Cytochrome p450bm3-based peroxygenase mutant v78i / f87g / t268d / a328f and use thereof

Abstract

The present application relates to a kind of overoxygenase mutant V78I / F87G / T268D / A328F based on cytochrome P450BM3 and its application, belong to enzymology field, the mutant uses cytochrome P450BM3 (CYP102A1) of bacillus megaterium origin as skeleton, with T268D as core activation site, and carry out combination mutation in F87, V78, A328, L75, A264 site;The mutant uses H2O2 as only oxidant, without NAD (P) H cofactor, realizes the selective O-demethylation of lignin aromatic monomer, realizes the O-demethylation of different size lignin monomer high efficiency, provides efficient and economical biological catalysis solution for lignin monomer green value-added.

Description

[0001] This patent is a divisional application based on the application filed on July 27, 2026, with application number 2026111141805, entitled "A peroxygenase mutant based on cytochrome P450BM3 and its application". Technical Field

[0002] This invention belongs to the field of biotechnology, specifically relating to a cytochrome P450BM3-based peroxygenase mutant V78I / F87G / T268D / A328F and its applications. Background Technology

[0003] Lignin accounts for 15%–30% of the dry weight of lignocellulose biomass and is the most abundant source of renewable aromatic compounds on Earth. The abundant methoxy groups (–OCH3) in lignin and its depolymerization products (such as guaiacol and syringol, among other aromatic monomers) lead to high oxygen content and uneven reactivity, causing excessive hydrogen consumption and catalyst carbon buildup during subsequent upgrading processes, severely limiting the economic viability of lignin-based chemicals and fuels. Selectively removing methoxy groups from aromatic rings while retaining the benzene ring structure (i.e., demethoxylation) can convert these monomers into high-value platform compounds such as phenols and catechols, which is a key step in upgrading lignin depolymerization products. Therefore, systematic research on the demethoxylation of lignin aromatic monomers is of significant scientific and practical value for developing high-value conversion technologies for lignin.

[0004] Traditional chemical O-demethylation methods rely on Lewis acid reagents such as boron tribromide (BBr3), require anhydrous and low-temperature conditions, and generate numerous byproducts, making them fundamentally incompatible with aqueous biorefining processes. In nature, bacterial cytochrome P450 GcoA / GcoB from Pseudomonas can regioselectively demethylate guaiacol to catechol, but this enzyme is strictly dependent on the NAD(P)H cofactor and multi-component redox chaperone proteins, resulting in high cost and complex operation, limiting cell-free applications. The peroxide bypass pathway offers a cofactor-free alternative: H2O2 can directly generate the active iron-oxygen intermediate (Compound I) without the need for NAD(P)H. Natural peroxygenase-type P450s (such as SPα and OleTJE) utilize this pathway by replacing conserved I-helical threonine residues with carboxylic acid residues. P450BM3 (CYP102A1), derived from Bacillus megaterium, is the fastest-progressing self-sufficient P450 monooxygenase, efficiently expressed in Escherichia coli, making it an ideal chassis for engineering modification. However, its natural I-helical threonine T268 participates in the proton-transfer water network, hindering efficient H2O2 utilization. Previous studies have attempted to convert P450BM3 into a peroxygenase using T268D / N substitution and decoy molecule strategies, but the efficient catalytic mechanism targeting the full range of lignin monomers from guaiacol to eugenol remains unclear, and the catalytic activity of large-volume clove substrates needs further improvement. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a highly efficient P450BM3 peroxygenase mutant that uses H2O2 as the sole oxidant and does not require NAD(P)H cofactors to achieve selective O-demethylation of lignin aromatic monomers. This enables efficient O-demethylation of lignin monomers of different sizes, providing an efficient and economical biocatalytic solution for the green value-added of lignin monomers.

[0006] The technical solution of the present invention is as follows: One objective of this invention is to provide a peroxygenase mutant based on cytochrome P450BM3, said mutant being derived from Bacillus megaterium. Using cytochrome P450BM3 (CYP102A1) derived from megaterium as the backbone, with T268D as the core activation site, and combined mutations at F87, V78, A328, L75, and A264 sites; the mutants include: F87G / T268D, F87G / T268D / A328F, V78F / F87A / T268D / A328F, V78I / F87G / T268D / A328F, L75H / F87G / T268D / A328F, V78A / F87G / T268D / A328F, V78A / F87A / T268D / A328F, or F87G / A264G / T268D / A328F; The F87G / T268D is based on the amino acid sequence shown in SEQ ID NO. 1, where phenylalanine at position 87 is mutated to glycine and threonine at position 268 is mutated to aspartic acid. The F87G / T268D / A328F is based on the amino acid sequence shown in SEQ ID NO.1, where phenylalanine at position 87 is mutated to glycine, threonine at position 268 is mutated to aspartic acid, and alanine at position 328 is mutated to phenylalanine. The V78F / F87A / T268D / A328F is based on the amino acid sequence shown in SEQ ID NO.1, where valine at position 78 is mutated to phenylalanine, 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. The V78I / F87G / T268D / A328F is a mutation in the amino acid sequence shown in SEQ ID NO.1, where valine at position 78 is mutated to isoleucine, phenylalanine at position 87 is mutated to glycine, threonine at position 268 is mutated to aspartic acid, and alanine at position 328 is mutated to phenylalanine. The L75H / F87G / T268D / A328F is a sequence in which leucine at position 75 is mutated to histidine, phenylalanine at position 87 is mutated to glycine, threonine at position 268 is mutated to aspartic acid, and alanine at position 328 is mutated to phenylalanine. The V78A / F87G / T268D / A328F is a mutation in the amino acid sequence shown in SEQ ID NO.1, where valine at position 78 is mutated to alanine, phenylalanine at position 87 is mutated to glycine, threonine at position 268 is mutated to aspartic acid, and alanine at position 328 is mutated to phenylalanine. The V78A / F87A / T268D / A328F is a mutation in the amino acid sequence shown in SEQ ID NO.1, where valine at position 78 is mutated to alanine, 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. The F87G / A264G / T268D / A328F is a sequence in which phenylalanine at position 87 is mutated to glycine, alanine at position 264 is mutated to glycine, threonine at position 268 is mutated to aspartic acid, and alanine at position 328 is mutated to phenylalanine.

[0007] A second objective of this invention is to provide an expression vector containing any one of the aforementioned mutants.

[0008] A third objective of this invention is to provide a genetically engineered bacterium containing the aforementioned expression vector.

[0009] The fourth objective of this invention is to provide an application of the mutant, which uses H2O2 as an oxidant to catalyze the O-demethylation reaction of guaiacol, eugenol or 4-methyleugenol to generate the corresponding catechol products without the need for NAD(P)H cofactor and electron transport protein.

[0010] Furthermore, when catalyzing guaiacol, the optimal mutant was V78F / F87A / T268D / A328F, with a total transformation number (TON) as high as 1607; when catalyzing eugenol, the optimal mutant was V78F / F87A / T268D / A328F, with a TON of 334; and when catalyzing 4-methyleugenol, the optimal mutant was V78I / F87G / T268D / A328F, with a TON of 1135.

[0011] Furthermore, in the aforementioned application, different concentrations of substrate and 1 μM MP450BM3 mutant were added to 50 mM, pH 7.5 potassium phosphate buffer. After incubation, H2O2 was added to start the reaction, and the reaction was terminated with 1 M HCl after 60 min.

[0012] Furthermore, the H2O2 concentration for catalyzing guaiacol was 20 mM, and the optimal temperature was 25℃; the H2O2 concentration for catalyzing eugenol and 4-methyleugenol was 30 mM, the optimal temperature for catalyzing eugenol was 25℃, and the optimal temperature for catalyzing 4-methyleugenol was 30℃.

[0013] The amino acid sequence of WT P450BM3 (SEQ ID NO.1) is as follows: T I K E M P Q P K T F G E L K N L P L L N T D K P V Q A L M K I A D EL G E I F K F E A P G R V T R Y L S S Q R L I K E A C D E S R F D K N L S Q AL K F V R D F A G D G L F T S W T H E K N W K K A H N I L L P S F S Q Q A M KG Y H A M M V D I A V Q L V Q K W E R L N A D E H I E V P E D M T R L T L D TI G L C G F N Y R F N S F Y R D Q P H P F I T S M V R A L D E A M N K L Q R AN P D D P A Y D E N K R Q F Q E D I K V M N D L V D K I I A D R K A S G E Q SD D L L T H M L N G K D P E T G E P L D D E N I R Y Q I I T F L I A G H E T TS G L L S F A L Y F L V K N P H V L Q K A A E E A A R V L V D P V P S Y K Q VK Q L K Y V G M V L N E A L R L W P T A P A F S L Y A K E D T V L G G E Y P LE K G D E L M V L I P Q L H R D K T I W G D D V E E F R P E R F E N P S A I PQ H A F K P F G N G Q R A C I G Q Q F A L H E A T L V L G M M L K H F D F E DH T N Y E L D I K E T L T L K P E G F V V K A K S K K I P L

[0014] The nucleotide sequence of the wild-type P450BM3 (SEQ ID NO. 2) is:

[0015] Compared with the prior art, the present invention has the following beneficial effects: (1) The mutant of the present invention catalyzes the O-demethylation reaction of guaiacol, eugenol or 4-methyleugenol without the need for cofactors. The entire catalytic system only requires H2O2 as an oxidant, without the need for expensive NAD(P)H and electron transport chaperone proteins, which greatly reduces costs and simplifies the operation process. (2) The mutants of the present invention have high activity and high selectivity: the optimal mutant V78F / F87A / T268D / A328F has a TON of up to 1607 for guaiacol, which is about 67 times that of the single mutant T268D; V78I / F87G / T268D / A328F has a TON of 1135 for 4-methyleugenol, which is about 45 times that of the single mutant T268D; (3) The mutant of this invention has a wide substrate range: Through the size adaptive strategy, it achieves efficient catalysis of lignin monomers with a full range of sizes, from guaiacol (small volume) to eugenol (medium volume) and then to 4-methyleugenol (large volume), revealing the volume-dependent engineering law: small volume substrates rely on the precise confinement of rigid aromatic side chains, while large volume substrates rely on the synergistic adaptation of flexible hydrophobic residues and cavity release; (4) The catalytic reaction conditions of the mutant of the present invention are mild, green and scalable: the aqueous system and room temperature operation provide a scalable and environmentally friendly biocatalytic platform for the green value-added of lignin monomers. Attached Figure Description

[0016] Figure 1 The image shows the SDS-PAGE electrophoresis results of WT P450BM3 and a series of mutant proteins, with the target band appearing at approximately 55 kDa for each protein. Figure 2 CO difference spectrum of mutant V78A / F87A / T268D / A328F; Figure 3 The CO difference spectrum of mutant F87A / T268D; Figure 4 The CO difference spectrum of the mutant F87G / T268D; Figure 5 The CO difference spectrum of the mutant T268D / A328F; Figure 6 CO difference spectrum of mutant L75H / F87G / T268D / A328F; Figure 7 CO difference spectrum of mutant V78A / F87G / T268D / A328F; Figure 8CO difference spectrum of mutant F87A / T268D / A328F; Figure 9 The CO difference spectrum of the mutant F87G / T268D / A328F; Figure 10 The CO difference spectrum of the mutant F87G / A264G / T268D / A328F; Figure 11 CO difference spectrum of mutant V78I / F87G / T268D / A328F; Figure 12 The CO difference spectrum of the mutant T268D; Figure 13 CO difference spectrum of mutant V78F / F87A / T268D / A328F; Figure 14 The CO difference spectrum of WTP450BM3; Figure 15 To optimize the reaction temperature of guaiacol, the optimal temperature was found to be 25 °C. Figure 16 To optimize the reaction time of guaiacol, the optimal reaction time was 60 min. Figure 17 To optimize the pH of the guaiacol reaction, the optimal pH was 7.5. Figure 18 To optimize the H2O2 concentration for the guaiacol reaction, the optimal H2O2 concentration was 20 mM. Figure 19 The optimal reaction temperature for eugenol was determined to be 25 °C. Figure 20 To optimize the reaction time of eugenol, the optimal reaction time was found to be 60 min. Figure 21 The optimal pH for the eugenol reaction was 7.5. Figure 22 To optimize the H2O2 concentration for the eugenol reaction, the optimal H2O2 concentration was 30 mM. Figure 23 The optimal reaction temperature for 4-methyleugenol was determined to be 30 °C. Figure 24 The optimal reaction time for 4-methyleugenol was determined to be 60 min. Figure 25 The pH for the 4-methyleugenol reaction was optimized, and the optimal pH was 7.5. Figure 26 The optimal H2O2 concentration for the 4-methyleugenol reaction was found to be 30 mM. Figure 27The HPLC liquid chromatogram of the guaiacol reaction shows that the demethylation product can only be observed when the P450BM3 mutant coexists with H2O2; Figure 28 The HPLC chromatogram of the eugenol reaction shows that the demethylation product can only be observed when the P450BM3 mutant coexists with H2O2; Figure 29 The HPLC chromatogram of the reaction of 4-methyleugenol shows that the demethylation product can only be observed when the P450BM3 mutant coexists with H2O2. Figure 30 The mass spectrometry results for 4-methyleugenol catalyzed by P450BM3 are as follows: the demethoxylated product 3-methoxy-5-methylbenzene-1,2-diol was observed at m / z=153.0585, while the expected m / z=154. Detailed Implementation

[0017] The principles and features of the present invention are further described below. The listed embodiments are only for explaining the present invention and are not intended to limit the scope of protection. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods in the art; the materials and reagents used are commercially available unless otherwise specified.

[0018] Example 1 Construction of mutants Using the recombinant plasmid pET-28a(+)-BMP containing the heme domain gene of wild-type P450BM3 (amino acid sequence as shown in SEQ ID NO.1, nucleotide sequence as shown in SEQ ID NO.2) as a template, the Q5 site-directed mutagenesis kit (New England) was used. Biolabs and the primers listed in Table 1 were used for site-directed PCR mutagenesis, and DNA sequencing confirmed the mutations. The specific mutagenesis strategy is as follows: T268D was used as the core activation mutation to replace threonine at position 268 with aspartic acid, activating the peroxide bypass mechanism; F87A or F87G was used to expand the substrate binding cavity, with F87A reducing the side chain volume to make the methoxy group of the small aromatic ring substrate closer to the iron-oxygen active species, and F87G further expanding the cavity to accommodate large-volume substrates such as eugenol; A328F was used to enhance the hydrophobic binding affinity of the substrate; V78 (V78A / V78I / V78F) was used to fine-tune the internal stereoenvironment and hydrophobicity of the pocket, regulating the orientation of the methoxy group relative to the iron-oxygen species; L75H was used to regulate the polarity of the substrate channel; and A264G was used to optimize the precise orientation of the substrate at the active site.

[0019] The specific mutation involves replacing phenylalanine at position 87 of the amino acid sequence shown in SEQ ID NO.1 with alanine and glycine, valine at position 78 with alanine, phenylalanine, and isoleucine, leucine at position 75 with histidine, alanine at position 264 with glycine, threonine at position 268 with aspartic acid, and alanine at position 328 with phenylalanine. The resulting 12 combined mutants are: T268D, F87A / T268D, F87G / T268D, and T268D / A. 328F, F87A / T268D / A328F, F87G / T268D / A328F, V78F / F87A / T268D / A328F, V78I / F87G / T268D / A328F, L75 H / F87G / T268D / A328F, V78A / F87G / T268D / A328F, V78A / F87A / T268D / A328F, F87G / A264G / T268D / A328F.

[0020] The total PCR site-directed mutagenesis system was 25 μL, containing 12.5 μL Q5 hot-start ultra-fidelity 2×Master Mix, 1.25 μL forward primer (10 μM), 1.25 μL reverse primer (10 μM), 1 μL template DNA, and 9 μL sterile water. After KLD ligation, the DNA was transformed into E. coli DH5α competent cells and cultured overnight at 37°C on LB agar plates containing 50 μg / mL kanamycin. Single clones were picked and sequenced for verification, and 12 mutants were successfully constructed. Table 1. Primers used in P450BM3 gene mutagenesis experiments .

[0021] Example 2: Induction and purification of mutants The recombinant plasmid containing the P450BM3 mutant was transformed into E. coli BL21(DE3) competent cells, and seeded into LB liquid medium containing 50 μg / mL kanamycin. The cells were then cultured at 37°C and 200 rpm until OD600 was reached. 600Approximately 0.8 μL of 5-aminolevulinate (0.5 mM) and FeCl3 (0.5 mM) were added. After 30 min, 1 mM IPTG was added, and the mixture was induced at 30°C and 200 rpm for 20 h. Bacterial cells were collected by centrifugation at 4°C and 8000 rpm for 10 min, resuspended in buffer A (100 mM KPi, 500 mM NaCl, pH 7.4), sonicated, and centrifuged at 10000 rpm for 50 min. The supernatant was collected, filtered through a 0.22 μm filter, and purified using the AKTA protein purification system and a 5 mL HisTrap HP affinity chromatography column (eluted with buffer B 100 mM KPi, 500 mM NaCl, 500 mM imidazole, pH 7.4). The purified protein was dialyzed overnight in buffer C (100 mM KPi, 100 mM NaCl, pH 7.4). The target protein was placed in an ultrafiltration concentration cup for concentration and desalting, with a filter membrane size of 30 kDa; an equal volume of glycerol was added to the concentrated protein, and it was stored at -80 °C.

[0022] Example 3 Protein purity determination and protein characterization (1) Protein purity determination: The purity of the purified P450BM3 protein was determined by SDS-PAGE. The mass concentration of the separating gel used was 12%. The protein was diluted to 2 μM and mixed with the loading buffer at a ratio of 4:1 (v / v). After denaturation by boiling in a water bath for 10 min, 5 μL was added to the electrophoresis gel tank, and 5 μL of protein marker was added to another electrophoresis gel tank at the same time. The initial voltage was set to 80 V. When the band reached the middle position, the voltage was increased to 120 V until the end. After electrophoresis, the gel was stained with Coomassie Brilliant Blue for 2 h, and then destained with destaining solution (pure water:methanol:acetic acid = 67:25:8) until the desired effect was achieved. Finally, the gel was observed and analyzed in a gel imaging system (e.g., Figure 1 (As shown).

[0023] (2) Ultraviolet absorption spectroscopy characterization of proteins P450BM3 protein characterization was performed using a UV spectrophotometer. First, WT P450BM3 and its mutants were diluted to 5 μM. 10 mg of Na₂S₂O₄ was added to 3 mL of the P450BM3 protein dilution solution. Then, CO₂ was bubbled into the solution until saturation (approximately 3 min). The absorption spectra of P450BM3 protein before and after CO₂ reduction were measured in the 350–700 nm range. The shift of the characteristic absorption peaks in the two spectra was used to characterize the P450BM3 protein. The heme cofactor of cytochrome P450BM3 can be reduced to ferrous heme by Na₂S₂O₄, and then CO₂ combines with it to form a ferrous-CO₃ complex (Fe₂O₃ + CO₂). 2+ (-P450-CO), therefore, P450BM3's characteristic absorption peak at 418 nm shifts to 450 nm. Thus, P450BM3 can be characterized by its 450 nm characteristic absorption peak, such as... Figure 2-14 As shown, the reduced protein exhibits a characteristic Soret absorption peak at 450 nm, confirming successful expression of the P450 protein.

[0024] Example 4: Optimization of Reaction Conditions and Control Experiment Using potassium phosphate buffer (50 mM) as the system, the concentration of H2O2 (5–40 mM), temperature (15–40 °C), pH (6.0–8.5), and reaction time (10–60 min) were systematically optimized. The results showed that the optimal H2O2 concentration for guaiacol was 20 mM, and for eugenol and 4-methyleugenol, it was 30 mM; the optimal temperature for guaiacol and eugenol was 25 °C, and for 4-methyleugenol, it was 30 °C; the formation of the demethylation product tended to stabilize at a reaction time of 60 min (e.g., ...). Figure 15-26 (As shown). Simultaneously, control experiments confirmed that the formation of demethylation products could only be detected when the P450BM3 mutant and H2O2 were present simultaneously (e.g., Figure 27-29 As shown), the demethoxylated products of guaiacol and eugenol are catechol and 3-methoxycatechol, respectively, which was confirmed by purchasing standards; while the demethoxylated product of 4-methyleugenol, 3-methoxy-5-methylphenyl-1,2-diol, was confirmed by mass spectrometry (m / z 153.0585) (as shown). Figure 30(As shown); the reaction products were analyzed using a Shimadzu LC20AD-AB SCIEX QTRAP 5000 UPLC-MS system. The chromatographic column was a reversed-phase C18 column (Agilent Eclipse Plus, 2.1 × 50 mm, 1.8 μm), with ESI negative ion mode detection and gradient elution (mobile phase A: 5 mM ammonium acetate + 0.1% formic acid aqueous solution; mobile phase B: acetonitrile). The total analysis time was 10 min.

[0025] Example 5: Dynamics Study 1. Kinetic study of guaiacol by the P450BM3 mutant: (1) Kinetic measurement In 1 mL potassium phosphate buffer (50 mM, pH 7.5), guaiacol and 1 μM MP450BM3 mutant were incubated at 25 °C, followed by the addition of 20 mM H2O2 to initiate the reaction. The reaction was terminated by adding 1 M HCl after incubation. The reaction solution was filtered through a 0.22 μm polyamide membrane and detected by high-performance liquid chromatography (HPLC, LC-20A, Shimadzu) using a Shim-pack Scepter HD-C18-80 column (5.0 μm, 4.6 mm × 150 mm) at 30 °C. The injection volume was 20 μL. The mobile phase was acetonitrile-water (v / v = 20:80), with isocratic elution at a flow rate of 0.6 mL / min for 25 min. Steady-state kinetic parameters were fitted according to the Michaelis-Menten equation. k cat , K m , k cat / K m As shown in Table 2; Table 2. Reaction kinetic data of guaiacol catalyzed by P450BM3 ; (2) Kinetic analysis Wild-type P450BM3 showed no detectable measurable activity against guaiacol. The mutant T268D / A328F exhibited the highest catalytic efficiency (58.26 mM). -1 ·s -1 ),That K m Minimum (0.19 mM). V78F / F87A / T268D / A328F k cat Highest (48.34 s -1 ),but Km Maximum (1.35 mM), efficiency is 35.80 mM -1 ·s -1 The catalytic efficiency of the F87G variant is generally higher than that of the F87A variant, for example, F87G / T268D (43.04 mM). -1 ·s -1 (28.09 mM) is higher than F87A / T268D. -1 ·s -1 The introduction of the A328F enables the T268D to... K m The efficiency increased from 29.88 mM to 58.26 mM when the flux density decreased from 0.90 mM to 0.19 mM. -1 ·s -1 The catalytic efficiencies of all variants ranged from 13.87 to 58.26 mM. -1 ·s -1 .

[0026] 2. Kinetic study of eugenol by the P450BM3 mutant: (1) Kinetic measurement In 1 mL potassium phosphate buffer (50 mM, pH 7.5), eugenol at different concentration gradients and 1 μM MP450BM3 mutant were incubated at 25 °C, followed by the addition of 30 mM H2O2 to initiate the reaction; after the reaction was completed, 1 M HCl was added to terminate it. The reaction solution was filtered through a 0.22 μm polyamide membrane and detected by high-performance liquid chromatography (HPLC, LC-20A, Shimadzu) using a Shim-pack Scepter HD-C18-80 column (5.0 μm, 4.6 mm × 150 mm) at 30 °C and an injection volume of 20 μL. The mobile phase was acetonitrile-water (v / v = 20:80), with isocratic elution at a flow rate of 0.6 mL / min and an elution time of 20 min. Steady-state kinetic parameters were fitted according to the Michaelis-Menten equation. k cat , K m , k cat / K m As shown in Table 3; Table 3. Kinetic data of eugenol catalysis by P450BM3 ; (2) Kinetic analysis Wild-type P450BM3 showed no detectable measurable activity against eugenol. The mutant F87G / A264G / T268D / A328F exhibited the highest catalytic efficiency (55.71 mM).-1 ·s -1 ),That K m The lowest (0.14 mM), but k cat Only 7.80 s -1 F87A / T268D / A328F is the second best (47.11 mM). -1 ·s -1 ), k cat It is 16.96 s -1 , K m The value is 0.36 mM. The introduction of A328F improves the T268D's... K m The efficiency increased from 20.97 mM to 24.06 mM as the molecular weight decreased from 1.07 mM to 0.72 mM. -1 ·s -1 The efficiency of the V78I / F87G / T268D / A328F is 29.29mM. -1 ·s -1 ) higher than the corresponding variant of V78A (21.90 mM) -1 ·s -1 The catalytic efficiencies of all variants ranged from 17.90 to 55.71 mM. -1 ·s -1 .

[0027] 3. Kinetic study of 4-methyleugenol by the P450BM3 mutant: (1) Kinetic measurement In 1 mL of potassium phosphate buffer (50 mM, pH 7.5), 4-methyleugenol and 1 μM MP450BM3 mutant were incubated at 30 °C, followed by the addition of 30 mM H2O2 to initiate the reaction. The reaction was terminated by adding 1 M HCl after incubation. The reaction solution was filtered through a 0.22 μm polyamide membrane and detected by high-performance liquid chromatography (HPLC, LC-20A, Shimadzu) using a Shim-pack Scepter HD-C18-80 column (5.0 μm, 4.6 mm × 150 mm) at 30 °C. The injection volume was 20 μL. The mobile phase was an acetonitrile-water system with gradient elution: acetonitrile concentration from 10% to 50% for 0-20 min, from 50% to 10% for 20-25 min, from 50% to 90% for 25-35 min, and from 90% to 10% for 35-40 min. The flow rate was 1 mL / min, and the elution time was 40 min. Steady-state kinetic parameters were fitted using the Michaelis-Menten equation. kcat , K m , k cat / K m As shown in Table 4; Table 4. Kinetic data of 4-methyleugenol catalyzed by P450BM3 .

[0028] (2) Dynamic analysis Wild-type P450BM3 showed no detectable measurable activity against 4-methyleugenol. The mutant V78A / F87A / T268D / A328F exhibited the highest catalytic efficiency (65.25 mM). -1 ·s -1 ), k cat It is 17.62 s -1 , K m The value is 0.27 mM. V78F / F87A / T268D / A328F is next (50.60 mM). -1 ·s -1 T268D single mutation k cat Highest (38.87 s -1 ),but K m Maximum (2.17 mM), efficiency only 17.91 mM -1 ·s -1 F87A / T268D K m The lowest (0.06 mM), but k cat Only 1.89 s -1 The F87G variant generally exhibits higher efficiency than the F87A variant. The catalytic efficiencies of all variants range from 17.90 to 65.25 mM. -1 ·s -1 .

[0029] Example 6: P450BM3 mutant catalyzes demethylation of lignin aromatic ether monomers 1. Demethoxylation of guaiacol by the P450BM3 mutant In 1 mL potassium phosphate buffer (50 mM, pH 7.5), containing 4 mM guaiacol and 1 μM P450BM3 mutant, the reaction was incubated at 25°C, and then 20 mM H2O2 was added to start the reaction; after 60 min, 1 M HCl was added to terminate the reaction. The liquid chromatography-HPLC detection method was the same as above. TON was calculated according to the total transformation number TON = product concentration / enzyme concentration, as shown in Table 5. Table 5. TON determination of P450BM3-catalyzed guaiacol to catechol conversion .

[0030] The results showed that the four mutants V78F / F87A / T268D / A328F had the highest TON, reaching 1607±18, far exceeding other mutants; the TON of V78A / F87G / T268D / A328F was 1081±15; and the TON of the three mutant F87A / T268D / A328F was 832±11.

[0031] 2. Demethoxylation of eugenol by the P450BM3 mutant In 1 mL of potassium phosphate buffer (50 mM, pH 7.5), containing 4 mM eugenol and 1 μM P450BM3 mutant, the reaction was incubated at 25°C, followed by the addition of 30 mM H2O2 to initiate the reaction; after the reaction was completed, 1 M HCl was added to terminate the reaction. The liquid chromatography-HPLC detection method was the same as above. TON was calculated according to the total transformation number TON = product concentration / enzyme concentration, as shown in Table 6. Table 6. Determination of TON in the P450BM3-catalyzed production of 3-methoxycatechol from eugenol .

[0032] The results showed that when catalyzing eugenol, the increased methoxy group of the substrate caused greater steric hindrance, and the overall TON of each mutant decreased compared with guaiacol. V78F / F87A / T268D / A328F still performed the best, with a TON of 334±5. The three mutants F87A / T268D / A328F (236±4) and F87G / T268D / A328F (203±5) were the next best.

[0033] 3. Demethoxylation of 4-methyleugenol by the P450BM3 mutant In 1 mL of potassium phosphate buffer (50 mM, pH 7.5), containing 2 mM 4-methyleugenol and 1 μM P450BM3 mutant, the reaction was incubated at 30 °C, followed by the addition of 30 mM H2O2 to initiate the reaction; after the reaction was completed, 1 M HCl was added to terminate the reaction. The liquid chromatography-HPLC detection method was the same as above. Using the internal standard method, TON was calculated according to the formula Total Transformation Number (TON) = Product Concentration / Enzyme Concentration, as shown in Table 7. Table 7. TON determination of P450BM3 catalysis for the conversion of 4-methyleugenol to 3-methoxy-5-methylbenzene-1,2-diol .

[0034] The results showed that when catalyzing 4-methyleugenol, the TON of most double, triple, and tetra mutants remained stable in the high range of 700–1150. The tetra mutant V78I / F87G / T268D / A328F had the highest TON, reaching 1135±42. The TON of the double mutant T268D / A328F also reached 1053±35. The TON of the single mutant T268D was 917±33, which was comparable to that of most tetra mutants. It is speculated that the large substrate itself can fill the wild-type pocket channel and naturally play a role in repelling water molecules, thereby reducing the need for helper mutations.

[0035] In summary, the P450BM3 mutant provided by this invention exhibits significantly enhanced catalytic performance for three aromatic lignin monomers, offering a promising biotechnological pathway for the O-demethylation of lignin monomers. Furthermore, the detailed and reproducible embodiments of this invention provide important reference for research and applications in related fields.

Claims

1. A peroxygenase mutant V78I / F87G / T268D / A328F based on cytochrome P450BM3, characterized in that, The amino acid sequence of the mutant V78I / F87G / T268D / A328F is based on the amino acid sequence shown in SEQ ID NO.1, where valine at position 78 is mutated to isoleucine, phenylalanine at position 87 is mutated to glycine, threonine at position 268 is mutated to aspartic acid, and alanine at position 328 is mutated to phenylalanine.

2. An expression carrier, characterized in that, The expression vector contains the mutant V78I / F87G / T268D / A328F as described in claim 1.

3. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria contain the expression vector described in claim 2.

4. The application of the mutant V78I / F87G / T268D / A328F as described in claim 1, characterized in that, The mutant V78I / F87G / T268D / A328F uses H2O2 as an oxidant to catalyze the O-demethylation reaction of guaiacol, eugenol or 4-methyleugenol to generate the corresponding catechol products without the need for NAD(P)H cofactor and electron transport protein.

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