A cytochrome P450BM3 mutant and its application in degrading 2-chlorophenol

Abstract

The present invention discloses a cytochrome P450BM3 mutant and its application in the degradation of 2-chlorophenol. The mutant is obtained by replacing at least one amino acid in the seven sites of the amino acid sequence shown in SEQ ID No.1, namely, positions 47, 51, 81, 82, 184, 330, and 401. By modifying the key amino acids in the active center of P450BM3, a P450BM3 mutant with high degradation efficiency and high selectivity capable of catalyzing the oxidation of the pollutant 2-chlorophenol is obtained, overcoming the problems of low degradation efficiency and poor selectivity of 2-chlorophenol in the prior art. The mutant can be efficiently degraded at room temperature without the need for external temperature control equipment, has the advantages of being green and low-carbon, and has stronger application significance in engineering practice.

Description

Technical Field

[0001] The present invention relates to the technical field of environmental remediation microorganisms, in particular to a cytochrome P450BM3 mutant and application thereof in degrading 2-chlorophenol. Background Art

[0002] 2-Chlorophenol is an intermediate in the production of phenol, phenolic resins and dyes. It is often used as an insecticide and fungicide. It is a common and difficult-to-degrade organic pollutant in wastewater. It is highly toxic, carcinogenic and difficult to biodegrade.

[0003] At present, the treatment methods for 2-chlorophenol in sewage are mainly divided into physical, chemical and biological methods. Physical methods mainly include adsorption and reverse osmosis, which require the use of expensive treatment materials such as activated carbon and filter membranes, and the filter materials and membrane materials need to be replaced frequently, resulting in high treatment costs. Chemical methods mainly include ion exchange, UV / TiO2 / H2O2 advanced oxidation, etc., which use a large amount of chemical reagents and produce a large amount of iron sludge, which easily leads to secondary pollution. Compared with physical and chemical methods, biological methods have the advantages of low cost, green and low carbon, and environmental friendliness, and are widely used in sewage treatment. However, due to the weak degradation ability of microorganisms for difficult-to-degrade organic pollutants such as 2-chlorophenol, biological methods are not widely used in the degradation of 2-chlorophenol.

[0004] Currently, with the continuous development of environmental microbiology technology, the identification and screening of microorganisms capable of degrading difficult-to-degrade organic pollutants has achieved relatively mature results in the bioremediation of various pollutants. Conventional methods generally involve screening, isolating, and purifying strains capable of degrading specific pollutants from environmental samples containing pollutants. Genes with pollutant degradation functions are discovered through whole-genome sequencing, and the required enzymes are expressed in specific vectors. However, there are problems such as a small number of degradation genes and a long degradation cycle for the original enzymes. For example, Chinese patent CN 108823122 A discloses a strain of 2,4-dichlorophenol-degrading bacteria. A strain HD-1, belonging to the genus Pseudomonas mosselii, was screened from soil in a pesticide chemical plant and showed a high ability to degrade 2,4-dichlorobenzene. The strain achieved a degradation rate of 59.65% in 10 days, achieving initial biodegradation and removal of 2,4-dichlorophenol. However, the degradation time was as long as 10 days, resulting in low degradation efficiency. Chinese patent CN109337877 B discloses a dichlorophenol-degrading enzyme TcpA, which encodes a dichlorophenol-degrading enzyme TcpA, which is cloned from the Cupriavidustaiwanensis X1 strain with dichlorophenol-degrading ability. 8It takes 24 hours for a CFU to degrade 4 mg of 2,4-dichlorophenol, and the degradation efficiency needs to be further improved. Cytochrome P450 is an important superfamily of detoxification metabolic enzymes, and it has been clearly involved in the detoxification metabolism of various agents. Chinese patent CN 101659955B discloses a human liver cytochrome P450 gene that can metabolize chlorpyrifos and pirimiphos-methyl in vitro. The application of P450 in the degradation of organophosphorus pesticides has been preliminarily explored, but there are currently no reports of P450 degrading the pollutant 2-chlorophenol. At the same time, none of the above-mentioned microbial remediation technologies have evolved or modified the enzymes that play a key role in the pollutant degradation process. They have only explored and reorganized the original enzymes, and the degradation efficiency and selectivity of the biological enzymes have not been further improved. Therefore, the development of a highly selective and efficient P450BM3 mutant for the degradation of 2-chlorophenol is of great significance for the efficient and precise biological degradation of difficult-to-degrade organic pollutants in wastewater. Summary of the Invention

[0005] Purpose of the invention: The first purpose of the present invention is to provide a cytochrome P450BM3 mutant with high degradation efficiency and selectivity for 2-chlorophenol, overcome the shortcomings of low degradation efficiency and lack of selectivity of existing biodegradation methods for 2-chlorophenol, and achieve efficient and precise biodegradation of 2-chlorophenol; the second purpose of the present invention is to provide the application of the cytochrome P450BM3 mutant in the degradation of 2-chlorophenol.

[0006] Technical solution: The present invention relates to a cytochrome P450BM3 mutant, wherein the mutant is a SEQ At least one amino acid is substituted at positions 47, 51, 81, 82, 184, 330, and 401 of the amino acid sequence shown in ID No. 1; the arginine Arg at position 47 is mutated to leucine Leu, the tyrosine Tyr at position 51 is mutated to any one of phenylalanine Phe, tryptophan Trp, or proline Pro; the phenylalanine Phe at position 81 is mutated to any one of isoleucine Ile, leucine Leu, valine Val, methionine Met, or alanine Ala; the alanine Ala at position 82 is mutated to any one of methionine Met and lysine Lys; the alanine Ala at position 184 is mutated to any one of isoleucine Ile, leucine Leu, or methionine Met; the alanine Ala at position 330 is mutated to any one of leucine Leu or glycine Gly; and the isoleucine Ile at position 401 is mutated to leucine Leu.

[0007] Cytochrome P450 BM3 (PDB ID: 1FAG) is from Priestia megaterium, and its amino acid sequence is shown in SEQ ID No. 1, and its gene sequence is shown in SEQ ID No. 2.

[0008] Preferably, the mutants include R47L, Y51F, Y51W, Y51P, F81I, F81L, F81V, F81M, F81A, A82M, A82K, A184I, A184L, A184M, A330L, A330G, I401L, R47L+Y51F, R47L+Y51F+F81I, R47L+Y51F+F81V, R47L +Y51F+F81M, R47L+Y51F+F81I+A82M, R47L+Y51F+F81I+A82K, R47L+Y51F+F81I+A82M+A184I, R47L+Y51F+F81I+A82M+A184I+A330L or R47L+Y51F+F81I+A82M+A184I+A330L+I401L.

[0009] The mutant R47L, i.e., the 47th arginine Arg is mutated to the leucine Leu. The mutant Y51F, i.e., the 51st tyrosine Tyr is mutated to the phenylalanine Phe.

[0010] The mutant Y51W is a mutant in which the 51st tyrosine Tyr is mutated to tryptophan Trp.

[0011] The mutant Y51P is a mutation of tyrosine Tyr at position 51 to proline Pro.

[0012] The mutant F81I, namely, the phenylalanine Phe at position 81 is mutated to isoleucine Ile.

[0013] The mutant F81L, namely, the phenylalanine Phe at position 81 is mutated to leucine Leu.

[0014] The mutant F81V, namely, the phenylalanine Phe at position 81 is mutated to valine Val.

[0015] The mutant F81M, namely, the phenylalanine Phe at position 81 is mutated to methionine Met.

[0016] The mutant F81A, namely, the phenylalanine Phe at position 81 is mutated to alanine Ala.

[0017] The mutant A82M, that is, the alanine Ala at position 82 is mutated to methionine Met.

[0018] The mutant A82K is a mutant in which the alanine Ala at position 82 is mutated to lysine Lys.

[0019] In the mutant A184I, the 184th alanine Ala is mutated to isoleucine Ile.

[0020] In the mutant A184L, the 184th alanine Ala is mutated to leucine Leu.

[0021] In the mutant A184M, the 184th alanine Ala is mutated to methionine Met.

[0022] In the mutant A330L, alanine Ala at position 330 is mutated to leucine Leu.

[0023] In the mutant A330G, the 330th alanine Ala is mutated to glycine Gly.

[0024] In the mutant I401L, the 401st isoleucine Ile is mutated to leucine Leu.

[0025] The mutant R47L+Y51F, namely, the 47th arginine Arg is mutated to leucine Leu, and the 51st tyrosine Tyr is mutated to phenylalanine Phe.

[0026] The mutant R47L+Y51F+F81I, that is, the 47th arginine Arg is mutated to leucine Leu, the 51st tyrosine Tyr is mutated to phenylalanine Phe, and the 81st phenylalanine Phe is mutated to isoleucine Ile.

[0027] The mutant R47L+Y51F+F81V, that is, the 47th arginine Arg is mutated to leucine Leu, the 51st tyrosine Tyr is mutated to phenylalanine Phe, and the 81st phenylalanine Phe is mutated to valine Val.

[0028] The mutant R47L+Y51F+F81M, namely, the 47th arginine Arg is mutated to leucine Leu, the 51st tyrosine Tyr is mutated to phenylalanine Phe, and the 81st phenylalanine Phe is mutated to methionine Met.

[0029] The mutant R47L+Y51F+F81I+A82M, that is, the arginine Arg at position 47 is mutated to leucine Leu, the tyrosine Tyr at position 51 is mutated to phenylalanine Phe, the phenylalanine Phe at position 81 is mutated to isoleucine Ile, and the alanine Ala at position 82 is mutated to methionine Met.

[0030] The mutant R47L+Y51F+F81I+A82K, that is, the 47th arginine Arg is mutated to leucine Leu, the 51st tyrosine Tyr is mutated to phenylalanine Phe, the 81st phenylalanine Phe is mutated to isoleucine Ile, and the 82nd alanine Ala is mutated to lysine Lys.

[0031] The mutant R47L+Y51F+F81I+A82M+A184I, that is, the 47th arginine Arg mutated to leucine Leu, the 51st tyrosine Tyr mutated to phenylalanine Phe, the 81st phenylalanine Phe mutated to isoleucine Ile, the 82nd alanine Ala mutated to methionine Met, and the 184th alanine Ala mutated to isoleucine Ile.

[0032] The mutant R47L+Y51F+F81I+A82M+A184I+A330L, that is, the 47th arginine Arg mutated to leucine Leu, the 51st tyrosine Tyr mutated to phenylalanine Phe, the 81st phenylalanine Phe mutated to isoleucine Ile, the 82nd alanine Ala mutated to methionine Met, the 184th alanine Ala mutated to isoleucine Ile, and the 330th alanine Ala mutated to leucine L.

[0033] The mutant R47L+Y51F+F81I+A82M+A184I+A330L+I401L, that is, the arginine Arg at position 47 mutates to leucine Leu, the tyrosine Tyr at position 51 mutates to phenylalanine Phe, the phenylalanine Phe at position 81 mutates to isoleucine Ile, the alanine Ala at position 82 mutates to methionine Met, the alanine Ala at position 184 mutates to isoleucine Ile, and the alanine Ala at position 330 mutates to leucine L, and the isoleucine Ile at position 401 mutates to leucine Leu.

[0034] The gene described in the present invention is a gene encoding the cytochrome P450BM3 mutant protein.

[0035] The recombinant plasmid of the present invention is a recombinant plasmid containing the gene.

[0036] Preferably, the expression vector of the recombinant plasmid is a PET series expression vector.

[0037] The recombinant bacteria of the present invention are recombinant bacteria carrying the mutant gene or the recombinant plasmid.

[0038] Preferably, the host is Escherichia coli C43 (DE3) or BL21 (DE3).

[0039] Application of the cytochrome P450BM3 mutant, the recombinant plasmid or the recombinant bacteria in degrading 2-chlorophenol.

[0040] 2-Chlorophenol is a typical environmentally toxic and difficult-to-degrade organic pollutant. It is toxic to aquatic organisms and may cause long-term adverse effects in the aquatic environment. For example, its half-lethal concentration (LC50) for goldfish reaches 12.37 mg / L. It also bioaccumulates and can accumulate in aquatic organisms, such as the bluegill sunfish, with a bioconcentration factor (BCF) of 214. 2-Chlorophenol has the potential to irritate the eyes and skin and damage the nervous and respiratory systems in humans, and is listed as a priority pollutant. Compared to phenol, the introduction of chlorine atoms in 2-chlorophenol increases the compound's lipophilicity, making it easier for it to pass through cell membranes, enter organisms, and accumulate. At the same time, chlorine atoms can improve chemical stability, extending its half-life in the environment and thus increasing its persistence in ecosystems. Due to its increased lipophilicity and stability, 2-chlorophenol is more likely to bioaccumulate and biomagnify in the food chain, leading to higher concentrations in organisms at the top of the food chain, posing more serious water quality risks.

[0041] In terms of biodegradability, the chlorine atom is a strong electron-withdrawing group. When attached to a benzene ring, it reduces the electron density of the benzene ring through a conjugation effect, thereby reducing its nucleophilicity for substitution reactions. Furthermore, the chlorine atom is larger than a hydrogen atom, which may increase steric hindrance, making substitution reactions more difficult. In phenol, the ortho and para positions of the hydroxyl group are positions with high electron density because the conjugation effect of the hydroxyl group makes these positions more susceptible to electrophilic substitution. In 2-chlorophenol, the presence of chlorine atoms reduces the electron density of the meta position of the chlorine substituent, making the para-hydroxylation of 2-chlorophenol more difficult than that of phenol. Therefore, studying the regioselective oxidative degradation of 2-chlorophenol is of great significance for its removal and also provides a reference for water quality risk control of chlorophenol pollutants.

[0042] The application comprises the following steps: using a cytochrome P450BM3 mutant as a catalyst and 2-chlorophenol as a substrate to catalyze the conversion of 2-chlorophenol into 2-chlorohydroquinone. The catalytic mechanism is as follows:

[0043]

[0044] Preferably, the catalytic reaction has a pH of 6 to 8 and a temperature of 20 to 37°C.

[0045] Specifically, a crude enzyme solution obtained by fermenting and culturing a recombinant genetically engineered bacterium containing a P450 BM3 mutant encoding gene and then ultrasonically crushing the resulting solution is used as a catalyst, 2-chlorophenol is used as a substrate, and a reaction system is formed using a buffer solution with a pH of 6 to 8 (preferably a Kpi buffer solution of 7.5). The reaction is carried out at 300 to 500 rpm (preferably 400 rpm) and 20 to 37° C. (preferably 25° C.). After the reaction is completed, a reaction solution containing 2-chlorophenol and 2-chlorohydroquinone is obtained. The reaction solution is extracted with ethyl acetate, and the substance content in the organic phase is detected by gas chromatography.

[0046] Preferably, the Kpi buffer system is a 200 mM potassium phosphate buffer solution, which is composed of a mixture of potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and the pH of the Kpi solution is configured to 7.5.

[0047] Preferably, the amount of the catalyst is 10-40 g / L buffer (preferably 20 g / L) based on the weight of the wet cells, and the initial concentration of the substrate is 0.05-2 mM (preferably 1 mM).

[0048] Preferably, the wet bacteria are prepared as follows: the recombinant engineered bacteria containing the cytochrome P450 BM3 mutant encoding gene are inoculated into LB culture medium containing kanamycin at a final concentration of 30 μg / mL, and cultured at 37°C for 8 hours to obtain a seed solution; the seed solution is then inoculated into a sterile TB liquid culture medium containing kanamycin at a final concentration of 30 μg / mL at an inoculum volume concentration of 0.5%-5% (preferably 1%), and cultured at 37°C for about 16-24 hours until the bacterial concentration OD600 is 0.4-0.8, and then the culture solution is added. Isopropylthio-β-D-galactopyranoside (IPTG) and 0.2-0.3 mM (preferably 0.3 mM) of δ-aminolevulinic acid (δ-ALA) were used to induce expression at 20°C for 24-48 hours. Wet cells were then harvested by centrifugation at 4000 rpm for 10-20 minutes at 4°C. LB liquid medium consisted of 10 g / L peptone, 5 g / L yeast extract, and 10 g / L sodium chloride in deionized water, pH 7.0. TB liquid medium consisted of 2% tryptone, 2.4% yeast extract, 72 mM K2HPO4, 17 mM KH2PO4, and 0.4% glycerol.

[0049] The P450 BM3 mutant of the present invention is catalyzed by using a crude enzyme solution obtained by cell disruption or an isolated and purified enzyme. In addition, the P450 BM3 mutant can also be prepared into an immobilized enzyme or an enzyme in the form of immobilized cells using a specific immobilization technology.

[0050] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) By modifying the key amino acids in the active center of P450BM3, a P450BM3 mutant with high degradation efficiency and high selectivity that can catalyze the oxidation of the pollutant 2-chlorophenol is obtained, which overcomes the problems of low degradation efficiency and poor selectivity of 2-chlorophenol by existing means, and can be efficiently degraded at room temperature without the need for external temperature control equipment. It has the advantages of being green and low-carbon, and has stronger application significance in engineering practice; (2) It is the first to introduce the directed evolution method to improve the degradation efficiency of cytochrome P450 BM3 into the development of 2-chlorophenol degradation technology, and for the first time construct the optimal mutant R47L / Y51F / F81I / A82M / A184I / A330L / I401L. The P450BM3 mutant has a 2-chlorophenol degradation efficiency of 65.2% within 2 hours, which is higher than that of the wild-type P450 BM3 increased by 5.2 times, producing a single degradation product 2-chlorohydroquinone (product selectivity> 99%), which provides a way to achieve efficient and precise biodegradation of the difficult-to-degrade toxic organic pollutant 2-chlorophenol, and provides a reference for the efficient biodegradation of pollutants with similar structures; (3) The mutant R47L / Y51F / F81I / A82M / A184I / A330L / I401L, its K m and K cat 1.24 mM and 0.017 s, respectively -1 . BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Gas chromatograms of 2-chlorophenol standard (a), 2-chlorohydroquinone standard (b), and the reaction solution after degradation of 2-chlorophenol by the P450 BM3 mutant (c) (retention times: Rt(2-chlorophenol)=4.167min, Rt(2-chlorohydroquinone)=7.835min);

[0052] Figure 2 This is the effect diagram of iterative saturation mutation degradation of 2-chlorophenol to produce 2-chlorohydroquinone;

[0053] Figure 3 This is a graph showing the degradation kinetics of 2-chlorophenol by the mutant R47L / Y51F / F81I / A82M / A184I / A330L / I401L;

[0054] Figure 4 The figure shows the effect of the mutant R47L / Y51F / F81I / A82M / A184I / A330L / I401L on the degradation of 2-chlorophenol to produce 2-chlorohydroquinone at different pH values;

[0055] Figure 5This is a diagram showing the effect of the P450 BM3 mutant R47L / Y51F / F81I / A82M / A184I / A330L / I401L degrading 2-chlorophenol to produce 2-chlorohydroquinone at different temperatures. DETAILED DESCRIPTION

[0056] The technical solution of the present invention will be further described below in conjunction with embodiments.

[0057] Example 1

[0058] 1. Construction of pET28a(+)-P450 BM3 plasmid

[0059] The cytochrome P450 BM3 gene from Priestia megaterium (PDB ID: 1FAG) was synthesized by Jinweizhi (Suzhou) and constructed on the pET28a(+) vector (provided by Jinweizhi (Suzhou)). The constructed plasmid was transformed into competent E. coli DH5α cells (purchased from Shenzhen Kangti Life Science Technology Co., Ltd.). The transformed mixture was evenly spread on a plate containing LB solid medium and incubated upside down at 37°C for 16 hours. A single colony was picked and inoculated into 2 mL of sterile LB liquid medium. After incubation at 37°C and 150 rpm for 8–12 hours, the pET28a(+)-P450 BM3 plasmid was extracted from E. coli DH5α using a column-based plasmid extraction kit and used as a template for iterative saturation mutagenesis.

[0060] Example 2: Construction of P450 BM3 Iterative Saturation Library

[0061] According to the conclusion of Example 1, primers were designed based on the gene sequence of wild-type cytochrome P450 BM3 included in PDB (amino acid sequence shown in SEQ ID NO. 1, nucleotide sequence shown in SEQ ID NO. 2) (see Table 1). The parental P450 BM3 gene (nucleotide sequence is SEQ ID NO. 2) was subjected to site-directed saturation mutagenesis using primers R47NNK-F / R47NNK-R, Y51NNK-F / Y51NNK-R, F81NNK-F / F81NNK-R, F81I_A82NNK-F / F81I_A82NNK-R, A184NNK-F / A184NNK-R, A330NNK-F / A330NNK-R, and I401NNK-F / I401NNK-R (NNK degenerate codons cover 32 codon combinations (N=A / C / G / T, K=G / T) and can encode all 20 amino acids). pET-28a(+) was used as the expression vector to obtain mutant plasmids carrying the target genes, and the mutant plasmids carrying the target genes were transformed into E. coli. Mutants of recombinant bacteria containing the P450 BM3 mutant gene were obtained in BL21(DE3). The obtained recombinant bacterial mutants were screened for 2-chlorophenol degradation effects according to the method of Example 3. The selected best mutant R47L was used as a template, and the NNK primers Y51NNK-F / Y51NNK-R, F81NNK-F / F81NNK-R, F81I_A82NNK-F / F81I_A82NNK-R, A184NNK-F / A184NNK-R, A330NNK-F / A330NNK-R, and I401NNK-F / I401NNK-R with active sites having beneficial effects were used for iterative saturation mutagenesis. pET-28a(+) was used as the 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 double mutants of recombinant bacteria containing the P450 BM3 mutant gene. The recombinant bacterial mutants obtained in the second round were screened for 2-chlorophenol degradation effects according to the method of Example 3, and the best double-mutated recombinant bacterial mutant R47L+Y51F was selected as the template for the next round of saturation mutagenesis. Similarly, triple, quadruple, pentamutant, and sextumutant with gradually improving 2-chlorophenol degradation effects were obtained, as well as the seven-mutant R47L+Y51F+F81I+A82M+A184I+A330L+I401L with the best degradation effect.

[0062] Table 1: Primer design for construction of P450 BM3 iterative saturation mutagenesis library

[0063]

[0064]

[0065] The PCR amplification system was as follows: 50 μL reaction system;

[0066] ddH2O: 30 μL;

[0067] 10× Buffer: 5 μL;

[0068] dNTP: 5 μL;

[0069] MgSO4: 3 μL;

[0070] DMSO: 2 μL;

[0071] Upstream primer (50 μM): 1.5 μL;

[0072] Downstream primer (50 μM): 1.5 μL;

[0073] KOD enzyme: 1 μL;

[0074] Template DNA (plasmid): 1 μL;

[0075] PCR reaction conditions were as follows: initial denaturation at 95°C for 3 minutes, followed by 30 cycles of 95°C for 20 seconds, 55°C for 10 seconds, and 72°C for 30 seconds, with a final extension at 72°C for 10 minutes, and a stop temperature of 4°C. After verification of the PCR product by 1% agarose gel electrophoresis, 1 μL of DpnI was added to the PCR product and digested at 37°C for 2 hours to remove the template plasmid DNA. Transform into competent cells E. coli DH5α, spread the transformed mixture evenly on a plate containing kanamycin (30 μg / mL) LB solid medium, and culture inverted in a 37°C incubator for 16 hours. Pick a single colony and inoculate it into 2 mL of sterile LB liquid medium (containing kanamycin 30 μg / mL). After culturing at 37°C and 150 rpm for 8-12 hours, use a column plasmid extraction kit to extract the saturated mutation library plasmid from E. coli DH5α, and then transform into E. coli BL21 (DE3) competent cells, spread on an LB plate containing kanamycin (30 μg / mL), and culture at 37°C overnight to obtain the P450 BM3 mutation library. At this time, many single colonies with different mutations appear on the LB plate, and these single colonies are used for subsequent screening of the mutation library.

[0076] The parent strain: E. coli BL21 (DE3)-P450 BM3 WT was constructed using the same method.

[0077] Example 3: Screening of P450 BM3 mutant library

[0078] S1. Screening of P450 BM3 mutant library Using P450 BM3 WT as a reference, single colony clones (mutation library constructed by Example 2) were picked and cultured in 1 mL deep 96-well plates. 400 μL of LB culture medium containing a final concentration of 30 μg / mL kanamycin was added in advance. At the same time, three parent strains were picked and cultured in the last three wells of the 96-well plate as controls. The 1 mL 96-well plate was placed at 37 ° C and cultured for 8 h as seed liquid. Then, 100 μL of seed liquid was added to a new 2 mL deep 48-well plate for culture. Sterile TB culture medium containing a final concentration of 30 μg / mL kanamycin was added in advance. After being cultured at 37 ° C for 24 h, IPTG at a final concentration of 40 μM and 0.3 mM δ-aminolevulinic acid (δ-ALA) were added. After induced expression at 20 ° C for 48 h, the supernatant was discarded and the wet cells were collected for the next step of screening. TB liquid medium formula: 2% tryptone, 2.4% yeast extract, 72 mM K2HPO4, 17 mM KH2PO4, 0.4% glycerol.

[0079] S2. Add 500 μL of 200 mM KPi buffer to each well, resuspend the cells, and ultrasonically disrupt them for 3 minutes at 4°C, with an ultrasonic power of 65% and ultrasound on for 2 seconds and off for 6 seconds. Then centrifuge at 4000 rpm for 5 minutes at 4°C, and add glucose to a final concentration of 100 mM, glucose dehydrogenase to a final concentration of 5 mg / mL, 2-chlorophenol to a final concentration of 1 mM, and NADP to a final concentration of 80 μM. + Then, the mixture was reacted at 25°C and 400 rpm for 2 h, extracted with 500 μL of ethyl acetate, centrifuged at 12,000 rpm for 1 min, and 300 μL of the supernatant was detected by gas chromatography.

[0080] Gas phase analysis conditions: Agilent-8860GC and Cyclosil-B column, gas phase program: 60℃ to 250℃ at 20℃ / min, hold for 4 minutes. Retention time: Rt(2-chlorophenol) = 4.167min, Rt(2-chlorohydroquinone) = 7.835min ( Figure 1 The amount of 2-chlorohydroquinone produced by the wild type and the remaining amount of 2-chlorophenol were used as controls. The degradation data of 2-chlorophenol by the mutants are shown in Tables 2 and Figure 2 .

[0081] Table 2 Degradation effect of P450 BM3 mutant on 2-chlorophenol

[0082]

[0083]

[0084] From Table 2 and Figure 2 The data showed that all mutants were more effective than wild-type P450BM3 at degrading 2-chlorohydroquinone. The optimal mutant, R47L / Y51F / F81I / A82M / A184I / A330L / I401L, produced 65.2% of 2-chlorohydroquinone from 2-chlorophenol within 2 hours, 5.2 times that of wild-type P450 BM3.

[0085] Example 4: Degradation kinetics of 2-chlorophenol by mutant R47L / Y51F / F81I / A82M / A184I / A330L / I401L

[0086] According to the conclusion of Example 3, the mutant E. coli BL21 (DE3) -R47L / Y51F / F81I / A82M / A184I / A330L / I401L obtained by the above screening was inoculated into a 10 mL sterile test tube containing LB medium containing a final concentration of 30 μg / mL kanamycin in advance, and cultured at 37 ° C. 150 rpm shaking for 6-8 h, and then added to a 2 L conical flask according to a 1% inoculum size. 1 L of sterile TB medium containing a final concentration of 30 μg / mL kanamycin was added in advance, and cultured at 37 ° C. for 24 h, and then IPTG with a final concentration of 40 μM and 0.3 mM δ-aminolevulinic acid (δ-ALA) were added. After induced expression at 20 ° C. for 48 h, the supernatant was discarded and the wet cells were collected. Add 20mL of 200mM KPi buffer to the wet cells to resuspend the cells, and ultrasonically disrupt them for 5 minutes at 4℃, with an ultrasonic power of 65%, with ultrasound on for 2s and off for 6s. Centrifuge at 4℃ and 12000rpm for 15 minutes, take 200μL of the supernatant to determine the P450 concentration, and add 200mM KPi buffer to dilute the P450 concentration to 20μM. Take 5mL of the diluted supernatant in a 10mL glass reaction bottle, add glucose at a final concentration of 100mM, glucose dehydrogenase at a final concentration of 5mg / mL, 2-chlorophenol at a final concentration of 1mM, and NADP at a final concentration of 80μM. + , put in a magnetic stirring bar, stir and mix using a magnetic stirrer, react at 25°C and 400 rpm for 120 min, take 500 μL of the supernatant after the reaction at 5 min, 10 min, 20 min, 60 min, and 120 min, extract with 500 μL of ethyl acetate, then centrifuge at 12000 rpm for 1 min, take 300 μL of the supernatant and detect by gas chromatography.

[0087] The degradation kinetics curve of the P450 BM3 mutant R47L / Y51F / F81I / A82M / A184I / A330L / I401L was obtained ( Figure 4), as the reaction proceeded, 2-chlorophenol was degraded and its concentration gradually decreased, while the concentration of the degradation product 2-chlorohydroquinone gradually increased, and the yield reached 65.2% at 120 min.

[0088] Example 5: Screening of the optimal pH for degradation of 2-chlorophenol by mutant R47L / Y51F / F81I / A82M / A184I / A330L / I401L

[0089] According to the conclusion of Example 4, the mutant E.coli BL21 (DE3) -R47L / Y51F / F81I / A82M / A184I / A330L / I401L obtained by the above screening was inoculated into a sterile test tube of 10mL LB medium containing a final concentration of 30μg / mL kanamycin in advance, and cultured in a shaking table at 37°C, 150rpm for 6-8h. Then, according to an inoculum size of 1%, a 2L conical flask was added and cultured. 1L of sterile TB medium containing a final concentration of 30μg / mL kanamycin was added in advance. After being placed under 37°C and cultured for 24h, IPTG with a final concentration of 40μM and 0.3mM δ-aminolevulinic acid (δ-ALA) were added. After induced expression at 20°C for 48h, the supernatant was discarded and the wet cells were collected. 20mL 200mM KPi buffer was added to the wet cells to resuspend the cells. 1 mL of the resuspended mixture was added to each of 5 2 mL sterile centrifuge tubes, and the mixture was centrifuged at 4000 rpm for 10 min at 4°C. The supernatant was discarded, and 900 μL of buffer solutions of different pH values ​​(sodium hydrogen phosphate-citrate buffer at pH = 5, sodium hydrogen phosphate-citrate buffer at pH = 6, KPi buffer at pH = 7.5, sodium hydrogen phosphate-citrate buffer at pH = 8, and borate buffer at pH = 10) were added to each of the 5 centrifuge tubes to resuspend the bacterial solution. The cells were ultrasonically disrupted at 4°C for 3 min, with an ultrasonic power of 65%, with the ultrasound on for 2 s and off for 6 s. The cells were centrifuged at 12000 rpm for 1 min at 4°C. 500 μL of the supernatant was added to each of 5 5 mL glass reaction bottles, and then glucose at a final concentration of 100 mM, glucose dehydrogenase at a final concentration of 5 mg / mL, 2-chlorophenol at a final concentration of 1 mM, and NADP at a final concentration of 80 μM were added, respectively. + , stir with a magnetic stirrer and react at 25°C, 400 rpm for 2 h. Take 500 μL of the reaction solution from each reaction bottle, extract with 500 μL of ethyl acetate, centrifuge at 12,000 rpm for 1 min, and collect 300 μL of the supernatant for gas chromatography.

[0090] The experimental results are as follows Figure 4As shown, the optimum pH of the mutant R47L+Y51F+F81I+A82M+A184I+A330L+I401L is 7.5, and it can maintain good activity in the pH range of 6 to 8, with a degradation rate of 2-chlorophenol greater than 50%.

[0091] Example 6: Screening of the optimal temperature for degradation of 2-chlorophenol by the mutant R47L / Y51F / F81I / A82M / A184I / A330L / I401L

[0092] According to the conclusion of Example 4, the mutant E.coli BL21 (DE3) -R47L / Y51F / F81I / A82M / A184I / A330L / I401L obtained by the above screening was inoculated into a sterile test tube of 10mL LB medium containing a final concentration of 30μg / mL kanamycin in advance, and cultured on a shaking table at 37°C, 150rpm for 6-8h. Then, a 2L conical flask was added according to a 1% inoculum size to culture. 1L of sterile TB medium containing a final concentration of 30μg / mL kanamycin was added in advance. After being cultured at 37°C for 24h, IPTG at a final concentration of 40μM and 0.3mM δ-aminolevulinic acid (δ-ALA) were added. After induced expression at 20°C for 48h, the supernatant was discarded and the wet cells were collected. 20mL of 200mM KPi buffer, pH 7.5, was added to the wet cells to resuspend the cells. Ultrasonic disruption was performed at 4°C for 5 minutes, with an ultrasonic power of 65%, with ultrasound on for 2 seconds and off for 6 seconds. The cells were then centrifuged at 4°C at 12,000 rpm for 15 minutes. 200 μL of the supernatant was taken to determine the P450 concentration, and 200 mM KPi buffer at pH 7.5 was added to dilute the P450 concentration to 20 μM. 1 mL of the diluted supernatant was taken into four 5 mL glass reaction bottles, and glucose was added to a final concentration of 100 mM, glucose dehydrogenase to a final concentration of 5 mg / mL, 2-chlorophenol to a final concentration of 1 mM, and NADP to a final concentration of 80 μM. + , put in a magnetic stirring bar, stir and mix using a magnetic stirrer, react at 20℃, 25℃, 37℃, and 45℃ at 400rpm for 2h, take 500μL of reaction solution, extract with 500μL of ethyl acetate, then centrifuge at 12000rpm for 1min, take 300μL of supernatant and detect by gas chromatography.

[0093] The experimental results are as follows Figure 5As shown in the results, different temperatures have a significant effect on the catalytic effect of the mutant R47L+Y51F+F81I+A82M+A184I+A330L+I401L. The optimum temperature is 25°C, which means it has the highest degradation activity at room temperature. Increasing or decreasing the temperature will reduce the activity of the mutant. This enables the mutant to efficiently degrade 2-chlorophenol at room temperature without the need for external temperature control equipment, saving energy, and having the characteristics of being green and low-carbon.

[0094] Example 7: Determination of Michaelis-Menten kinetic parameters for degradation of 2-chlorophenol by mutant R47L / Y51F / F81I / A82M / A184I / A330L / I401L

[0095] According to the conclusion of Example 4, the mutant E.coli BL21 (DE3) -R47L / Y51F / F81I / A82M / A184I / A330L / I401L obtained by the above screening was inoculated into a sterile test tube of 10mL LB medium containing a final concentration of 30μg / mL kanamycin in advance, and cultured in a shaking table at 37°C, 150rpm for 6-8h. Then, according to an inoculum size of 1%, a 2L conical flask was added and cultured. 1L of sterile TB medium containing a final concentration of 30μg / mL kanamycin was added in advance. After being placed under 37°C and cultured for 24h, IPTG with a final concentration of 40μM and 0.3mM δ-aminolevulinic acid (δ-ALA) were added. After induced expression at 20°C for 48h, the supernatant was discarded and the wet cells were collected. 20mL 200mM KPi buffer was added to the wet cells to resuspend the cells. Ultrasonic disruption was performed at 4°C for 5 minutes, with an ultrasonic power of 65%, with ultrasound on for 2 seconds and off for 6 seconds. Centrifugation was then carried out at 4°C at 12000 rpm for 15 minutes. 200 μL of the supernatant was taken to determine the P450 concentration, and 200 mM KPi buffer was added to dilute the P450 concentration to 20 μM. 1 mL of the diluted supernatant was taken into 5 5 mL glass reaction bottles, and glucose with a final concentration of 100 mM, glucose dehydrogenase with a final concentration of 5 mg / mL, different concentrations of 2-chlorophenol, and 80 μM NADP were added. + 2-Chlorophenol was added to 5 glass reaction bottles with final concentrations of 0.2mM, 0.25mM, 0.5mM, 1.0mM, and 2mM, respectively. The reaction was stirred at 25℃ and 400rpm for 30min. 500μL of reaction solution was taken from each reaction bottle, extracted with 500μL of ethyl acetate, and then centrifuged at 12000rpm for 1min. 300μL of supernatant was taken for gas chromatography detection. The enzymatic reaction rate was determined, and a double reciprocal curve was drawn based on the reciprocal of the reaction rate and substrate concentration to calculate the Michaelis-Menten kinetic parameters. The Michaelis-Menten kinetic parameters of P450 BM3 for 2-chlorophenol were determined to be K, m =1.24mM, K cat＝0.017s -1 。

Claims

1. A cytochrome P450BM3 mutant, characterized in that The mutant is a mutant in which at least three amino acids are replaced at positions 47, 51, 81, 82, 184, 330 and 401 of the amino acid sequence shown in SEQ ID No. 1; the replacement of the mutant is selected from the following combinations: R47L + Y51F + F81I, R47L + Y51F + F81I + A82M, R47L + Y51F + F81I + A82K, R47L + Y51F + F81I + A82M + A184I, R47L + Y51F + F81I + A82M + A330L or R47L + Y51F + F81I + A82M + A330L + I401L.

2. A gene encoding the cytochrome P450BM3 mutant 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 according to claim 2 or the recombinant plasmid according to claim 3.

6. The recombinant bacterium according to claim 5, characterized in that The host 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 degrading 2-chlorophenol.

8. The use according to claim 7, characterized in that The application comprises the following steps: using a cytochrome P450BM3 mutant as a catalyst and 2-chlorophenol as a substrate to catalyze 2-chlorophenol to generate 2-chlorohydroquinone.

9. The use according to claim 8, characterized in that The catalytic reaction conditions are: pH 6-8, temperature 20-37°C.

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