A Cytochrome P450BM3 Mutant and Its Application in Region-Selective and Highly Efficient Degradation of 4-Chlorophenol

Abstract

The present invention discloses a cytochrome P450BM3 mutant and its application in the regioselective and highly efficient degradation of 4-chlorophenol. In the mutant, at least one amino acid at positions 51, 82, and 330 of the amino acid sequence shown in SEQ ID No.1 is replaced; tyrosine Tyr at position 51 is mutated to valine Val; alanine Ala at position 82 is mutated to methionine Met, and alanine Ala at position 330 is mutated to phenylalanine Phe. The present invention uses directed evolution to modify cytochrome P450BM3, and obtains a P450BM3 mutant with high regioselectivity (>99%) and improved 4-chlorophenol degradation activity.

Description

Technical Field

[0001] The present invention relates to the technical field of environmental remediation microorganisms, and particularly relates to a cytochrome P450BM3 mutant and its application in the regioselective and efficient degradation of 4-chlorophenol. Background Art

[0002] 4-Chlorophenol is a persistent environmental pollutant and is discharged in large quantities with industrial wastewater during processes such as pulp, wood preservation, flame retardants, pharmaceuticals, and coking and steelmaking. 4-Chlorophenol is not easily degraded naturally and has carcinogenic, mutagenic, and cytotoxic properties. It has been listed as a priority pollutant and potential carcinogen for humans by the World Health Organization. Physical and chemical methods such as adsorption, flotation, and advanced oxidation have been used for the treatment of 4-chlorophenol, but there are problems such as high energy consumption and the formation of more toxic intermediate products. Under the current technical requirements of energy conservation, emission reduction, green and low-carbon, the biological treatment technology based on microbial metabolism has more obvious advantages. However, due to the weak recognition ability and limited degradation ability of the degradation enzymes expressed by microorganisms in the sewage treatment system for 4-chlorophenol, and the lack of regioselectivity, some metabolites cannot be recognized by downstream enzymes, resulting in low treatment efficiency of the existing sewage biological treatment system for 4-chlorophenol, and the effluent containing 4-chlorophenol is discharged into the environment, endangering human health and ecological safety. Therefore, developing degradation enzymes with high regioselectivity and high degradation efficiency for 4-chlorophenol and strengthening the biodegradation of persistent pollutants such as 4-chlorophenol has broad application prospects and practical value.

[0003] At present, by separating and purifying microorganisms in contaminated site samples, strains with the ability to degrade 4-chlorophenol have been obtained, such as Pseudomonas, Streptomyces albus, Rhodococcus, Arthrobacter chlorophenolicus, etc. A halogenated phenol-degrading strain Bosea sp. DCP-2 disclosed in Chinese Patent CN 111378601A was isolated and screened from the soil of a pesticide chemical plant and can be applied to degrade 2,4-dichlorophenol (2,4-DCP), o-chlorophenol, p-chlorophenol or m-chlorophenol. After 5 days, the degradation rate of 2,4-DCP by strain DCP-2 reached 95.6%, but the degradation period was long and p-chlorophenol was not its preferred degradation substrate. Analysis of the microbial degradation pathway of 4-chlorophenol found that the bacterial degradation of 4-chlorophenol proceeds through the chlorocatechol pathway or the hydroquinone pathway. In the chlorocatechol pathway, 4-chlorophenol is first converted to 4-chlorocatechol by a monooxygenase, and then proceeds through the ortho-ring cleavage or meta-ring cleavage pathway. The monooxygenase in the first step of the reaction plays a key role in the degradation process of 4-chlorophenol, and its catalytic efficiency restricts the degradation efficiency of microorganisms on 4-chlorophenol. At the same time, the regioselectivity of the monooxygenase determines whether the first metabolite can be recognized by downstream enzymes, so as to achieve the complete degradation and mineralization of 4-chlorophenol. Chinese Patent CN 107619832 A discloses a gene cluster cnpAB of a chloronitrophenol compound oxidoreductase and its application, which mainly consists of a 2,6-dichloro-4-nitrophenol monooxygenase gene cnpA and a reductase gene cnpB. The CnpA and CnpB expressed by the engineered strain constructed using this gene cluster can completely degrade 0.1 mM of 2,6-dichloro-4-nitrophenol within 30 min, but the suitable degradation substrate concentration is relatively low, only 0.1 mM, and the treatment capacity for high-concentration chlorophenol wastewater is limited. The monooxygenase cytochrome P450 BM3 plays an important role in degrading exogenous environmental pollutants and the metabolic detoxification of organisms themselves and can be used as the first-step oxidase for 4-chlorophenol degradation. However, the naturally occurring P450 BM3 has insufficient recognition ability for 4-chlorophenol and low degradation efficiency. Therefore, there is an urgent need to develop a P450 BM3 mutant with high degradation efficiency and high regioselectivity for 4-chlorophenol. Summary of the Invention

[0004] Object of the Invention: The first object of the present invention is to provide a cytochrome P450BM3 mutant that can improve the degradation efficiency and regioselectivity of key degrading enzymes and enhance the high degradation efficiency and selectivity for 4-chlorophenol; the second object of the present invention is to provide the application of the cytochrome P450BM3 mutant in the regioselective and efficient degradation of 4-chlorophenol.

[0005] Technical solution: The cytochrome P450BM3 mutant of the present invention is a mutant in which at least one amino acid at positions 51, 82, and 330 of the amino acid sequence shown in SEQ ID No. 1 is replaced; tyrosine Tyr at position 51 is mutated to valine Val; alanine Ala at position 82 is mutated to methionine Met, and alanine Ala at position 330 is mutated to phenylalanine Phe.

[0006] Cytochrome P450 BM3 (PDB ID: 1FAG) is derived from Bacillus megaterium, and its amino acid sequence is shown in SEQ ID NO.1, and its nucleotide sequence is shown in SEQ ID NO.2.

[0007] Preferably, the mutant includes Y51V, A82M, A330F, Y51V+A82M, Y51V+A330F, A82M+A330F or Y51V+A82M+A330F.

[0008] The mutant Y51V means that tyrosine Tyr at position 51 is mutated to valine Val.

[0009] The mutant A82M means that alanine Ala at position 82 is mutated to methionine Met.

[0010] The mutant A330F means that alanine Ala at position 330 is mutated to phenylalanine Phe.

[0011] The mutant includes Y51V+A82M, that is, tyrosine Tyr at position 51 is mutated to valine Val, and alanine Ala at position 82 is mutated to methionine Met.

[0012] The mutant includes Y51V+A330F, that is, tyrosine Tyr at position 51 is mutated to valine Val, and alanine Ala at position 330 is mutated to phenylalanine Phe.

[0013] The mutant includes A82M+A330F, that is, alanine Ala at position 82 is mutated to methionine Met, and alanine Ala at position 330 is mutated to phenylalanine Phe.

[0014] The mutant includes Y51V+A82M+A330F, that is, tyrosine Tyr at position 51 is mutated to valine Val, alanine Ala at position 82 is mutated to methionine Met, and alanine Ala at position 330 is mutated to phenylalanine Phe.

[0015] The gene of the present invention is: the gene encoding the cytochrome P450BM3 mutant protein.

[0016] The recombinant plasmid described in the present invention is: a recombinant plasmid containing the said gene.

[0017] Preferably, the expression vector of the recombinant plasmid is a PET series expression vector.

[0018] The recombinant bacterium described in the present invention is a recombinant bacterium carrying the gene of the said mutant or the said recombinant plasmid.

[0019] Preferably, the host is Escherichia coli C43(DE3) or BL21(DE3).

[0020] Application of the said cytochrome P450BM3 mutant or the said recombinant plasmid or the said recombinant bacterium in regioselectively and efficiently degrading 4-chlorophenol to generate 4-chlorocatechol.

[0021] The said application comprises the following steps: using the cytochrome P450BM3 mutant as a catalyst and 4-chlorophenol as a substrate to catalyze 4-chlorophenol to generate 4-chlorohydroquinone. The catalytic mechanism is as follows:

[0022]

[0023] Preferably, the reaction conditions of the said catalysis are: pH 6 - 8, temperature 20 - 45°C.

[0024] The specific application method is: adding the pollutant 4-chlorophenol to the crude enzyme solution obtained by ultrasonic disruption after fermentation culture of the recombinant genetic engineering bacterium containing the cytochrome P450 BM3 mutant coding gene, using a buffer solution with pH 6 - 8 (preferably 7.5 Kpi buffer solution) to form a reaction system, reacting under the conditions of 300 - 500 rpm (preferably 400 rpm), 20 - 45°C (preferably 25°C). After the reaction, a reaction solution containing 4-chlorophenol and 4-chlorocatechol is obtained. Adding an equal volume of methanol to the reaction solution to precipitate proteins, centrifuging at 12000 rpm for 1 min, taking the supernatant after centrifugation and filtering it through a 0.22 μm filter membrane to obtain a sample to be tested, and detecting the contents of various substances in the sample using liquid chromatography.

[0025] Preferably, the Kpi buffer solution system is a 200 mM potassium phosphate buffer solution, composed of a mixture of potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and the pH of the Kpi solution is adjusted to 7.5.

[0026] Preferably, the wet bacterial cells are prepared as follows: The recombinant engineering bacteria containing the cytochrome P450 BM3 mutant encoding gene are inoculated into LB culture medium containing kanamycin with a final concentration of 30 μg / mL, and cultured at 37 °C for 8 h to obtain a seed solution; then the seed solution is inoculated into sterile TB liquid medium containing kanamycin with a final concentration of 30 μg / mL at an inoculation amount of 0.5%-5% (preferably 1%) by volume, and cultured at 37 °C for about 16-24 h until the cell concentration OD600 is 0.4-0.8. Then, isopropylthio-β-D-galactoside (IPTG) with a final concentration of 30-50 μM (preferably 40 μM) and δ-aminolevulinic acid (δ-ALA) with a final concentration of 0.2-0.4 mM (preferably 0.3 mM) are added to the culture solution, and induced to express at 20 °C for 24-48 h. After that, centrifuged at 4 °C and 4000 rpm for 15-30 min to collect the wet bacterial cells; LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, the solvent is deionized water, pH 7.0. TB liquid medium: 2% tryptone, 2.4% yeast extract, 72 mM K2HPO4, 17 mM KH2PO4, 0.4% glycerol.

[0027] The P450 BM3 mutant of the present invention is catalyzed by the crude enzyme solution obtained by cell disruption or the purified enzyme. In addition, the P450 BM3 mutant can be prepared into an immobilized enzyme or an enzyme in the form of immobilized cells by using a specific immobilization technique.

[0028] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages: (1) Using the means of directed evolution to modify cytochrome P450 BM3, improving the regioselectivity and degradation activity of the enzyme towards 4-chlorophenol; (2) The mutant Y51V+A82M+A330F, its K m and K cat are 0.1 mM and 0.003 s -1 respectively; (3) The mutant Y51V+A82M+A330F can degrade the pollutant 4-chlorophenol under the conditions of aqueous phase and normal temperature. The degradation rate of 2 mM 4-chlorophenol reaches 56% in 4 h, which is 6.5 times the degradation efficiency of the wild-type P450BM3, and produces a single degradation product 4-chlorocatechol (regioselectivity > 99%). The applicable pH range is 6-8, and it has the characteristics of high efficiency, low energy consumption and high regioselectivity, thus being more conducive to industrial application. Description of the Drawings

[0029] Figure 1Liquid chromatograms of 4-chlorophenol standard (a), 4-chlorocatechol standard (b), and reaction solution after degradation of 4-chlorophenol by P450 BM3 mutant (c) (retention times are Rt(4-chlorocatechol) = 5.1 min, Rt(4-chlorophenol) 6.7 min);

[0030] Figure 2 Effect diagrams of production of 4-chlorocatechol by single-point and iterative mutations of P450BM3 for degradation of 4-chlorophenol;

[0031] Figure 3 Degradation kinetic curve diagram of P450 BM3 mutant for degradation of 4-chlorophenol (2 mM);

[0032] Figure 4 Effect diagrams of production of 4-chlorocatechol by P450 BM3 mutant for degradation of 4-chlorophenol (1 mM) at different pH values;

[0033] Figure 5 Effect diagrams of production of 4-chlorocatechol by P450 BM3 mutant for degradation of 4-chlorophenol (2 mM) at different temperatures. Detailed implementation manners

[0034] The technical solutions of the present invention will be further described below in conjunction with embodiments.

[0035] Example 1: Heterologous expression and effect verification of P450 BM3-WT

[0036] The cytochrome P450 BM3 gene (derived from Bacillus megaterium, PDB ID: 1FAG) was synthesized by GenScript (Suzhou) Co., Ltd. and cloned onto the pET28a(+) vector (provided by GenScript (Suzhou) Co., Ltd.). The recombinant plasmid was transformed into competent E. coli DH5α cells (purchased from Shenzhen Kangti Life Technology Co., Ltd.), and the transformation product was evenly spread on an LB solid plate (containing kanamycin at a final concentration of 30 μg / mL) and incubated upside down in a 37°C incubator for 16 hours. A single colony was picked and inoculated into 5 mL of sterile LB liquid medium (containing kanamycin at a final concentration of 30 μg / mL), cultured at 37°C and 150 rpm for 8 - 12 hours, and then the pET28a(+)-P450 BM3-WT plasmid was extracted from E. coli DH5α using a column plasmid extraction kit.

[0037] The pET28a(+)-P450 BM3-WT plasmid obtained in the above steps was transformed into E. coli BL21(DE3) cells (purchased from Shenzhen Kangti Life Technology Co., Ltd.). The transformed product was evenly spread on an LB solid plate (containing kanamycin at a final concentration of 30 μg / mL) and incubated inverted in a 37°C incubator for 20 hours. Single colonies were picked and inoculated into 10 mL of sterile LB liquid medium (containing kanamycin at a final concentration of 30 μg / mL). The cells were cultured at 37°C and 150 rpm until the cell concentration OD600 = 0.6 - 0.8. 0.5 mL of the bacterial solution was taken into a 1.5 mL sterile centrifuge tube, and 0.5 mL of 50% glycerol was added. After mixing, it was stored in a -80°C refrigerator. The remaining bacterial solution was added to 1 L of sterile TB culture medium containing kanamycin at a final concentration of 30 μg / mL at an inoculation amount of 1%. After culturing at 37°C and 150 rpm for 12 - 24 hours, IPTG at a final concentration of 40 μM and δ-aminolevulinic acid (δ-ALA) at a final concentration of 0.3 mM were added. After inducing expression at 20°C for 48 h, centrifugation was carried out at 4°C and 4000 rpm for 5 min. The supernatant was discarded, and the wet bacterial cells were collected.

[0038] The wet bacterial cells were resuspended with 20 mL of 200 mM KPi buffer at pH = 7.5, and sonicated at 4°C for 5 min with a sonication power of 65%, sonication on for 2 s and off for 6 s. Then, centrifugation was carried out at 4°C and 12000 rpm for 15 min. 200 μL of the supernatant was taken to measure the P450 concentration, and it was diluted with 200 mM KPi buffer at pH = 7.5 to a P450 concentration of 50 μM. 5 mL of the diluted supernatant was taken into a 10 mL glass reaction flask, and glucose at a final concentration of 100 mM, glucose dehydrogenase at a final concentration of 5 mg / mL, 4-chlorophenol at a final concentration of 2 mM, and NADP at a final concentration of 80 μM were added. + Using a magnetic stirrer to mix evenly, after reacting at 25°C and 400 rpm for 4 hours, 500 μL of the supernatant after the reaction was taken, and the protein was precipitated with 500 μL of methanol. Then, centrifugation was carried out at 12000 rpm for 1 min. 300 μL of the supernatant was taken, filtered through a 0.22 μm filter membrane, and detected by liquid chromatography. 4-chlorophenol and 4-chloro-1,2-benzenediol (4-chlorocatechol) with concentrations of 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 2 mM, and 5 mM were prepared respectively using the same treatment method as the sample, and detected using the same liquid chromatography method.

[0039] The liquid chromatography detection method is: Agilent 1260 Infinity II liquid chromatography system, TC-C18 chromatographic column (4.6×250mm, 5μm), mobile phase is methanol: 0.15% phosphoric acid water = 65:35, column temperature is 25°C, flow rate is 1mL / min, UV-visible absorption detector, detection wavelength is 280nm. The elution time of 4-chlorophenol is 6.7min, and the elution time of 4-chlorocatechol is 5.1min.

[0040] LB medium formula: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride (adding 15 g / L agar powder to it makes LB solid medium); TB liquid medium formula: 2% tryptone, 2.4% yeast extract, 72 mM K2HPO4, 17 mM KH2PO4, 0.4% glycerol.

[0041] Example 2: Construction of a P450 BM3 site-directed saturation mutation library

[0042] According to the conclusion of Example 1, primers were designed based on the gene sequence of wild-type cytochrome P450 BM3 included in PDB (the amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2) (see Table 1). The parental P450 BM3-WT gene (nucleotide sequence is SEQ ID NO.2) was subjected to site-directed saturation mutagenesis using primers Y51NNK-F / Y51NNK-R, A82NNK-F / A82NNK-R, and A330NNK-F / A330NNK-R (NNK degenerate codons cover 32 codon combinations (N=A / C / G / T, K=G / T) and can encode all 20 amino acids), respectively. pET-28a(+) was used as an expression vector to obtain mutant plasmids carrying the target gene, and the mutant plasmids carrying the target gene were transformed into E. coli BL21(DE3) to obtain mutants of recombinant bacteria containing the P450 BM3 mutant gene.

[0043] Table 1 Primer design table for construction of P450 BM3 site-directed saturation mutagenesis library

[0044]

[0045]

[0046] PCR amplification system: 50μL reaction system:

[0047] ddH2O: 30μL;

[0048] 10×Buffer: 5 μL;

[0049] dNTP: 5 μL;

[0050] MgSO4: 3 μL;

[0051] DMSO: 2 μL;

[0052] Forward primer (50 μM): 1.5 μL;

[0053] Reverse primer (50 μM): 1.5 μL;

[0054] KOD enzyme: 1 μL;

[0055] Template DNA (plasmid): 1 μL;

[0056] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 3 min, followed by 30 cycles of temperature cycling at 95°C for 20 s, 55°C for 10 s, and 72°C for 30 s, and finally extension at 72°C for 10 min, with the termination temperature at 4°C. After the PCR products were analyzed and verified by 1% agarose gel electrophoresis, 1 μL of DpnI was added to the PCR products and digested at 37°C for 2 h to remove the template plasmid DNA. The mixture was transformed into competent cells of E. coli DH5α, and the transformed mixture was evenly spread on a plate of LB solid medium containing kanamycin (30 μg / mL) and incubated inverted in a 37°C incubator for 16 h. Single colonies were picked and inoculated into 2 mL of sterile LB liquid medium (containing 30 μg / mL of kanamycin), and cultured at 37°C and 150 rpm for 8 - 12 h. Then, the saturated mutant library plasmid was extracted from E. coli DH5α using a column plasmid extraction kit, and then transformed into competent cells of E. coli BL21(DE3). The cells were spread on an LB plate containing kanamycin (30 μg / mL) and cultured overnight at 37°C to obtain the mutant library of P450 BM3. At this time, many single colonies with different mutations appeared on the LB plate, and these single colonies were used for the subsequent screening of the mutant library.

[0057] Example 3: Screening of the P450 BM3 Mutant Library

[0058] The screening of the P450 BM3 mutant library was carried out with P450 BM3 WT as a reference. Single colony clones (the mutant library constructed in Example 2) were picked into a 1 mL deep 96-well plate for cultivation. 400 μL of LB culture medium containing kanamycin with a final concentration of 30 μg / mL was added in advance. At the same time, 3 parental strains were picked into the last 3 wells of the 96-well plate as controls. The 1 mL 96-well plate was cultured at 37 °C for 8 h as a seed solution. Then, 100 μL of the seed solution was taken and added to a new 2 mL deep 48-well plate for cultivation. Sterile TB culture medium containing kanamycin with a final concentration of 30 μg / mL was added in advance. After culturing at 37 °C for 24 h, IPTG with a final concentration of 40 μM and δ-aminolevulinic acid (δ-ALA) with a final concentration of 0.3 mM were added. After inducing expression at 20 °C for 48 h, centrifugation was carried out at 4 °C and 4000 rpm for 5 min. The supernatant was discarded, and the wet bacterial cells were collected for the next step of screening.

[0059] 500 μL of 200 mM KPi buffer was added to each well, and the bacterial cells were resuspended. Ultrasonic disruption was carried out at 4 °C for 3 min, with an ultrasonic power of 65%, ultrasonic on for 2 s, and off for 6 s. Then, centrifugation was carried out at 4000 rpm and 4 °C for 5 min. Glucose with a final concentration of 100 mM, glucose dehydrogenase with a final concentration of 5 mg / mL, 4-chlorophenol with a final concentration of 1 mM, and NADP with a final concentration of 80 μM were added. + After that, the reaction was carried out at 25 °C and 400 rpm for 4 h. Protein was precipitated with 500 μL of methanol, and then centrifugation was carried out at 12000 rpm for 1 min. 300 μL of the supernatant was taken, filtered through a 0.22 μm filter membrane, and detected by liquid chromatography. The test results are shown in Table 2 and Figure 2 。

[0060] Table 2 Degradation effect of P450 BM3 single mutants on 4-chlorophenol

[0061]

[0062] Taking the amount of 4-chlorocatechol produced by the wild type and the remaining amount of 4-chlorophenol as controls. The degradation effect of all single mutants on 4-chlorophenol was better than that of the wild type P450 BM3. The best single mutant Y51V was obtained, and the yield of 4-chlorocatechol produced by degrading 4-chlorophenol within 4 h was 15%, which was 1.76 times that of the wild type P450 BM3.

[0063] Example 4: Construction of the P450 BM3 iterative saturation mutant library

[0064] Based on the conclusion of Example 3, using the mutant Y51V obtained by the above screening as a template, mutant primers were designed. The mutant Y51V gene was subjected to iterative saturation mutagenesis with primers A82NNK-F / A82NNK-R and A330NNK-F / A330NNK-R respectively, and mutant plasmids carrying the target gene were obtained. The mutant plasmids carrying the target gene were transformed into E. coli BL21(DE3), and mutants of recombinant bacteria containing the P450 BM3 mutant gene were obtained. According to the method of Example 3, the recombinant bacteria mutants obtained in the second round were screened for the degradation effect of 4-chlorophenol, and the best double-mutant recombinant bacteria mutant Y51V+A82M was selected as the template for the next round of saturation mutagenesis, and so on, to obtain triple mutants with gradually improved 4-chlorophenol degradation effect.

[0065] Table 3 Primer design table for the construction of the P450 BM3 iterative saturation mutagenesis library

[0066]

[0067]

[0068] The PCR amplification system was: 50 μL reaction system:

[0069] ddH2O: 30 μL;

[0070] 10×Buffer: 5 μL;

[0071] dNTP: 5 μL;

[0072] MgSO4: 3 μL;

[0073] DMSO: 2 μL;

[0074] Upstream primer (50 μM): 1.5 μL;

[0075] Downstream primer (50 μM): 1.5 μL;

[0076] KOD enzyme: 1 μL;

[0077] Template DNA (plasmid): 1 μL;

[0078] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 3 min, followed by temperature cycling at 95°C for 20 s, 55°C for 10 s, 72°C for 30 s for a total of 30 cycles, and finally extension at 72°C for 10 min, with the termination temperature at 4°C. After verification by 1% agarose gel electrophoresis analysis of the PCR products, 1 μL of DpnI was added to the PCR products and digested at 37°C for 2 h to remove the template plasmid DNA. It was transformed into the competent cell E. coli DH5α, and the transformed mixture was evenly spread on a plate of LB solid medium containing kanamycin (30 μg / mL), and cultured inverted in a 37°C incubator for 16 h. Single colonies were picked and inoculated into 2 mL of sterile LB liquid medium (containing 30 μg / mL of kanamycin), and cultured at 37°C and 150 rpm for 8 - 12 h. Then, a column plasmid extraction kit was used to extract the saturated mutant library plasmid from E. coli DH5α, and then transformed into the competent cell E. coli BL21(DE3). It was spread on an LB plate containing kanamycin (30 μg / mL) and cultured overnight at 37°C to obtain the mutant library of P450 BM3. At this time, many single colonies with different mutations appeared on the LB plate. The screening of the mutant library was the same as in Example 3. The test results are shown in Table 4 and Figure 2 。

[0079] Table 4 Degradation effects of P450 BM3 double mutants and triple mutants on 4-chlorophenol

[0080]

[0081] The amounts of 4-chlorocatechol produced and the remaining amounts of 4-chlorophenol by the wild type and mutant Y51V were used as controls. The degradation effects of all double mutants and triple mutants on 4-chlorophenol were better than those of the wild type P450 BM3. After three rounds of iterative saturation mutagenesis, an excellent mutant strain was obtained, denoted as mutant Y51V / A82M / A330F, whose yield of 4-chlorocatechol produced from the degradation of 4-chlorophenol within 4 h was 56%, which was 6.5 times that of the wild type P450 BM3 ( Figure 2 ).

[0082] Example 5: Degradation kinetics of P450 BM3 mutants on 4-chlorophenol

[0083] According to the conclusion of Example 4, the screened mutant E. coli BL21(DE3)-Y51V / A82M / A330F was inoculated into a sterile test tube containing 10 mL of LB medium with a final concentration of 30 μg / mL kanamycin, and cultured in a shaker at 37°C and 150 rpm for 6 - 8 h. Then, it was added to 1 L of sterile TB medium containing a final concentration of 30 μg / mL kanamycin at an inoculation amount of 1%, and cultured at 37°C for 24 h. After that, IPTG with a final concentration of 40 μM and δ-aminolevulinic acid (δ-ALA) with a final concentration of 0.3 mM were added, and induced expression was carried out at 20°C for 48 h. Then, it was centrifuged at 4000 rpm for 30 min, the supernatant was discarded, and the wet cells were collected. 20 mL of 200 mM KPi buffer with pH = 7.5 was added to the wet cells to resuspend the cells, and ultrasonic disruption was carried out at 4°C for 5 min with an ultrasonic power of 65%, ultrasonic on for 2 s, off for 6 s. Then, it was centrifuged at 12000 rpm at 4°C for 15 min, 200 μL of the supernatant was taken to measure the P450 concentration, and it was diluted to a P450 concentration of 20 μM with 200 mM KPi buffer with pH = 7.5. 5 mL of the diluted supernatant was taken into a 10 mL glass reaction flask, and glucose with a final concentration of 100 mM, glucose dehydrogenase with a final concentration of 5 mg / mL, 4-chlorophenol with a final concentration of 2 mM, and NADP with a final concentration of 80 μM were added. + , and it was stirred and mixed evenly using a magnetic stirrer, and reacted at 25°C and 400 rpm for 240 min. At 10 min, 30 min, 60 min, 90 min, 120 min, 180 min, and 240 min, 500 μL of the supernatant after the reaction was taken, 500 μL of methanol was added to precipitate the protein, and then it was centrifuged at 12000 rpm for 1 min. 300 μL of the supernatant was taken, filtered through a 0.22 μm filter membrane, and detected by liquid chromatography.

[0084] The degradation kinetic curve of the P450 BM3 mutant Y51V / A82M / A330F was obtained ( Figure 3 ). As the reaction proceeded, 4-chlorophenol was degraded and its concentration gradually decreased, while the concentration of the degradation product 4-chlorocatechol gradually increased, and the yield reached 56% at 240 min.

[0085] Example 6: Optimization of the Optimal pH Condition for the Degradation of 4-Chlorophenol by the P450 BM3 Mutant

[0086] According to the conclusion of Example 4, the screened mutant E. coli BL21(DE3)-Y51V / A82M / A330F was inoculated into a sterile test tube containing 10 mL of LB medium with a final concentration of 30 μg / mL kanamycin, and cultured in a shaker at 37 °C and 150 rpm for 6 - 8 h. Then, according to an inoculation amount of 1%, it was added to 1 L of sterile TB medium containing a final concentration of 30 μg / mL kanamycin, and cultured at 37 °C for 24 h. Then, IPTG with a final concentration of 40 μM and δ-aminolevulinic acid (δ-ALA) with a final concentration of 0.3 mM were added. After inducing expression at 20 °C for 48 h, it was centrifuged at 4 °C and 4000 rpm for 30 min, the supernatant was discarded, and the wet cells were collected. 20 mL of 200 mM KPi buffer with pH = 7.5 was added to the wet cells to resuspend the cells. 1 mL of the resuspended mixture was added to each of 5 sterile 2-mL centrifuge tubes, and centrifuged at 4 °C and 4000 rpm for 10 min. The supernatant was discarded, and 900 μL of buffers with different pH values (sodium hydrogen phosphate-citric acid buffer with pH = 6, KPi buffer with pH = 7.5, sodium hydrogen phosphate-citric acid buffer with pH = 8, borate buffer with pH = 10) were added to the 5 centrifuge tubes to resuspend the cell suspension. It was ultrasonically broken at 4 °C for 3 min, the ultrasonic power was 65%, the ultrasound was on for 2 s and off for 6 s, and centrifuged at 4 °C and 12,000 rpm for 1 min. 500 μL of the supernatant was taken and added to 5 5-mL glass reaction flasks, and then glucose with a final concentration of 100 mM, glucose dehydrogenase with a final concentration of 5 mg / mL, 4-chlorophenol with a final concentration of 1 mM, and NADP with a final concentration of 80 μM were added respectively. + It was stirred and mixed evenly using a magnetic stirrer, and reacted under the conditions of 25 °C and 400 rpm for 3 h. 500 μL of the reaction solution was taken from each reaction flask, 500 μL of methanol was added to precipitate proteins, and then centrifuged at 12,000 rpm for 1 min. 300 μL of the supernatant was filtered through a 0.22-μm filter membrane and detected by liquid chromatography.

[0087] The experimental results are as Figure 4 shown. The optimal pH of the P450 BM3 mutant is 7.5, and it can maintain good activity within the pH range of 6 - 8, and the degradation rate of 1 mM 4-chlorophenol > 50%.

[0088] Example 7: Optimization of the Optimal Temperature Condition for the Degradation of 4-Chlorophenol by the P450 BM3 Mutant

[0089] According to the conclusion of Example 4, the mutant E. coli BL21(DE3)-Y51V / A82M / A330F obtained by the above screening was inoculated into a sterile test tube containing 10 mL of LB medium with a final concentration of 30 μg / mL kanamycin added in advance, and cultured in a shaker at 37 °C and 150 rpm for 6 - 8 h. Then, according to an inoculation amount of 1%, it was added to 1 L of sterile TB medium containing a final concentration of 30 μg / mL kanamycin, and cultured at 37 °C for 24 h. Then, IPTG with a final concentration of 40 μM and δ-aminolevulinic acid (δ-ALA) with a final concentration of 0.3 mM were added. After inducing expression at 20 °C for 48 h, it was centrifuged at 4 °C and 4000 rpm for 30 min. The supernatant was discarded, and the wet cells were collected. 20 mL of 200 mM KPi buffer at pH 7.5 was added to the wet cells to resuspend the cells. Ultrasonic disruption was carried out at 4 °C for 5 min, with an ultrasonic power of 65%, ultrasonic on for 2 s, and off for 6 s. Then, it was centrifuged at 12000 rpm at 4 °C for 15 min. 200 μL of the supernatant was taken to measure the P450 concentration, and it was diluted with 200 mM KPi buffer at pH 7.5 to a P450 concentration of 50 μM. 1 mL of the diluted supernatant was taken and placed into 4 5-mL glass reaction flasks, and glucose with a final concentration of 100 mM, glucose dehydrogenase with a final concentration of 5 mg / mL, 4-chlorophenol with a final concentration of 2 mM, and NADP with a final concentration of 80 μM were added. + A magnetic stir bar was placed, and the mixture was stirred and mixed evenly using a magnetic stirrer. The reaction was carried out at 20 °C, 25 °C, 37 °C, and 45 °C at 400 rpm for 3 h. 500 μL of the reaction solution was taken, 500 μL of methanol was added to precipitate the protein, and then it was centrifuged at 12000 rpm for 1 min. 300 μL of the supernatant was taken, filtered through a 0.22-μm filter membrane, and detected by liquid chromatography.

[0090] The experimental results are as Figure 5 shown. Different temperatures have a significant impact on the catalytic effect of the P450 BM3 mutant. The optimal temperature of the P450BM3 mutant is 25 °C, that is, it has the highest degradation activity at room temperature. An increase or decrease in temperature will reduce the activity of the P450 BM3 mutant. This enables the P450 BM3 mutant to efficiently degrade 4-chlorophenol at room temperature, without the need for additional temperature control equipment, saving energy consumption, and having the characteristics of being green and low-carbon.

[0091] Example 8: Determination of the Michaelis kinetic parameters of the P450 BM3 mutant for degrading 4-chlorophenol

[0092] According to the conclusion of Example 4, the screened mutant E. coli BL21(DE3)-Y51V / A82M / A330F was inoculated into a sterile test tube containing 10 mL of LB medium with a final concentration of 30 μg / mL kanamycin in advance, and cultured in a shaker at 37 °C and 150 rpm for 6 - 8 h. Then, according to an inoculation amount of 1%, it was added to 1 L of sterile TB medium containing a final concentration of 30 μg / mL kanamycin, and cultured at 37 °C for 24 h. After that, IPTG with a final concentration of 40 μM and δ-aminolevulinic acid (δ-ALA) with a final concentration of 0.3 mM were added, and induced expression was carried out at 20 °C for 48 h. Then, it was centrifuged at 4 °C and 4000 rpm for 30 min, the supernatant was discarded, and the wet cells were collected. 20 mL of 200 mM KPi buffer with pH = 7.5 was added to the wet cells to resuspend the cells. Ultrasonic disruption was carried out at 4 °C for 5 min, the ultrasonic power was 65%, the ultrasound was on for 2 s and off for 6 s. Then, it was centrifuged at 4 °C and 12,000 rpm for 15 min, 200 μL of the supernatant was taken to measure the P450 concentration, and it was diluted with 200 mM KPi buffer to a P450 concentration of 50 μM. 1 mL of the diluted supernatant was taken into 5 5-mL glass reaction flasks respectively, glucose with a final concentration of 100 mM, glucose dehydrogenase with a final concentration of 5 mg / mL, 4-chlorophenol with different concentrations, and NADP with a final concentration of 80 μM were added respectively. + , where 4-chlorophenol with final concentrations of 0.2 mM, 0.25 mM, 0.5 mM, 1.0 mM, and 2 mM were added to the 5 glass reaction flasks respectively, and the reaction was magnetically stirred at 25 °C and 400 rpm for 30 min. 500 μL of the reaction solution was taken from each reaction flask, 500 μL of methanol was added to precipitate the protein, and then it was centrifuged at 12,000 rpm for 1 min. 300 μL of the supernatant was taken, filtered through a 0.22-μm filter membrane, and detected by liquid chromatography. The enzymatic reaction rate was measured, and a double-reciprocal curve was made based on the reaction rate and the reciprocal of the substrate concentration to calculate the Michaelis kinetic parameters. The Michaelis kinetic parameters of the P450 BM3 mutant E. coli BL21(DE3)-Y51V / A82M / A330F for 4-chlorophenol were K m = 0.1 mM, K cat = 0.003 s -1 .

Claims

1. A cytochrome P450BM3 mutant, characterized in that, The mutant is obtained by substituting at least two amino acids at positions 51, 82, and 330 of the amino acid sequence shown in SEQ ID No. 1; tyrosine (Tyr) at position 51 is mutated to valine (Val); alanine (Ala) at position 82 is mutated to methionine (Met), and alanine (Ala) at position 330 is mutated to phenylalanine (Phe). The mutant is selected from Y51V+A330F, A82M+A330F, or Y51V+A82M+A330F.

2. A gene encoding the cytochrome P450BM3 mutant protein according to claim 1.

3. A recombinant plasmid containing the gene according to claim 2.

4. The recombinant plasmid according to claim 3, characterized in that, The expression vector of the recombinant plasmid is a PET series expression vector.

5. A recombinant bacterium carrying the gene of the mutant according to claim 2 or the recombinant plasmid according to claim 3.

6. The recombinant bacterium according to claim 5, wherein The host of the gene or recombinant plasmid is Escherichia coli C43(DE3) or BL21(DE3).

7. Use of the cytochrome P450BM3 mutant according to claim 1, the recombinant plasmid according to claim 3, or the recombinant bacterium according to claim 5 in the degradation of 4-chlorophenol to produce 4-chlorocatechol.

8. The application according to claim 7, characterized in that, The use comprises the following steps: using the cytochrome P450BM3 mutant as a catalyst and 4-chlorophenol as a substrate to catalyze the production of 4-chlorohydroquinone from 4-chlorophenol.

9. The application according to claim 8, characterized in that, The reaction conditions for the catalysis are: pH 6 - 8, temperature 20 - 45 °C.

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