A mutant catalyzing O-demethylation of lignin aromatic ether monomers and its application

Abstract

The invention relates to a catalytic lignin aromatic ether monomer O‑ Demethylated mutants and applications thereof belong to the technical field of biocatalysis. The mutants are F87A, F87G, F87A / V78A, F87A / V78G or F87A / T268D, F87A / V78A / T268P, F87A / V78G / T268P, F87A / T268D / A328F or F87A / V78A / T268D / A328F. The invention also provides expression vectors and genetically engineered bacteria containing the mutant genes, and catalysis of lignin aromatic ether monomers by using the mutants, expression vectors or engineered bacteria. O‑ Demethylation, where the F87A / V78A / T268D / A328F mutant had a selectivity of 99.9% for guaiacol and the highest turnover number. The F87A / T268D mutant had the highest conversion rate for 2,6-dimethoxyphenol and successfully achieved double demethylation.

Description

Technical Field

[0001] The invention belongs to the technical field of biocatalysis, and particularly relates to a mutant catalyzing O-demethylation of lignin aromatic ether monomers and application thereof. Background Art

[0002] Lignin is an amorphous aromatic polymer found in terrestrial plants that is abundant but underutilized. Its structural unit is 4-hydroxyphenylpropane (monophenol), which can be divided into 4-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) according to the number of methoxy groups and substitution positions on the aromatic ring. As the most abundant renewable aromatic polymer in nature, plants on Earth produce about 500-3600 million tons of lignin each year. In addition, as the largest chemical process industry using plant biomass as raw materials, the pulp and paper industry produces about 60 million tons of lignin "waste" each year. Such a huge output of lignin has an unignorable impact on the carbon cycle in nature. However, due to the heterogeneity of lignin, most organisms find it difficult to degrade it. Therefore, finding an efficient lignin degradation mechanism is of great significance to environmental protection and economic development.

[0003] At present, the methods for lignin degradation are mainly physical and chemical methods and biological methods. Among them, the physical and chemical degradation process has the disadvantages of high energy consumption and "secondary pollution", which cannot meet the development needs of the green economy. In contrast, biodegradation has stood out due to its advantages such as mild action conditions, wide sources, and no pollution, and has become a hot field for lignin degradation in recent years. Microbial degradation has become a popular strategy for lignin degradation and reuse in recent years due to its advantages such as mild action conditions, wide sources, and no pollution. This strategy uses powerful oxidases secreted by decaying fungi and certain bacteria to decompose lignin polymers into small fragments, and then further degrades them into carbon and energy through at least four decomposition and metabolism methods. Lignin is mainly composed of two structural units, guaiacyl (G) and syringyl (S), which have one and two methoxy groups on the benzene ring, respectively. Therefore, almost all lignin aromatic ether monomers must first undergo demethylation reactions to generate corresponding diol intermediates, and then they can be oxidized and cleaved to generate open-ring compounds, thereby participating in the carbon cycle process. Therefore, demethylation is a key reaction for the utilization of lignin in natural carbon cycles and emerging biotechnologies. In 2018, Mallinson et al. fully characterized a two-component protein P450 demethylase, which is composed of a cytochrome P450 enzyme (GcoA) of the CYP255A family and a three-domain reductase (GcoB). It can effectively catalyze the demethylation of various lignin derivatives. However, GcoAB consumes expensive NAD(P)H during the reaction. If H 2 O 2As an oxygen source, it can not only greatly shorten the catalytic cycle, but also reduce the cost investment. The cong research group innovatively proposed a bifunctional small molecule (DFSM) driven H 2 O 2 P450BM3-dependent peroxygenase system can utilize H 2 O 2 Replace O 2 As an oxygen atom donor, it catalyzes the oxidation reaction of non-natural substrates. In the large family of cytochrome P450 enzymes, only a few specific P450s can selectively catalyze the demethylation reaction of aromatic ethers, and most P450s exhibit multiple oxidation functions. Previously, the active space of the artificial P450BM3 peroxygenase system promoted by a bifunctional small molecule (DFSM) was modified to construct a P450BM3 / DFSM system to achieve an improvement in the demethylation activity of lignin G-lignin. However, there is still a disadvantage of catalyzing the single demethylation of S-lignin (2,6-dimethoxyphenol). Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a P450 mutant enzyme that catalyzes the O-demethylation of lignin aromatic ether monomers. The present invention uses a P450BM3-AldO system to study the P450 mutant enzyme catalyzing the O-demethylation of lignin aromatic ether monomers.

[0005] The technical solution of the present invention is as follows:

[0006] A mutant catalyzing O-demethylation of lignin aromatic ether monomers, wherein the mutant is a site mutation at the 87th phenylalanine of cytochrome P450BM3 oxidase; the site mutation at the 87th phenylalanine is alanine or glycine; the amino acid sequence of the cytochrome P450BM3 oxidase is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2.

[0007] The mutant can also be superimposed on one or more of the following amino acid positions of cytochrome P450BM3 oxidase for mutation: the 78th valine, the 328th alanine and the 268th threonine.

[0008] Furthermore, the bifunctional small molecule compound Im-C6-Phe is used to induce the oxidation domain mutant of the P450 enzyme to achieve the use of H generated by glycerol oxidation. 2 O 2Highly efficient catalysis of O-demethylation of lignin aromatic ether monomers; wherein the mutant is a single, double, triple or quadruple mutant containing mutations at positions 78, 87, 268 and 328, wherein the phenylalanine at position 87 is mutated to glycine or alanine, the valine at position 78 is mutated to glycine or alanine, the threonine at position 268 is changed to proline or aspartic acid, and the alanine at position 328 is mutated to phenylalanine; the specific mutant is: the phenylalanine at position 87 of the amino acid sequence shown in SEQ ID NO.1 is replaced by alanine or glycine; the obtained mutants are named F87A (SEQ ID NO.3) and F87G (SEQ ID NO.4) respectively.

[0009] The phenylalanine at position 87 of the amino acid sequence shown in SEQ ID NO.1 was replaced by alanine, the valine at position 78 was replaced by glycine or alanine, and the threonine at position 268 was replaced by aspartic acid; the mutants were named F87A / V78A (SEQ ID NO.5), F87A / V78G (SEQ ID NO.6), and F87A / T268D (SEQ ID NO.7), respectively.

[0010] The phenylalanine at position 87 of the amino acid sequence shown in SEQ ID NO.1 was replaced by alanine, the valine at position 78 was replaced by glycine or alanine, the alanine at position 268 of the amino acid sequence was replaced by proline or aspartic acid, and the alanine at position 328 was replaced by phenylalanine; the mutants were named F87A / V78A / T268P (SEQ ID NO.8), F87A / V78G / T268P (SEQ ID NO.9), and F87A / T268D / A328F (SEQ ID NO.10), respectively.

[0011] The phenylalanine at position 87 of the amino acid sequence shown in SEQ ID NO.1 was replaced with alanine, the valine at position 78 was mutated to alanine, the alanine at position 268 of the amino acid sequence was mutated to aspartic acid, and the alanine at position 328 was mutated to phenylalanine; the mutant was named F87A / V78A / T268D / A328F (SEQ ID NO.11).

[0012] An expression vector comprising a gene of any one of the mutants described above.

[0013] A genetically engineered bacterium contains the expression vector.

[0014] The invention discloses an application of a mutant, wherein the mutant catalyzes the O-demethylation of lignin aromatic ether monomers.

[0015] The catalytic system comprises a bifunctional small molecule compound, the mutant, glycerol, sugar alcohol oxidase (AldO) and a substrate; wherein the bifunctional small molecule compound is an N-(ω-imidazole, fatty acyl)-L-phenylalanine derivative.

[0016] As a specific embodiment, the mutant is used to selectively catalyze the O-demethylation of guaiacol and 2,6-dimethoxyphenol as substrates, and a bifunctional small molecule N-(ω-imidazolyl, fatty acyl)-L-phenylalanine derivative is used to induce the oxidation domain mutant of the P450 enzyme to achieve continuous H oxidation of glycerol by AldO. 2 O 2 It catalyzes the demethylation of guaiacol and 2,6-dimethoxyphenol to generate catechol, pyrogallic acid and 3-methoxycatechol respectively.

[0017] Furthermore, the bifunctional small molecule Im-C6-Phe was used to induce the oxidation domain mutant of the P450 enzyme to achieve continuous utilization of H by oxidizing glycerol through alcohol oxidase (AldO). 2 O 2 The invention catalyzes the demethylation of guaiacol and 2,6-dimethoxyphenol to generate catechol, pyrogallic acid and 3-methoxycatechol respectively; wherein the mutant is a single, di, tri or quadruple mutant containing mutations at positions 87, 78, 328 and 268, wherein the phenylalanine at position 87 is mutated to glycine or alanine, the valine at position 78 is mutated to glycine or alanine, the threonine at position 268 is changed to proline or aspartic acid, and the alanine at position 328 is mutated to phenylalanine; the catalytic reaction system is to sequentially add 4 mM substrate, 0.5 μM P450BM3 series mutants, 0.5 mM bifunctional small molecules and 1% v / v glycerol to a reaction system of 1 mL sodium phosphate buffer (100 mM, pH 8.0), incubate at 25°C for 5 min, then add 2.5 μM AldO and react for 90 min.

[0018] The substrates are guaiacol and 2,6-dimethoxyphenol.

[0019] The present invention combines the dual regulatory capabilities of protein engineering technology with the synergistic strategy of bifunctional small molecules to achieve green and efficient conversion of guaiacol and 2,6-dimethoxybenzene, optimize the P450BM3 catalytic system and expand the substrate range, providing new ideas and methods for further research in the field of biocatalysis.

[0020] Specifically, the oxidase part of cytochrome P450-BM3 monooxygenase from Bacillus megaterium was used as a template, and the mutant primer containing the 87 mutation site was used for PCR mutation, and the F87 single mutant was obtained by sequencing; then the single mutant gene was used as a template, and the F87-V78 combination and the F87-T268 combination double mutant were PCR amplified in the same way; then the double mutant gene was used as a template, and the F87-V78-T268 combination and the F87-T268-A328 triple mutant were PCR amplified in the same way; then the triple mutant gene was used as a template, and the F87-V78A-T268-A328 was PCR amplified in the same way, and the P450BM3-AldO system catalyzed the O-demethylation effect of lignin aromatic ether monomers. Experiments have confirmed that compared with P450BM3-H 2 O 2 Compared with the original system, the catalytic efficiency is up to 5 times that of the original system.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention uses random mutation high-throughput screening to obtain dominant mutants that catalyze the O-demethylation of lignin aromatic ether monomers through directed evolution of P450BM3. Through semi-rational design of dominant mutation sites, a "small and fine" mutant library is constructed to improve the screening efficiency. Under the P450BM3-AldO system, the F87A / V78A / T268D / A328F mutant has a selectivity of up to 99.9% (TON=1895) for guaiacol and has the highest turnover number. At the same time, the F87A / T268D mutant has the highest conversion rate for 2,6-dimethoxyphenol (TON=483) and successfully achieves double demethylation. In short, we believe that the catalytic activity can be further improved through protein engineering, providing a more efficient and economical method for the decomposition and utilization of lignin. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Protein electrophoresis diagrams of the P450BM3 series mutants and sugar alcohol oxidase provided in the embodiments of the present invention. Lanes 1-10 are: F87A, F87G, F87A / V78A, F87A / V78G, F87A / T268D, F87A / V78A / T268P, F87A / V78G / T268P, F87A / V78A / T268I / A264G, F87A / T268D / A328F, F87A / V78A / T268D / A328F. (b) Lane 1 is: AldO;

[0024] Figure 2 The bifunctional small molecule provided in the embodiment of the present invention cooperates with the typical mutant of P450BM3 to utilize H2 O 2 HPLC analysis chart of catalytic guaiacol, wherein (a) is the standard; (b) is F87A / V78G / T268P; (c) is F87A; (d) is F87A.T268D / A328F; (e) is F87A / V78A / T268P; (f) is F87A / V78G; (g) is F87AG; (h) is F87A / V78A / T268D / A328F; (i) is F87A / T268D; (j) is F87A / V78A;

[0025] Figure 3 The bifunctional small molecule provided in the embodiment of the present invention cooperates with the typical mutant of P450BM3 to utilize H 2 O 2 HPLC analysis chart of catalytic 2,6-dimethoxyphenol, where (a) is the standard; (b) is F87G; (c) is F87A; (d) is F87A / T268D; (e) is F87A / V78G; (f) is F87A / V78A; (g) is F87A / T268D / A328F; (h) is F87A / V78A / T268P; (i) is F87A / V78A / T268D / A328F; (j) is F87A / V78G / T268P. DETAILED DESCRIPTION

[0026] The principles and features of the present invention are further described below. The examples listed are only used to explain the present invention and are not used to limit the scope of protection of the present invention. In addition, the experimental methods used in the following examples are conventional methods in the art unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0027] The present invention firstly uses protein engineering and chemical modification strategies to perform semi-rational amino acid mutations and construct a P450BM3-AldO peroxygenase system to catalyze the conversion of two substrates, guaiacol and 2,6-dimethoxyphenol, into catechol and pyrogallic acid, 3-methoxycatechol. The system is simple in composition and can achieve efficient biocatalysis without relying on expensive cofactors NAD(P)H and electron transfer chaperone proteins, and avoids the direct addition of H 2 O 2 It solves the problem of protein structure destruction and provides a green and efficient alternative method for the demethylation of lignin aromatic ether monomers.

[0028] Meanwhile, the small molecule compounds of the following examples can be purchased commercially or synthesized according to the prior art.

[0029] The following methods were used for the construction of engineering bacteria, induced expression and purification of enzymes, and determination of catalytic activity involved in the following examples, as detailed below.

[0030] 1. Construction of mutants: The wild-type cytochrome P450BM3 oxidase gene (the gene sequence is shown in SEQ ID NO.1) was subjected to site-directed mutagenesis using PCR mutagenesis technology. Using the recombinant plasmid pET-28a(+)-BMP connected to the P450BM3 oxidase part (BMP) gene as a template, a pair of forward and reverse primers containing the mutation site were designed, and PCR was performed to obtain the recombinant gene; wherein, the PCR amplification program is shown in Table 1;

[0031] Table 1 PCR amplification procedure

[0032] .

[0033] The amplified PCR product was detected by 0.7% agarose gel electrophoresis, and a band with the same length as the P450BM3 gene was observed, which indicated that the gene encoding the target mutant had been amplified. The PCR product was transformed into E. coli DH5ɑ competent cells after KLD connection, and evenly coated on a solid LB plate containing 50 μl / mg kanamycin after adding culture medium for 1 hour. After overnight culture at 37 °C, a single clone was selected and cultured in 5 mL LB liquid culture medium containing 50 μg / mL kanamycin, and then sent to a sequencing company for sequencing to obtain the correct mutant.

[0034] 2. Induced expression and purification of mutants: Pick a single colony with successful mutation, randomly pick a single colony and put it into 5 mL LB medium containing Kana. After activation for a period of time, transfer 500 μL of bacterial solution to 50 mL LB liquid medium (Kana). After activation for a period of time, transfer 5 mL of bacterial solution to 500 mL LB liquid medium (Kana). Wait for OD 600 nm When the value is 0.6-0.8, add 0.5 mM FeCl 3 , 5' - ALA (5-aminolevulinic acid) was cultured at 37°C and 180 rpm for 30 min, and then 1 mM IPTG was added and induced at 30°C and 180 rpm for 20 h.

[0035] The expressed bacterial solution was centrifuged at 8000 rpm and 4°C for 10 min, the supernatant was discarded, and the cells were washed with loading buffer (100 mM K 2 HPO 4 -KH 2 PO 4, 500 mM NaCl, pH7.4) and resuspended and washed twice to obtain resting cells. The resting cells were resuspended in a loading buffer with one-tenth volume of the bacterial liquid, mixed and then disrupted by ultrasonic wave. The disrupted product was centrifuged at 10000 rpm and 4°C for 90 min to remove cell debris and obtain the crude enzyme solution of the mutant.

[0036] The crude enzyme solution was purified by Ni column affinity chromatography. The crude enzyme solution was filtered through a 0.22 μm filter membrane. The pretreated series P450BM3 crude protein was further purified using the AKTAexplorer 10S protein rapid purification instrument, and dual wavelength detection was performed using the 280nm protein absorption wavelength and the 418nm P450 protein characteristic absorption wavelength. Specific steps: First, balance the His Trap with 100% loading solution TM HP affinity chromatography column, flow rate of 5 mL / min, remove the impurities that may remain in the column, and start loading after the baseline is stable, with a loading flow rate of 3 mL / min; at this time, most of the impurities will directly pass through the column to show a penetration peak, and the target protein P450BM3 and a small amount of impurities will bind to the column due to ion interaction; when the baseline is stable again, add 96% loading solution and 4% eluent (100 mM K 2 HPO 4 -KH 2 PO 4 , 500 mM NaCl, 500 mM imidazole, pH 7.4) to elute the impurities. After the baseline stabilized, wash the impurities again with 100 mL (20 column volumes) of 94% loading solution and 6% eluent. After the baseline stabilized again, start eluting the target protein, adjust the eluent concentration ratio to 40%, and collect the target protein from the outlet line when the characteristic absorption peak appeared at 418 nm. At this time, P450BM3 no longer interacts with His Trap TM The HP affinity chromatography column binds specifically and is displaced by high concentration of imidazole; the concentration ratio of the eluent is adjusted to 100%, and the protein in the column is washed with high concentration of imidazole. After the baseline is stable, the system and pump are washed with ultrapure water and 20% ethanol respectively; the high concentration of imidazole in the target protein is removed by dialysis, first dialyzing once with pure water 100 times the volume of the target protein, and then dialyzing the target protein twice with dialysate, each time for 2 hours; the target protein is placed in an ultrafiltration concentration cup for concentration and desalination, and the filter membrane size is 30 kDa; an equal volume of glycerol is added to the concentrated protein and stored at -80 ℃.

[0037] 3. P450BM3-AldO system catalyzes the demethylation of lignin aromatic ether monomers: In 1 mL of sodium phosphate buffer (100 mM, pH 8.0), P450BM3 and its mutants (0.5 μM), substrate (4 mM), Im-C6-Phe (0.5 mM), 1% v / v glycerol, AldO (2.5 μM), and 2.5% methanol as a cosolvent were added in sequence. The reaction was carried out at 25 °C in the dark for 90 min, and 100 μL of HCl solution (1 M) was added to terminate the reaction. Then, an equal volume of NaOH solution (1 M) was added for neutralization. After filtering with a 0.22 μm disposable nylon filter membrane, HPLC detection was performed to qualitatively and quantitatively analyze the catalytic products of different mutants.

[0038] 4. HPLC detection conditions: The oxidation products were qualitatively and quantitatively detected by HPLC using a Shim-pack ScepterHD-C18-80 column (5.0 μm, 4.6 mm × 150 mm), an injection volume of 20 μL, and a column temperature of 30 °C. The specific detection conditions are as follows:

[0039] Guaiacol: flow rate: 0.6 mL / min; mobile phase: acetonitrile: water (containing 0.05% acetic acid) = 17:83; detector: 210 nm; running time 30 min;

[0040] 2,6-Dimethoxyphenol: flow rate: 0.5 mL / min; mobile phase: acetonitrile: water (containing 0.05% acetic acid) = 2:8; detector: 210 nm; running time 30 min.

[0041] 5. Primers for mutation of sites are shown in Table 2;

[0042] Table 2 Primer sequences for mutations at sites 78, 268, 87, and 328

[0043] .

[0044] Example 1 Preparation of F87 single mutant

[0045] Using the recombinant plasmid pET-28a(+)-BMP connected to the P450BM3 oxidase part (BMP) gene as a template, and using forward and reverse primers containing different mutation sites (see Table 1) as primer pairs, PCR was performed to obtain the recombinant genes of F87 single mutants F87A and F87G. The amplification system and PCR conditions are shown in the general method. The induced expression and purification of the mutants are shown in the general method. The target protein is shown in Figure 1 .

[0046] Example 2 Preparation of F87-V78 and F87-T268 double mutations, F87A / V78A (SEQ ID NO.5), F87A / V78G (SEQ ID NO.6), F87A / T268D (SEQ ID NO.7) were prepared using the recombinant plasmid pET-28a(+)-F87 connected with the P450BMP-F87 single mutation as a template, and forward and reverse primers containing different mutation sites as primer pairs (see Table 2), respectively, to perform PCR, and obtain the recombinant genes of different F87A-V78A, F87A-V78G and F87A-T268D double mutants. The amplification system and PCR conditions are shown in the general method. The induced expression and purification of the mutants are shown in the general method, and the target protein is shown in Figure 1 .

[0047] Example 3 Preparation of F87-V78-T268 and F87-T268-A328 triple mutations

[0048] Using the recombinant plasmids pET-28a(+)-F87-V78 and pET-28a(+)-F87-T268 connected with the two mutations of P450BMP-F87-V78 and P450BMP-F87-T268 as templates, and the forward and reverse primers containing different mutation sites as primer pairs (see Table 2), PCR was performed to obtain the recombinant genes of different F87A / V78A / T268P, F87A / V78G / T268P and F87A / T268D / A328F triple mutants. The amplification system and PCR conditions are shown in the general method. The induced expression and purification of the mutants are shown in the general method, and the target protein is shown in Figure 1 .

[0049] Example 4 Preparation of F87-V78-T268-A328 quadruple mutation

[0050] Using the recombinant plasmid pET-28a(+)-F87-T268-A328 connected with the P450BMP-F87-T268-A328 triple mutation as a template, and using forward and reverse primers containing different mutation sites as primer pairs (see Table 2), PCR was performed to obtain the recombinant genes of different F87A / V78A / T268D / A328F quadruple mutants. The amplification system and PCR conditions are shown in the general method. The induced expression and purification of the mutants are shown in the general method, and the target protein is shown in Figure 1 .

[0051] Example 5 Detection of reaction products of lignin aromatic ether monomer derivatives by HPLC

[0052] In this example, different combinations of F87-T268 double mutations were obtained according to the above preparation method, and the catalytic activity and selectivity of lignin aromatic ether monomers were determined. The reaction conditions were as described in General Method 4. All mutants were prepared with P450BM3-H 2 O 2 The system was used as a reaction control; the specific activity is shown in the following table:

[0053] 1) Guaiacol

[0054] In the P450BM3-AldO system, the turnover number (TON) and selectivity of the guaiacol demethylation products catalyzed by the P450BM3 series mutants are shown in Table 3 below;

[0055] Table 3 shows the turnover number (TON) and selectivity of the guaiacol demethylation products catalyzed by the P450BM3 series mutants

[0056] ;

[0057] a: The amount of product converted per P450BM3 molecule within 90 min; b: No product was detected.

[0058] F87A and F87G demethylated guaiacol to catechol, and the catalytic activity was significantly improved (TON: 754,736), with a selectivity of 100% for catechol. The double mutants F87A / V78A and F87A / V78G also had 99.9% regioselectivity, but the catalytic activity was not significantly improved. The introduction of non-polar proline at position 268 of the F87A / V78G mutant greatly improved the catalytic ability, bringing the catalytic activity to 1593, which was 4.03 times higher than that of F87A / V78G. By mutating the alanine at position 328 to phenylalanine, the F87A / V78A / T268D / A328F mutant exhibited the highest O-demethylation catalytic ability, with a turnover number of 1895, which was 2.51 times that of F87A.

[0059] With P450BM3-H 2 O 2 Compared with the P450BM3-AldO system, the P450BM3-AldO system improved the catalytic efficiency and regioselectivity of various P450BM3 mutants. The catalytic efficiency of these nine mutants increased by 1.15 to 5 times, while maintaining 99.9% regioselectivity. This indicates that the introduced AldO has a synergistic regulatory effect on catalytic activity and regioselectivity. In addition, the P450BM3-AldO system has a significant effect on the catalytic activity and regioselectivity. 2 O 2 Adjustment of concentration inhibited enzyme inactivation and further improved the catalytic activity and regioselectivity of P450BM3.

[0060] 2) In the P450BM3-AldO system, the turnover number (TON) and selectivity of the demethylation products of 2,6-dimethoxyphenol catalyzed by the P450BM3 series mutants are shown in Table 4 below;

[0061] Table 4 Mutant catalysis 2,6-dimethoxyphenol demethylation product turnover number (TON) and selectivity

[0062] .

[0063] With P450BM3 / H 2 O 2 Compared with the previous systems, the P450BM3-AldO system successfully achieved double demethylation to obtain the hydroxylated product pyrogallic acid. The catalytic performance of the P450BM3-AldO system was significantly improved by strategic amino acid substitutions, and the arrangement of amino acids in the active site of the protein has a great influence on the catalytic ability. The turnover numbers of the single mutants F87A and F87G were 331 and 284, respectively, and the regioselectivities were 88% and 87%, respectively. The turnover number of the double mutant F87A / V78A was 318 and the regioselectivity was 88%, while the turnover number of F87A / V78G was 279 and the selectivity was 89%. Although the catalytic activities of these double mutants were not significantly improved compared with the single mutants, their regioselectivities were improved. The TONs of the triple mutants F87A / V78A / T268P and F87A / V78G / T268P were 270 and 397, respectively, and the regioselectivities were 87% and 89%, respectively. The F87A / T268D mutant had the best catalytic effect, with a TON of 483 and a regioselectivity of 92%. The catalytic efficiency of the F87A / T268D mutant was 1.46 times that of F87A and 1.52 times that of F87A / V78A. The quadruple mutant F87A / V78A / T268D / A328F had a turnover number of 471 and a regioselectivity of 91%, further demonstrating the potential of strategic amino acid substitutions in improving the catalytic performance of the P450BM3-AldO system. See the specific HPLC graph for details. Figure 2 and Figure 3 shown.

[0064] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

[0065] SEQ ID NO.1: Amino acid sequence of Bacillus megaterium P450BM3:

[0066] TIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDKNLSQALKFVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYFLVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLKHFDFEDHTNYELDIKETLTLKPEGFVVKAKSKKIPLGGIPSPSTEQSAKKVRKKAEN。

[0067] SEQ ID NO.2: Nucleotide sequence of Bacillus megaterium P450BM3

[0068]

[0069] SEQ ID NO.3: Amino acid sequence of P450BM3-F87A:

[0070] TIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDKNLSQALKFVRDFAGDGL A TSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYFLVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLKHFDFEDHTNYELDIKETLTLKPEGFVVKAKSKKIPLGGIPSPSTEQSAKKVRKKAEN。

[0071] SEQ ID NO.4: Amino acid sequence of P450BM3-F87G:

[0072] TIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDKNLSQALKFVRDFAGDGL GTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYFLVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLKHFDFEDHTNYELDIKETLTLKPEGFVVKAKSKKIPLGGIPSPSTEQSAKKVRKKAEN。

[0073] SEQ ID NO.5: Amino acid sequence of P450BM3 - F87A / V78A:

[0074] TIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDKNLSQALKF A RDFAGDGL A TSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYFLVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLKHFDFEDHTNYELDIKETLTLKPEGFVVKAKSKKIPLGGIPSPSTEQSAKKVRKKAEN。

[0075] SEQ ID NO.6: Amino acid sequence of P450BM3-F87A / V78G:

[0076] TIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDKNLSQALKF G RDFAGDGL A TSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYFLVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLKHFDFEDHTNYELDIKETLTLKPEGFVVKAKSKKIPLGGIPSPSTEQSAKKVRKKAEN.

[0077] SEQ ID NO.7: Amino acid sequence of P450BM3-F87A / T268D:

[0078] TIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDKNLSQALKFARDFAGDGL A TSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHE DTSGLLSFALYFLVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLKHFDFEDHTNYELDIKETLTLKPEGFVVKAKSKKIPLGGIPSPSTEQSAKKVRKKAEN。

[0079] SEQ ID NO.8: Amino acid sequence of P450BM3 - F87A / V78A / T268P:

[0080] TIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDKNLSQALKF A RDFAGDGL A TSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHE P TSGLLSFALYFLVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLKHFDFEDHTNYELDIKETLTLKPEGFVVKAKSKKIPLGGIPSPSTEQSAKKVRKKAEN。

[0081] SEQ ID NO.9: Amino acid sequence of P450BM3 - F87A / V78G / T268P:

[0082] TIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDKNLSQALKF GRDFAGDGL A TSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHE P TSGLLSFALYFLVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLKHFDFEDHTNYELDIKETLTLKPEGFVVKAKSKKIPLGGIPSPSTEQSAKKVRKKAEN。

[0083] SEQ ID NO.10: Amino acid sequence of P450BM3-F87A / T268D / A328F

[0084] TIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDKNLSQALKFVRDFAGDGL A TSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHE D TSGLLSFALYFLVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPT FPAFSLYAKEDTVLGGEYPLEKGDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLKHFDFEDHTNYELDIKETLTLKPEGFVVKAKSKKIPLGGIPSPSTEQSAKKVRKKAEN。

[0085] SEQ ID NO.11: Amino acid sequence of P450BM3 - F87A / V78A / T268D / A328F:

[0086] TIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDKNLSQALKF A RDFAGDGL A TSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHE D TSGLLSFALYFLVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPT F PAFSLYAKEDTVLGGEYPLEKGDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLKHFDFEDHTNYELDIKETLTLKPEGFVVKAKSKKIPLGGIPSPSTEQSAKKVRKKAEN。

Claims

1. A catalytic lignin aromatic ether monomer O- A demethylated mutant, characterized in that The mutants are 4 mutants F87A / V78A / T268D / A328F, wherein F87A indicates that the phenylalanine at the 87th position of the amino acid sequence of cytochrome P450BM3 is mutated to alanine; V78A indicates that the valine at the 78th position of the amino acid sequence of cytochrome P450BM3 is mutated to alanine, T268D indicates that the threonine at the 268th position of the amino acid sequence of cytochrome P450BM3 is mutated to aspartic acid, and A328F indicates that the alanine at the 328th position of the amino acid sequence of cytochrome P450BM3 is mutated to phenylalanine, and the amino acid sequence of the cytochrome P450BM3 is shown in SEQ ID NO.

1.

2. An expression vector, characterized in that: The expression vector contains the gene of the mutant according to claim 1.

3. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria contains the expression vector according to claim 2.

4. Use of the mutant according to claim 1, the expression vector according to claim 2 or the genetically engineered bacteria according to claim 3, characterized in that: The application is to use the mutant, expression vector or genetically engineered bacteria to catalyze lignin aromatic ether monomers O- Demethylation.

5. The use according to claim 4, characterized in that: The catalytic system is a bifunctional small molecule compound, the mutant according to claim 1, the expression vector according to claim 2 or the genetically engineered bacteria according to claim 3, glycerol, sugar alcohol oxidase and a substrate; wherein the bifunctional small molecule compound is Im-C6-Phe.

6. The use according to claim 5, characterized in that: The substrate is guaiacol and / or 2,6-dimethoxyphenol.

Images