Heme iron cytochrome P450 enzyme mutant with enhanced electrocatalytic rate

Abstract

This invention provides a mutant of heme iron cytochrome P450 enzyme with enhanced electrocatalytic rate and its application. By performing site-directed mutagenesis on the heme iron cytochrome enzyme with the amino acid sequence shown in SEQ ID NO:1 as the parent, leucine at position 82 of SEQ ID NO:1 is mutated to glutamine, phenylalanine at position 167 is mutated to methionine, or valine at position 280 is mutated to threonine, a mutant with enhanced electrocatalytic rate is obtained, thereby solving the technical problem of low electrocatalytic rate of heme iron cytochrome enzyme in the prior art.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a heme iron cytochrome P450 enzyme mutant with enhanced enzyme electrocatalytic rate and its application. Background Technology

[0002] Lignin is a complex aromatic polymer formed by the cross-linking of phenylpropane structural units through various chemical bonds. Its structural complexity and heterogeneity make the efficient degradation and economical conversion of lignin a global challenge. In recent years, studies have found that the unique non-specific redox system of microorganisms and their multi-functional metabolic degradation ability of aromatic compounds have provided new ideas for the resource utilization of lignin. Since most lignin-derived aromatic monomers contain methoxy groups at different positions on the benzene ring, the demethylation process has become the key rate-limiting step in the metabolism of these compounds. Among demethylating enzymes, heme iron cytochrome P450 enzymes (P450s) are widely used (Nat Commun 2018, 9: 2487; Eur. J. Biochem.1993, 213(1): 211-216; Proc. Natl. Acad. Sci. USA 2020, 117(41): 25771-25778). P450 enzymes are a class of heme-dependent monooxygenases with a typical iron porphyrin ring structure and a substrate-binding pocket nearby. These enzymes typically require ferredoxin-NADP. + Reductases act as redox chaperone proteins. FNRN contains an N-terminal [2Fe-2S] iron-sulfur cluster domain, a flavin adenine dinucleotide (FAD) binding site, and a NADH binding site. Its mechanism of action is as follows: when a substrate binds to a P450 enzyme, the chaperone protein FNRN acquires an electron from NAD(P)H and transfers it to the P450 enzyme to catalyze the substrate reaction.

[0003] In most P450 enzyme-catalyzed reactions, not only is expensive NAD(P)H required as an electron donor, but chaperone proteins are also needed, which significantly increases the complexity of the reaction process. In addition, NAD(P)H is expensive and difficult to regenerate. Therefore, there is an urgent need to develop new technologies that can replace NAD(P)H and chaperone proteins. Enzyme electrocatalysis technology, with its mild reaction conditions and excellent selectivity, can replace chaperone proteins and NAD(P)H-dependent systems. Currently, many P450 enzymes have been successfully applied to enzyme electrosynthesis (Colloids Surf. A Physicochem. Eng. Asp. 384 (1-3) (2011) 388-392) or enzyme sensor development (Biosens. Bioelectron. 121 (2018) 192-204; NanoConvergence 8 (1) (2021)). Although most P450 enzymes can directly acquire electrons from the electrode to achieve substrate conversion (i.e., direct electron transfer, DET), their electrocatalytic rates are generally low because their catalytic center—the iron porphyrin ring—is deeply embedded inside the enzyme molecule and is not itself an electron transfer center. In recent years, research on improving the electrocatalytic rate of P450 enzymes has mainly focused on material selection and optimization (Chem. Commun. 50 (90) (2014) 13896-13899; Chem. Commun. 53 (18) (2017) 2673-2676.) and chaperone protein fusion strategies (Angew. Chem. Int. Ed. 55 (48) (2016) 15002-15006.). These methods aim to improve the microenvironment of the enzyme electrode. If we can start from the protein crystal structure and rationally modify key amino acid residues to improve the enzyme electrocatalytic rate, it is expected to open up new research directions for breaking through the bottleneck of P450 enzyme electrocatalytic efficiency. Summary of the Invention

[0004] This invention provides a heme iron cytochrome enzyme mutant with improved electrocatalytic rate and its application. By performing site-directed mutagenesis on the heme iron cytochrome enzyme with the amino acid sequence shown in SEQ ID NO:1 as the parent, leucine at position 82 of SEQ ID NO:1 is mutated to glutamine, phenylalanine at position 167 is mutated to methionine, or valine at position 280 is mutated to threonine, a mutant with improved electrocatalytic rate is obtained, thereby solving the technical problem of low electrocatalytic rate of heme iron cytochrome enzyme in the prior art.

[0005] According to a first aspect of the present invention, a heme iron cytochrome P450 enzyme mutant with enhanced electrocatalytic rate is provided. The heme iron cytochrome P450 enzyme mutant uses the amino acid sequence shown in SEQ ID NO:1 as the parent, and mutates leucine at position 82 of SEQ ID NO:1 to glutamine, phenylalanine at position 167 to methionine, or valine at position 280 to threonine.

[0006] According to another aspect of the invention, a nucleotide is provided that encodes the heme iron cytochrome P450 enzyme mutant.

[0007] According to another aspect of the present invention, a recombinant plasmid is provided, the recombinant plasmid comprising the aforementioned nucleotides.

[0008] According to another aspect of the invention, a host cell is provided, the host cell comprising the recombinant plasmid described above.

[0009] According to another aspect of the present invention, an enzyme electrode for degrading lignin is provided, comprising a metal electrode and the heme iron cytochrome P450 enzyme mutant covering the surface of the metal electrode.

[0010] According to another aspect of the present invention, an enzyme electrocatalytic reactor is provided, the enzyme electrocatalytic reactor comprising the enzyme electrode for degrading lignin.

[0011] According to another aspect of the present invention, a method for demethoxylation is provided, wherein the enzyme electrode or enzyme electrocatalytic reactor for degrading lignin is contacted with a reaction solution and an electric current is generated to carry out the reaction, wherein the reaction solution includes a substrate, catalase and a buffer solution, wherein the substrate is 3,4-dimethoxybenzoic acid; a heme iron cytochrome P450 enzyme mutant on the enzyme electrode is used to demethoxylate 3,4-dimethoxybenzoic acid, thereby achieving the degradation of 3,4-dimethoxybenzoic acid.

[0012] According to another aspect of the present invention, the application of the heme iron cytochrome P450 enzyme mutant in the degradation of lignin is provided.

[0013] In summary, compared with the prior art, the above-described technical solutions conceived by this invention mainly possess the following technical advantages:

[0014] (1) The mutants in this invention significantly improve the electron transport rate. This invention optimizes the electron transport pathway from the active site to the electrode surface by site-directed mutagenesis of key amino acid sites on the substrate channel, reduces the interfacial electron transport resistance, and enables the enzyme electrode to obtain a higher electron transport rate under the same conditions.

[0015] (2) The mutants in this invention maintain the catalytic properties and stability of the enzyme. The amino acid mutations in this invention target the substrate channel rather than the active site, which optimizes electron transfer without significantly altering the enzyme's substrate recognition ability and overall stability, thereby extending the lifespan of the enzyme electrode.

[0016] (3) The catalytic efficiency of the P450 mutant provided by the present invention remains at 80-90% of that of the wild type, while the enzyme electrocatalytic efficiency is increased by 2-30 times. In the enzyme electrocatalytic reaction, its rate is mainly determined by two aspects: the electron transfer rate and the enzyme catalytic ability. Therefore, the catalytic efficiency of the mutant obtained by the present invention remains basically unchanged, while the main reason for the increase in enzyme electrocatalytic rate is the increase in electron transfer rate. Attached Figure Description

[0017] Figure 1 The graph shows the conversion results of amino acid residues related to the P450 substrate channel.

[0018] Figure 2 The graph shows the conversion results of key amino acid residues in the P450 substrate channel.

[0019] Figure 3 The graph shows the current efficiency results of key amino acid residues in the P450 substrate channel. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0021] This invention provides a method for improving the electrocatalytic rate of enzymes. Rhodococcus jostii RHA1 The P450 mutant maintains stable enzyme catalytic activity.

[0022] This invention discloses a heme iron cytochrome P450 mutant with enhanced enzyme electrocatalytic rate, using the amino acid sequence as shown in SEQ ID NO.1 as the parent. Specifically, the parent is mutated at position 82 (leucine) to glutamine Q (SEQ ID NO.2), position 167 (phenylalanine) to methionine M (SEQ ID NO.3), and position 280 (valine) to threonine T (SEQ ID NO.4).

[0023] The present invention also discloses recombinant plasmids containing the above-mentioned genes.

[0024] In one embodiment, the expression vector for the recombinant plasmid includes, but is not limited to, the pET series, pACYC series, or pGEX series. In one embodiment, pET-28a is used as the vector.

[0025] The present invention also discloses the above-mentioned mutants, or the host cells of the above-mentioned recombinant plasmids.

[0026] In one embodiment, Escherichia coli (E. coli) Escherichia coli, E. coli ( ) serves as the host, expressing the P450 mutant.

[0027] The present invention also discloses the above-mentioned P450 mutant with improved enzyme electrocatalysis rate, or the above-mentioned gene, or the above-mentioned recombinant plasmid, or the application of the above-mentioned P450 mutant in enzyme electrocatalysis.

[0028] The target mutant was obtained by amplification of plasmids using site-directed mutagenesis primers.

[0029] Specifically, the host cell monoclonal strain containing the recombinant plasmid was inoculated into 5 mL of liquid LB medium and cultured overnight at 37°C to obtain the seed culture.

[0030] The seed culture was inoculated into LB medium at a rate of 1%, and cultured at 37°C until the biomass OD 600 reached 0.6-0.8. Then, an inducer was added and cultured at 16°C for another 12-16 h. The cells were then collected by centrifugation.

[0031] The cells collected by centrifugation were homogenized under high pressure, and the lysate was centrifuged at 4℃ and 8000 rpm for 25 min to obtain crude enzyme solution.

[0032] The crude enzyme solution was subjected to nickel column affinity chromatography to obtain the pure enzyme of the P450 mutant.

[0033] This invention obtains a P450 mutant with an improved electrocatalytic rate by performing site-directed saturation mutagenesis on a P450 enzyme derived from Rhodococcus.

[0034] The specific experimental and verification process is as follows.

[0035] Example 1

[0036] Using the Caver website (https: / / caver.cz / ), key amino acid residues in the substrate channel of the P450 enzyme were predicted. Site-directed saturation mutagenesis was performed on key amino acid residues using L81, L82, F167, L230, L231, D236, T237, V280, and F283.

[0037] The required cells and reagents are as follows: expression plasmid pET-28a was obtained from our laboratory, and E. coli Top 10 and BL21(DE3) were prepared in-house or available commercially; nickel affinity chromatography packing material was purchased from Sigma-Aldrich, and column packing was performed as needed during protein purification; 5-aminolevulinic acid (ALA) and β-nicotinamide adenine dinucleotide coenzyme reduction (NADH) were purchased from Aladdin, and the remaining reagents were domestically produced or imported analytical grade.

[0038] The target mutant was obtained by PCR amplification using site-directed mutagenesis primers with the mutant plasmid pET-28a-P450 containing information from previous laboratory work as a template. The PCR amplification program was as follows: pre-denaturation at 98℃ for 2 min; denaturation at 98℃ for 10 s, annealing at 56℃ for 30 s, extension at 72℃ for 90 s, 25 cycles; extension at 72℃ for 5 min. The above products were digested with DpnI at 37℃ for 1 h, purified using a DNA purification kit, and then transformed into E. coli competent cells Top 10 to obtain positive clones. Single clones were selected and sequenced to obtain different mutants. Recombinant plasmids from 19 mutants were extracted for later use. The specific primer sequences are shown in Table 1.

[0039]

[0040] Example 2: Expression and screening of P450 mutants

[0041] The P450 mutant plasmid obtained in Example 1 was transformed into E. coli BL21(DE3) for P450 mutant expression. The specific steps are as follows:

[0042] (1) Preparation of Escherichia coli strains expressing P450 mutant. The screened positive strains were streaked on LB solid medium (100 μg / mL kanamycin) until single colonies grew. Single colonies were picked and inoculated into liquid LB medium and cultured in a shaker at 37℃ and 200 rpm for 12-16 h to obtain seed culture.

[0043] (2) Mutant enzyme induction and screening. The induction conditions for 57 mutant enzymes were the same as those for wild-type P450. The seed culture that had been cultured overnight was inoculated into LB medium at a 1% inoculum and cultured at 37°C until the biomass OD600 reached 0.6-0.8. Then, isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.2 mM and the cells were induced at 16°C for 12-16 h. The cells were collected by centrifugation at 4°C. The collected cells were homogenized by high pressure (1000 bar) for 1 min in lysis buffer (50 mM PBS, pH 7.0) and then centrifuged at 8000 rpm at 4°C for 25 min. The supernatant was collected. The supernatant was directly fed into a nickel affinity chromatography column. Impurities were eluted with equilibration buffer (50 mM PBS, pH 7.0, containing 20 mM imidazole), followed by elution with elution buffer (50 mM PBS, pH 7.0, containing 200 mM imidazole). The target protein was concentrated by centrifugation and diluted with imidazole to below 0.1 mM.

[0044] Example 3: Substrate conversion performance characterization of P450 mutant

[0045] The mutant from Example 2 was reacted with chaperone proteins (pudaoxorubicin reductase PdR and pudaoxorubicin Pdx) under the following conditions: 30 µM P450, 30 µM Pdx, 30 µM Pdr, 1000 U / L catalase, 1 mM 3,4-dimethoxybenzoic acid, 3 mM NADPH, and 20 mM MOPS buffer. After 1 hour of reaction, the reaction was terminated with 10% acetonitrile. 100 μL of the reaction solution was added to 100 μL of acetylacetone colorimetric solution and the reaction was carried out at 100 °C. The absorbance was read at 412 nm. One molecule of 3,4-dimethoxybenzoic acid was converted to one molecule of vanillic acid and one molecule of formaldehyde. The substrate conversion rate was calculated using the formaldehyde yield. Figure 1 and Figure 2 ).Depend on Figure 1 As can be seen from the data, when scanning the alanine content of the predicted key amino acids for the substrate channels, L82, F167, and V280 showed the lowest substrate conversion rates, indicating that these three sites may have a significant impact on enzyme catalytic activity. Subsequently, site-directed saturation mutagenesis was performed on these three sites, such as... Figure 2 As shown, the catalytic activity varies between 0% and 200% of that of the wild type.

[0046] Example 4: Electrocatalytic performance characterization of the P450 mutant

[0047] Glassy carbon electrode (GCE) was selected as the base electrode. The GCE was polished with Al₂O₃, then ultrasonically cleaned with ultrapure water and ethanol for 5 min each, and dried under nitrogen. 10 μL of 1 mg / mL graphene oxide (GO) solution was drop-coated onto the GCE surface, labeled GO / GCE. After air drying, 10 μL of the above-mentioned active mutant (2 g / L) was drop-coated onto the GO / GCE electrode surface, air-dried, and the unbound mutant was washed with ultrapure water and dried under N₂, labeled P450 / GO / GCE. Cyclic voltammetry was performed on all mutant electrodes, with a scan range of -0.8 V to 0.6 V and a scan rate of 1 mV / s. The electrolyte was 50 mM Tris-HCl (pH 7.0) with 5 mM 3,4-dimethoxybenzoic acid added. After stabilization, the last cycle was used as the final analytical basis. Compare the reduction currents of different mutant enzyme electrodes for 3,4-dimethoxybenzoic acid, and screen mutant electrodes based on the magnitude of the reduction current. Figure 3 ).Depend on Figure 3 It can be seen that V280T has the highest electrocatalytic rate, which is about 30 times that of the wild type, while its catalytic activity is 80% of that of the wild type.

[0048] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method of demethoxylation, characterized by, The lignin-degrading enzyme electrode or enzyme electrocatalytic reactor is contacted with a reaction solution comprising a substrate, a catalase and a buffer, and an electric current is generated, wherein the substrate is 3,4-dimethoxybenzoic acid, and the heme-iron cytochrome P450 enzyme mutant on the enzyme electrode is used to demethoxylate 3,4-dimethoxybenzoic acid, thereby achieving degradation of 3,4-dimethoxybenzoic acid; The lignin-degrading enzyme electrode metal electrode and the heme-iron cytochrome P450 enzyme mutant covering the surface of the metal electrode, wherein the heme-iron cytochrome P450 enzyme mutant has an amino acid sequence shown in SEQ ID NO:1 as a parent sequence, and the leucine at position 82, the phenylalanine at position 167 or the valine at position 280 is mutated to glutamine, methionine or threonine, respectively. The enzyme electrocatalytic reactor comprises a lignin-degrading enzyme electrode, wherein the lignin-degrading enzyme electrode metal electrode and the heme-iron cytochrome P450 enzyme mutant covering the surface of the metal electrode, wherein the heme-iron cytochrome P450 enzyme mutant has an amino acid sequence shown in SEQ ID NO:1 as a parent sequence, and the leucine at position 82, the phenylalanine at position 167 or the valine at position 280 is mutated to glutamine, methionine or threonine, respectively.

2. Use of a heme iron cytochrome P450 enzyme mutant in the degradation of lignin, characterized in that, The heme-iron cytochrome P450 enzyme mutant has an amino acid sequence shown in SEQ ID NO:1 as a parent sequence, and the leucine at position 82, the phenylalanine at position 167 or the valine at position 280 is mutated to glutamine, methionine or threonine, respectively.

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