Non-cofactor dependent p450bm3 mutants and their use in degrading lignin-derived phenolics

Abstract

The present application relates to non-cofactor-dependent P450BM3 mutants and their application in degrading lignin-derived phenols, belonging to the field of enzymology, and provides three new mutants, specifically F87A / T268D / V78A, F87A / T268D / A328H and F87A / T268D / A328H / V78A, wherein wild-type P450BM3 has almost no degrading function on lignin-derived phenols, and the mutants T268D, T268D / A328F, F87A / T268D, F87A / T268D / V78A, F87A / T268D / A328F, F87A / T268D / A328H, F87A / T268D / A328H / V78A or F87A / T268D / A328F / V78A at a series of sites based on the wild-type P450BM3 have degrading function on lignin-derived phenols.

Description

Technical Field

[0001] This invention belongs to the field of enzymology, specifically relating to a non-cofactor-dependent P450B M3 mutant and its application in the degradation of lignin-derived phenols. Background Technology

[0002] Lignin is one of the most abundant aromatic biopolymers in nature, accounting for 30% of the biosphere's organic carbon. It plays a crucial role in plant physiology, including structural integrity, water conduction, and defense against biotic and abiotic stresses. Industrially, approximately 50 million tons of lignin are produced annually as a byproduct of the pulp and paper industry. The inefficient stabilization of lignin is both a loss of renewable carbon resources and a driver of serious environmental problems, particularly in the pulp and paper industry. The high color and toxicity of industrial wastewater are primarily attributed to lignin-derived aromatic compounds (LDACs), which degrade water quality, threaten aquatic life, and exhibit strong resistance to traditional remediation strategies. Among LDACs, eugenol, isoeugenol, and coniferyl alcohol are of particular concern due to their persistence and biotoxicity. Coniferyl alcohol is a representative G-type lignin monomer, while eugenol and isoeugenol are phenylpropane derivatives with one or two methoxy substituents. These compounds are widely distributed in essential oils and are recognized for their persistence and adverse biological effects. Specifically, eugenol has been shown to cause liver toxicity and nerve damage, while isoeugenol, commonly used as a fish anesthetic, is associated with skin irritation in humans.

[0003] Traditional physicochemical methods for LDAC degradation, such as metal catalysis and pyrolysis, typically require harsh reaction conditions and have limited sustainability. Furthermore, these processes suffer from low efficiency, high cost, excessive energy consumption, and environmental incompatibility, highlighting the need for more environmentally friendly and practical alternatives. Biodegradation using microorganisms or enzymes offers a more environmentally friendly approach. However, microbial systems tend to be kineticly slow, have long reaction cycles, and limited scalability. In contrast, enzymes exhibit higher catalytic selectivity and can operate efficiently under mild conditions, making them attractive candidates for LDAC remediation. Overall, these drawbacks underscore the urgent need for more efficient, selective, and scalable oxidase systems capable of addressing the persistence and toxicity issues of LDACs.

[0004] Cytochrome P450BM3 (CYP102A1) is an attractive monooxygenase, but its strict dependence on the NAD(P)H cofactor and narrow substrate range limit its practical application. While low-cost H2O2 is an ideal alternative to the expensive cofactor NAD(P)H driving P450 catalysis, its bypass pathway is inefficient or absent in most natural enzymes, fundamentally limiting the application of P450 enzymes. Given the enormous potential of peroxidases as practical biocatalysts, the academic community has been dedicated to developing H2O2-driven P450 systems. Currently, there are two main strategies: one is the recently reported artificial system based on P450BM3, which introduces a bifunctional small molecule (DFSM) to achieve both anchoring and acid-base catalytic functions within the active site. However, the complex and costly synthesis of DFSM limits its application. The second approach is to directly introduce acid-base residues into myoglobin or P450 through site-directed mutagenesis, thereby converting it into a catalyst with peroxidase or peroxidase activity.

[0005] While these designs have demonstrated the feasibility of proof-of-concept, they are still limited by auxiliary molecule requirements, synthetic complexity, and poor scalability, which restricts their industrial relevance. Summary of the Invention

[0006] To address the technical problems existing in the prior art, this invention provides a cofactor-independent P450BM3 mutant and its application in the oxidative degradation of lignin-derived phenols. This invention employs a multi-site engineering strategy (F87, T268, A328, V78) to construct a cofactor-independent P450BM3 peroxidase capable of rapidly and nearly completely degrading lignin-derived aromatics under mild conditions. HPLC-MS pathway analysis revealed the formation of possible degradation products such as vanillin and caffeic acid, highlighting the dual potential of this system in the detoxification of lignin pollutants and the biotransformation of lignin in papermaking wastewater.

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

[0008] A non-cofactor-dependent P450BM3 mutant, wherein the mutant is F87A / T268D / V78A, F87A / T268D / A328H, or F87A / T268D / A328H / V78A; wherein F87A / T268D / V78A is based on the amino acid sequence of SEQ ID NO.1, where phenylalanine at position 87 is mutated to alanine, threonine at position 268 is mutated to aspartic acid, and valine at position 78 is mutated to alanine;

[0009] The F87A / T268D / A328H is based on the amino acid sequence of SEQ ID NO.1, where phenylalanine at position 87 is mutated to alanine, threonine at position 268 is mutated to aspartic acid, and alanine at position 328 is mutated to histidine.

[0010] The F87A / T268D / A328H / V78A is based on the amino acid sequence of SEQ ID NO.1, where phenylalanine at position 87 is mutated to alanine, threonine at position 268 is mutated to aspartic acid, alanine at position 328 is mutated to histidine, and valine at position 78 is mutated to alanine.

[0011] This invention also provides the application of the non-cofactor-dependent P450BM3 mutant in the degradation of lignin-derived phenols, wherein the mutant is T268D, T268D / A328F, F87A / T268D, F87A / T268D / V78A, F87A / T268D / A328F, F87A / T268D / A328H, F87A / T268D / A328H / V78A or F87A / T268D / A328F / V78A;

[0012] The T268D is a mutation of threonine at position 268 to aspartic acid based on the amino acid sequence of SEQ ID NO.1;

[0013] The T268D / A328F is based on the amino acid sequence of SEQ ID NO.1, where threonine at position 268 is mutated to aspartic acid and alanine at position 328 is mutated to phenylalanine.

[0014] The F87A / T268D is based on the amino acid sequence of SEQ ID NO.1, where phenylalanine at position 87 is mutated to alanine and threonine at position 268 is mutated to aspartic acid.

[0015] The F87A / T268D / V78A is based on the amino acid sequence of SEQ ID NO.1, where phenylalanine at position 87 is mutated to alanine, threonine at position 268 is mutated to aspartic acid, and valine at position 78 is mutated to alanine.

[0016] The F87A / T268D / A328F is based on the amino acid sequence of SEQ ID NO.1, where phenylalanine at position 87 is mutated to alanine, threonine at position 268 is mutated to aspartic acid, and alanine at position 328 is mutated to phenylalanine.

[0017] The F87A / T268D / A328H is based on the amino acid sequence of SEQ ID NO.1, where phenylalanine at position 87 is mutated to alanine, threonine at position 268 is mutated to aspartic acid, and alanine at position 328 is mutated to histidine.

[0018] The F87A / T268D / A328H / V78A is based on the amino acid sequence of SEQ ID NO.1, where phenylalanine at position 87 is mutated to alanine, threonine at position 268 is mutated to aspartic acid, alanine at position 328 is mutated to histidine, and valine at position 78 is mutated to alanine.

[0019] The F87A / T268D / A328F / V78A is based on the amino acid sequence of SEQ ID NO.1, where phenylalanine at position 87 is mutated to alanine, threonine at position 268 is mutated to aspartic acid, alanine at position 328 is mutated to phenylalanine, and valine at position 78 is mutated to alanine.

[0020] Furthermore, the application method involves using H2O2 as an oxidant and employing a P450BM3 peroxygenase system to biodegrade three common lignin derivatives: eugenol, isoeugenol, and coniferol.

[0021] The nucleotide sequence of WT P450BM3 is (SEQ ID NO.1):

[0022]

[0023] The degradation performance of WT P450BM3 and its mutants on three lignin derivatives was tested using high performance liquid chromatography (HPLC). Within 3 min, the F87A / T268D / A328F / V78A mutants showed degradation rates of 99.38%, 99.69%, and 99.25% for eugenol, isoeugenol, and coniferyl alcohol, respectively.

[0024] The catalytic efficiency of P450BM3 and its series of mutants for lignin derivatives was determined. The results showed that the F87A / T268D / A328H mutant exhibited excellent catalytic efficiency for eugenol and isoeugenol. k cat / K m The values ​​are 13808.63 M. -1 s -1 and 16129.42 M -1 s -1 The F87A / T268D / A328F / V78A mutant exhibits excellent catalytic efficiency for coniferyl alcohol. k cat / K m The value is 14657.83 M -1 s -1 .

[0025] The beneficial effects of this invention compared to the prior art are as follows:

[0026] This invention successfully developed a novel biocatalyst based on cytochrome P450BM3 and its various mutants, providing a new and efficient pathway for the degradation of lignin derivatives.

[0027] This invention clarifies the highly efficient catalytic effect of various mutants on different lignin derivatives, providing a scientific basis for selection and optimization in practical applications;

[0028] The expression vector containing mutants and the genetically engineered bacteria constructed in this invention provide a foundation for large-scale production and application, which is conducive to the promotion and popularization of the technology.

[0029] The degradation reaction system of this invention is simple and the conditions are mild, which reduces the difficulty and cost of operation and has good practical application feasibility.

[0030] This invention provides an innovative, efficient, and economically feasible solution for the pollution control of lignin derivatives in papermaking wastewater environments, which is of great significance for protecting ecosystems and human health. Attached Figure Description

[0031] Figure 1SDS-PAGE electrophoresis images of WT P450BM3 and a series of mutant proteins;

[0032] Figure 2 The degradation rate of eugenol by the P450BM3 series mutants;

[0033] Figure 3 The degradation rate of isoeugenol by the P450BM3 series mutants;

[0034] Figure 4 The degradation rate of coniferyl alcohol by the P450BM3 series mutants;

[0035] Figure 5A The image shows the UV absorption spectra of WT P450BM3 before and after the addition of Na2S2O4 to form the CO complex (black line: oxidized protein; red curve: reduced ferrous-CO complex).

[0036] Figure 5B The image shows the UV absorption spectra of T268D before and after the addition of Na2S2O4 to form the CO complex (black line: oxidized protein; red curve: reduced ferrous-CO complex).

[0037] Figure 5C The image shows the UV absorption spectra of T268D / A328F before and after the addition of Na2S2O4 to form a CO complex (black line: oxidized protein; red curve: reduced ferrous-CO complex).

[0038] Figure 5D The image shows the UV absorption spectra of F87A / T268D before and after the addition of Na2S2O4 to form a CO complex (black line: oxidized protein; red curve: reduced ferrous-CO complex).

[0039] Figure 5E The image shows the UV absorption spectra of F87A / T268D / V78A before and after the addition of Na2S2O4 to form a CO complex (black line: oxidized protein; red curve: reduced ferrous-CO complex).

[0040] Figure 5F The image shows the UV absorption spectra of F87A / T268D / A328F before and after the addition of Na2S2O4 to form a CO complex (black line: oxidized protein; red curve: reduced ferrous-CO complex).

[0041] Figure 5G The image shows the UV absorption spectra of F87A / T268D / A328H before and after the addition of Na2S2O4 to form a CO complex (black line: oxidized protein; red curve: reduced ferrous-CO complex).

[0042] Figure 5HThe image shows the UV absorption spectra of F87A / T268D / A328H / V78A before and after the addition of Na2S2O4 to form a CO complex (black line: oxidized protein; red curve: reduced ferrous-CO complex).

[0043] Figure 5I The image shows the UV absorption spectra of F87A / T268D / A328F / V78A before and after the addition of Na2S2O4 to form a CO complex (black line: oxidized protein; red curve: reduced ferrous-CO complex). Detailed Implementation

[0044] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0045] Example 1: P450BM3 site-directed mutagenesis:

[0046] (1) Design of mutation sites: Based on previous research and theoretical analysis, the 268, 328, 87 and 78 amino acid sites that may improve the catalytic activity of P450BM3 on lignin derivatives were identified. Table 1 lists the primers used for each site.

[0047] Table 1. Mutant Primer Sequences

[0048] ;

[0049] ;

[0050] Note: The underlined part is the primer sequence corresponding to the mutation site.

[0051] (2) Polymerase chain reaction (PCR) site-directed mutagenesis: The total reaction volume was 25 μL, which included 12.5 μL of Q5 hot-start ultra-fidelity 2×Master Mix, 1.25 μL of 10 μM forward primer, 1.25 μL of 10 μM reverse primer, 1 μL of template DNA, and 9 μL of sterile water. The above components were added to a sterile centrifuge tube, placed in a PCR instrument, and the amplification reaction was performed according to the preset program.

[0052] (3) KLD ligation reaction: KLD ligation reaction of ring-opening plasmid was carried out according to a specific reaction system. The reaction system was 10 μL. After the reaction was completed, it was placed at room temperature for 5 min.

[0053] (4) Plasmid transformation and bacterial culture acquisition: 10 μL of KLD product was added to 100 μL of DH5α competent cells, mixed well, and then incubated on ice for 30 min, followed by heat shock at 42℃ for 30 s, and then quickly transferred to ice and allowed to stand for 5 min. Then, 1 mL of antibiotic-free LB liquid medium was added, and the culture was carried out at 37℃ and 180 rpm for 1 h. After incubation, the bacterial culture was centrifuged at 12000 rpm for 1 min, 900 μL of supernatant was discarded, and the remaining bacterial culture was mixed and spread on LB agar plates containing 50 μg / mL kanamycin, and incubated at 37℃ for 12 h. Single colonies were picked from the plates and inoculated into 5 mL of LB liquid medium containing kanamycin, and incubated at 37℃ and 180 rpm for 12 h to obtain cells containing the P450BM3 plasmid. E. coli DH5α competent cells.

[0054] (5) Gene sequencing of bacterial culture and preservation of bacterial strain: 1 mL of the bacterial culture obtained above was sequenced, and the sequencing results were analyzed using ApE sequence alignment analysis software to screen out bacterial cultures that successfully underwent site-directed mutagenesis. 1000 μL of the bacterial culture that successfully underwent site-directed mutagenesis was mixed with 500 μL of 70% sterile glycerol and placed in an ultra-low temperature freezer at -80℃ for bacterial culture preservation.

[0055] Example 2: Expression of P450BM3 and its series of mutants:

[0056] (1) Plasmid extraction: Using a rapid plasmid extraction kit, plasmids that showed successful mutations after sequence comparison were extracted. E. coli The pET-28a(+)-BM3 recombinant plasmid was extracted from DH5α competent cells, and the operation steps were the same as those for conventional plasmid extraction.

[0057] (2) Plasmid transformation and strain preservation: Take 10 μL of the extracted recombinant plasmid and add it to 100 μL of the strain. E. coli BL21(DE3) competent cells were incubated on ice for 30 min, followed by heat shock at 42°C for 60 s, and then transferred to ice for 5 min. 1 mL of antibiotic-free LB liquid medium was added, and the cells were incubated at 37°C and 180 rpm for 1 h. The bacterial culture was centrifuged at 12000 rpm for 1 min, and 900 μL of the supernatant was discarded. The remaining bacterial culture was spread onto LB agar plates containing 50 μg / mL kanamycin and incubated at 37°C for 12 h. Single colonies from the plates were inoculated into 5 mL of LB liquid medium containing kanamycin and incubated at 37°C and 180 rpm for 12 h to obtain competent cells containing the P450BM3 recombinant plasmid. 1000 μL of the bacterial culture was mixed with 500 μL of 70% sterile glycerol and stored at -80°C.

[0058] (3) Induced expression: The preserved expression was expressed using the streak plate method. E. coli BL21(DE3) strain was activated by inoculation on LB solid medium and cultured at 37°C for 12 h. A single colony was picked from a plate and inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C and 180 rpm for 12 h. 500 μL of this bacterial culture (1% inoculum) was transferred to 50 mL of LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C and 180 rpm for 12 h. Another 5 mL of this bacterial culture (1% inoculum) was transferred to 500 mL of LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C and 200 rpm until the absorbance at 600 nm reached approximately 0.8. Finally, 0.5 mM 5'-Ala and FeCl3 were added sequentially to the bacterial culture, and the culture was incubated at 30°C and 200 rpm for 40 min. Then, 1 mM IPTG was added for further induction for 20 h.

[0059] Example 3: Purification of P450BM3 and its series of mutant proteins:

[0060] (1) Pretreatment before protein purification: The induced bacterial culture was centrifuged at 4℃ and 8000 rpm for 10 min, and the supernatant was discarded to obtain the bacterial cell pellet. The bacterial cell pellet was dissolved and resuspended thoroughly. The above centrifugation operation was repeated, and the bacterial culture was resuspended again. Then, the bacterial culture was sonicated for 15 min (5 s sonication, 15 s cooling) to fully release the intracellular protein. The disrupted bacterial culture was centrifuged at 4℃ and 10000 rpm for 90 min, the pellet was discarded, the supernatant was collected, and the crude protein was obtained after filtration through a 0.22 μm filter membrane.

[0061] (2) Protein purification: Given that the P450BM3 gene has a His tag at the end, His Trap was used. TMHP affinity chromatography column and AKTA explorer 10S protein rapid purification system were used. Dual wavelength detection was performed using the 280 nm protein absorption wavelength and the 418 nm P450 characteristic absorption wavelength. Pretreatment was performed by rinsing the instrument and pump with ultrapure water, solution A (100 mM KPi, 500 mM NaCl, pH 7.4), and solution B (100 mM KPi, 500 mM NaCl, 500 mM imidazole, pH 7.4). For sample loading, equilibration was performed with 100% solution A at a flow rate of 5 mL / min. After baseline stabilization, the flow rate was increased to 3 mL / min, allowing contaminating proteins to flow through and binding to the target protein. After baseline stabilization again, contaminating proteins were eluted with 96% solution A and 4% solution B, followed by rinsing with 100 mL of 94% solution A and 6% solution B. After baseline stabilization again, the concentration of solution B was adjusted to 40% to elute the target protein. The sample was collected when the characteristic peak appeared at 418 nm, indicating that the target protein had been replaced by a high concentration of imidazole. Then, adjust solution B to 100% and rinse, followed by rinsing the system and pump with ultrapure water and 20% ethanol. Dialyze to remove imidazole, first with pure water then with dialysate, for 2 hours each time. Finally, concentrate and remove salt by ultrafiltration, using a 30 kDa filter membrane, add glycerol, and store at -80°C.

[0062] Example 4: Purity detection of P450BM3 and its series of mutant proteins:

[0063] (1) Pretreatment: Dilute the purified protein to 2 μM, then mix it with protein loading buffer (5×) at a volume ratio of 4:1, boil for 10 min to completely denature and depolymerize the protein, and then centrifuge at 10000 rpm for 1 min.

[0064] (2) Sample loading: Insert the pre-cast gel into the electrophoresis tank and add the electrophoresis buffer. Take 5 μL of the supernatant after centrifugation and add it to each lane along with the protein marker.

[0065] (3) Electrophoresis: Set the initial voltage for electrophoresis to 80 V. When the bands run in a straight line, adjust the voltage to 120 V until the bands reach the bottom of the gel and stop electrophoresis.

[0066] (4) Staining: After electrophoresis, remove the gel and stain it with Coomassie brilliant blue staining agent for 2 h.

[0067] (5) Decolorization: After the gel is fully stained, it is placed in a decolorization solution (pure water: ethanol: acetic acid volume ratio of 7:2:1) for 2 hours, and then placed in pure water for decolorization until the desired effect is achieved.

[0068] (6) Observation results: The decolorized gel was observed and analyzed in a gel imaging system. The purity of the target protein was determined by comparing it with the protein marker, and the experimental results were recorded by taking pictures. Figure 1 As shown.

[0069] Example 5: Characterization and concentration determination of P450BM3 and its mutant proteins:

[0070] (1) Characterization of P450BM3 and its mutant proteins: P450BM3 protein was characterized using a UV-Vis spectrophotometer. Wild-type and mutant proteins were diluted to 5 μM. 10 mg of Na2S2O4 was added to 3 mL of the protein dilution buffer, and the mixture was sealed and purged with CO2 for 3 min until saturation. The absorbance in the 390-700 nm wavelength range before and after reduction was measured. Characterization was achieved by comparing the shifts in the positions of characteristic absorption peaks. The results were summarized in […]. Figures 5A-5I .

[0071] (2) Protein concentration determination of P450BM3 and a series of mutants: The protein concentration was indirectly obtained by measuring the concentration of heme cofactor in wild-type and a series of mutants based on the heme binding assay. First, 1.75 mL of pyridine was mixed with 0.75 mL of 1 mol / L NaOH and centrifuged at 8500 rpm for 1 min at room temperature. 0.25 mL of the supernatant was taken and mixed with the protein dilution buffer at a ratio of 3:1 to 1 mL. 2.5-3 mg of Na2S2O4 was added and mixed well. The UV spectrum in the 390-700 nm band was recorded. The blank group only replaced the protein solution. The protein absorbance was calculated based on the average absorbance at 390 nm and 450 nm, and the difference between the absorbance at 418 nm and the average absorbance at 418 nm was the protein absorbance. The protein concentrations of P450BM3 and a series of mutants were summarized in Table 2.

[0072] Table 2 Protein concentrations of P450BM3 and its mutant series

[0073] ;

[0074] .

[0075] Example 6: Degradation rate and kinetics of eugenol by the P450BM3 mutant:

[0076] (1) Degradation rate determination: P450BM3 and a series of mutants were added to the reaction system containing eugenol (substrate concentration 500 μM). After the reaction, the mixture was filtered through a 0.22 μm filter membrane and analyzed by high performance liquid chromatography (HPLC) with a UMISil WatersSunfire™ C18 column (4.6 mm × 250 mm, 5 μm). The column temperature was set at 30 °C, the injection volume was 20 μL, the mobile phase was water and acetonitrile (v:v = 40:60), the flow rate was 1 mL / min, and the detection wavelength was 280 nm. The degradation results are as follows: Figure 2 Under optimal conditions (35°C, pH 7.0, 25 mM H2O2), the degradation rate of the F87A / T268D / A328F / V78A mutant reached 99.38%.

[0077] (2) Kinetic analysis: The total volume of the reaction system was 1 mL, containing eugenol at different concentration gradients and 1.5 μM P450BM3, all dissolved in 50 mM KPi at pH 7.0. After incubation in a metal bath for 5 min, 25 mM H2O2 was added to initiate the reaction. The concentration of eugenol before and after the reaction was determined using high performance liquid chromatography. According to the Michaelis-Menten equation, v / [protein] = k cat [substrate] / ( K m The dynamic parameters are calculated using [substrate]). k cat , K m The catalytic efficiency of the T268D mutant for eugenol was determined. k cat / K m 7986.25 M -1 s -1 The F87A / T268D / A328H mutant k cat / K m 13808.63 M -1 s -1 It is approximately 1.73 times that of the T268D mutant.

[0078] Table 3 Kinetic parameters of eugenol catalysis by P450BM3 and its mutants

[0079] .

[0080] Example 7: Degradation rate and kinetics of isoeugenol by the P450BM3 mutant:

[0081] (1) Degradation rate determination: P450BM3 series mutants were added to the reaction system containing isoeugenol (substrate concentration 500 μM). After the reaction, the mixture was filtered through a 0.22 μm filter membrane and analyzed using high-performance liquid chromatography (HPLC) with a UMISil WatersSunfire™ C18 column (4.6 mm × 250 mm, 5 μm). The column temperature was set at 30℃, the injection volume was 20 μL, the mobile phase was water and acetonitrile (v:v = 40:60), the flow rate was 1 mL / min, and the detection wavelength was 280 nm. The degradation results are as follows: Figure 3Under optimal conditions (30°C, pH 7.0, 20 mM H2O2), the degradation rate of the F87A / T268D / A328F / V78A mutant reached 99.69%.

[0082] (2) Kinetic analysis: The total volume of the reaction system was 1 mL, containing isoeugenol at different concentration gradients and 1.5 μM P450BM3, all dissolved in 50 mM KPi at pH 7.0. After incubation in a metal bath for 5 min, 20 mM H2O2 was added to initiate the reaction. The concentration of isoeugenol before and after the reaction was determined using high performance liquid chromatography. According to the Michaelis-Menten equation, v / [protein] = k cat [substrate] / ( K m The dynamic parameters are calculated using [substrate]). k cat , K m Where v is the reaction rate and [protein] is the enzyme protein concentration. k cat Let be the catalytic constant, [substrate] be the substrate concentration, and K be the Michaelis constant. The catalytic efficiency of the T268D mutant for isoeugenol was determined. k cat / K m 13246.51 M -1 s -1 The F87A / T268D / A328H mutant k cat / K m 16129.42 M -1 s -1 It is approximately 1.22 times that of the T268D mutant;

[0083] Table 4 Kinetic parameters of isoeugenol catalyzed by P450BM3 and its mutants

[0084] .

[0085] Example 8: Degradation rate and kinetics of coniferyl alcohol by the P450BM3 mutant:

[0086] (1) Degradation rate determination: P450BM3 series mutants were added to the reaction system containing coniferyl alcohol (substrate concentration 500 μM). After the reaction, the mixture was filtered through a 0.22 μm filter membrane and analyzed using high-performance liquid chromatography (HPLC) with a UMISil Waters Sunfire™ C18 column (4.6 mm × 250 mm, 5 μm). The column temperature was set at 30℃, the injection volume was 20 μL, the mobile phase was water and acetonitrile (gradient elution), the flow rate was 1 mL / min, and the detection wavelength was 280 nm. The degradation results are as follows: Figure 4 Under optimal conditions (25°C, pH 7.0, 20 mM H2O2), the degradation rate of the F87A / T268D / A328F / V78A mutant reached 99.25%.

[0087] (2) Kinetic analysis: The total volume of the reaction system was 1 mL, containing coniferyl alcohol at different concentration gradients and 1.5 μM P450BM3, all dissolved in 50 mM KPi at pH 7.0. After incubation in a metal bath for 5 min, 20 mM H2O2 was added to initiate the reaction. The concentration of coniferyl alcohol before and after the reaction was determined using high performance liquid chromatography. According to the Michaelis-Menten equation, v / [protein] = k cat [substrate] / ( K m + [substrate]) calculates the dynamic parameters k cat , K m The catalytic efficiency of the T268D mutant for coniferyl alcohol was determined. k cat / K m 12670.26 M -1 s -1 The F87A / T268D / A328F / V78A mutant k cat / K m 14657.83 M -1 s -1 It is approximately 1.16 times that of the T268D mutant;

[0088] Table 5 Kinetic parameters of coniferol catalyzed by P450BM3 and its mutants

[0089] .

[0090] In summary, the P450BM3 mutant provided by this invention exhibits significantly enhanced catalytic performance against lignin derivatives, offering a promising biotechnological approach for the effective degradation of lignin derivatives in papermaking wastewater. Furthermore, the detailed and reproducible embodiments of this invention provide important reference for research and applications in related fields.

Claims

1. A non-cofactor dependent P450BM3 mutant, characterized in that, The mutant is F87A / T268D / V78A, F87A / T268D / A328H and F87A / T268D / A328H / V78A; the F87A / T268D / V78A is based on the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO. 1, the phenylalanine at position 87 is mutated to alanine, the threonine at position 268 is mutated to aspartic acid, and the valine at position 78 is mutated to alanine; The F87A / T268D / A328H is based on the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO. 1, the phenylalanine at position 87 is mutated to alanine, the threonine at position 268 is mutated to aspartic acid, and the alanine at position 328 is mutated to histidine; The F87A / T268D / A328H / V78A is based on the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO. 1, the phenylalanine at position 87 is mutated to alanine, the threonine at position 268 is mutated to aspartic acid, the alanine at position 328 is mutated to histidine, and the valine at position 78 is mutated to alanine; The sequence number of the mutant is counted from the 2nd position of the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO.

1.

2. The use of the non-cofactor-dependent P450BM3 mutant in claim 1 in degrading lignin-derived phenols, which is the use of P450BM3 peroxygenase system to biodegrade eugenol, isoeugenol and / or coniferyl alcohol with H2O2 as oxidant.

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