1) Degradation rate determination: The reaction system containing diclofenac: 50μM diclofenac, 3μM P450BM3, 0.5mM N-(ω-imidazolyl)-hexanoyl-l-phenylalanine (abbreviated as Im-C6-Phe), 15mM H2O2, the pH value of the reaction system is 7.0, and the reaction temperature is 25°C. WT P450BM3 and its mutants were added respectively. After the reaction, the mixture was filtered through a 0.22μm polyamide membrane and analyzed using a high performance liquid chromatography equipped with a Waters Sunfire™ C18 column (4.6mm×150mm, 5μm). The column temperature was set to 30°C, the injection volume was 20μL, the mobile phase was 25mM KPi and methanol (v:v=30:70), the flow rate was 1mL / min, and the detection wavelength was 280nm. The degradation results are shown in Figure 2 Under the optimal conditions (25°C, pH 7.0, 15mM H2O2, 0.5mM Im-C6-Phe), the degradation rate of diclofenac by WT P450BM3 was 61.62% within 10 min, while the degradation rate of the F87I / T268D mutant reached 99.22%.

[0058] (2) Kinetic analysis: The total volume of the reaction system was 1 mL, containing diclofenac at different concentration gradients, 1 μM P450BM3, and 0.5 mM Im-C6-Phe small molecules, all dissolved in 25 mM KPi at pH 7.0. After incubation in the metal bath for 5 min, H2O2 was added to start the reaction. The concentration of diclofenac 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 parameters k cat , K m The catalytic efficiency of WT P450BM3 for diclofenac was obtained. k cat / K m 1400.00M -1 s -1 , while the F87I / T268D double mutant k cat / K m 3625.00M -1 s -1 , about 2.6 times that of WT P450BM3;

[0059] Table 2 Kinetic parameters of diclofenac catalyzed by P450BM3 and its mutants

[0060] .

[0061] Example 7 Study on the degradation rate and kinetics of meloxicam by P450BM3 mutants:

[0062] (1) Degradation rate determination: The experimental process is the same as that of Example 6. The reaction system is: 50 μM meloxicam, 3 μM P450BM3, 0.5 mM Im-C6-Phe, 5 mM H2O2, the pH value of the reaction system is 7.0, and the reaction temperature is 35°C. The mobile phase is 25 mM KPi and methanol (v:v=50:50), the flow rate is 1 mL / min, and the detection wavelength is 365 nm. The degradation results are shown in Figure 3 The degradation rate of meloxicam by the F87I / T268D mutant could reach 98.86% within 5 minutes.

[0063] (2) Kinetic analysis: The reaction conditions and analysis methods were the same as those in Example 6. The results showed that the F87I / T268D mutant had a strong affinity for meloxicam. kcat / K m 5000.00M -1 s -1 ;

[0064] Table 3 Kinetic parameters of meloxicam catalyzed by P450BM3 and its mutants

[0065] .

[0066] Example 8 Study on the degradation rate and kinetics of phenylbutazone by P450BM3 mutants:

[0067] (1) Degradation rate determination: The experimental steps of Example 6 were repeated. The reaction system contained 50 μM phenylbutazone, 3 μM P450BM3, 0.5 mM Im-C6-Phe, and 15 mM H2O2. The pH value of the reaction system was 7.0 and the reaction temperature was 25°C. The mobile phase was 25 mM KPi and methanol (v:v = 30:70), the flow rate was 1 mL / min, and the detection wavelength was 280 nm. The degradation results are shown in Figure 4 The degradation rate of phenylbutazone by the F87I / T268D mutant was 90.51% within 5 min.

[0068] (2) Kinetic analysis: The reaction conditions and analysis methods were the same as those in Example 6. The results showed that the F87I / T268D mutant had a k cat / K m 3882.35M -1 s -1 ;

[0069] Table 4 Kinetic parameters of phenylbutazone catalyzed by P450BM3 and its mutants

[0070] .

[0071] Example 9 Study on the degradation rate and kinetics of acetaminophen by P450BM3 mutants:

[0072] (1) Degradation rate determination: According to the method of Example 6, the reaction system contained 50 μM acetaminophen, 3 μM P450BM3, 0.5 mM Im-C6-Phe, and 15 mM H2O2. The pH value of the reaction system was 7.0, and the reaction temperature was 25°C. The mobile phase was 25 mM KPi and methanol (v:v = 65:35), the flow rate was 0.4 mL / min, and the detection wavelength was 243 nm. The degradation results are shown in Figure 5 , the degradation rate of acetaminophen by the F87G mutant reached 99.08% within 2 min.

[0073] (2) Kinetic analysis: The reaction and analysis conditions were the same as those in Example 6. The total volume of the reaction system was 1 mL, and it contained substrates of different concentration gradients, 1 μM P450BM3, and 0.5 mM Im-C6-Phe. The above substances were dissolved in 25 mM KPi at pH 7.0. After incubation in a metal bath for 5 min, H2O2 was added to start the reaction. k cat / K m 3740.74M -1 s -1 , which is about 4.4 times the catalytic efficiency of WT P450BM3;

[0074] Table 5 Kinetic parameters of acetaminophen catalyzed by P450BM3 and its mutants

[0075] .

[0076] In summary, the P450BM3 mutant provided by the present invention exhibits significantly improved catalytic performance for non-steroidal anti-inflammatory drugs, providing a potential biotechnology approach for the effective degradation of non-steroidal anti-inflammatory drugs in the environment. At the same time, the implementation scheme of the present invention is detailed and repeatable, providing an important reference for research and application in related fields.

Claims

1. A mutant based on cytochrome P450BM3, an expression vector or a recombinant cell containing the mutant gene, characterized in that: The application method is to utilize the obtained mutant, expression vector or recombinant cell, use H2O2 as an oxidant, and biodegrade non-steroidal anti-inflammatory drugs including diclofenac, meloxicam, phenylbutazone and acetaminophen under the action of the P450BM3 catalase system promoted by the bifunctional small molecule Im-C6-Phe; The mutants are F87G, T268D, F87G / T268D, F87A / T268D, F87V / T268D, and F87I / T268D. The amino acid sequence of cytochrome P450BM3 is shown in SEQ ID NO.

1. F87G, F87A, F87V, and F87I respectively indicate that the phenylalanine at the 87th position of the amino acid sequence of cytochrome P450BM3 is mutated to glycine, alanine, valine, and isoleucine, and T268D indicates that the threonine at the 268th position of the amino acid sequence of cytochrome P450BM3 is mutated to aspartic acid.

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