Heme iron cytochrome P450 enzyme mutant with improved enzyme electrocatalysis rate

Abstract

The invention provides a heme iron cytochrome P450 enzyme mutant with improved enzyme electrocatalysis rate and application thereof. The heme iron cytochrome P450 enzyme mutant is obtained by performing site-directed mutagenesis on heme iron cytochrome enzyme with an amino acid sequence as shown in SEQ ID NO: 1 as a parent; according to the present invention, the 82nd leucine of SEQ ID NO: 1 is subjected to site-specific mutagenesis to obtain glutamine, the 167th phenylalanine of SEQ ID NO: 1 is subjected to site-specific mutagenesis to obtain methionine or the 280th valine of SEQ ID NO: 1 is subjected to site-specific mutagenesis to obtain threonine, such that the mutant with the improved enzyme electro-catalysis rate is obtained so as to solve the technical problem of low enzyme electro-catalysis rate of the heme iron cytochrome enzyme in the prior art;

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and particularly relates to a heme iron cytochrome P450 enzyme mutant with improved enzyme electrocatalytic rate and application. BACKGROUND

[0002] Lignin is a complex aromatic polymer formed by cross-linking of phenylpropane structural units through various chemical bonds. The complexity and heterogeneity of its structure make efficient degradation and economic conversion of lignin a worldwide problem. In recent years, research has found that the unique non-specific oxidation-reduction system of microorganisms and their multi-metabolic degradation ability of aromatic compounds provide a new way for the resource utilization of lignin. Because most lignin-derived aromatic monomers contain methoxy groups at different positions of the benzene ring, demethylation process becomes the key rate-limiting step of the metabolism of such compounds. Among the demethylation 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. U.S.A. 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 exists near it. This class of enzymes usually needs ferredoxin-NADPH reductase as an oxidation-reduction partner protein. FNRN contains an N-terminal [2Fe-2S] iron-sulfur cluster domain, a flavin adenine dinucleotide (FAD) binding site and an NADH binding site. Its mechanism of action is as follows: after the substrate binds to the P450 enzyme, the partner protein FNRN obtains electrons from NAD(P)H and transfers them to the P450 enzyme to catalyze the substrate reaction. + The reduction enzyme serves as an oxidation-reduction partner protein. FNRN contains an N-terminal [2Fe-2S] iron-sulfur cluster domain, a flavin adenine dinucleotide (FAD) binding site and an NADH binding site. Its mechanism of action is as follows: after the substrate binds to the P450 enzyme, the partner protein FNRN obtains electrons from NAD(P)H and transfers them to the P450 enzyme to catalyze the substrate reaction.

[0003] In most P450 enzyme-catalyzed reactions, not only does it need to rely on expensive NAD(P)H as an electron donor, but also needs the participation of chaperone proteins, which significantly increases the complexity of the reaction process. In addition, NAD(P)H is costly and difficult to regenerate. Therefore, it is urgent to develop new technologies that can replace NAD(P)H and chaperone proteins. Enzyme electrocatalysis technology can replace chaperone protein and NAD(P)H-dependent system due to its mild reaction conditions and excellent selectivity. At present, many P450 enzymes have been successfully applied to enzyme electro-synthesis (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)) through this technology. Although most of the current P450 enzymes can directly obtain electrons from the electrode to achieve substrate conversion (i.e. direct electron transfer, DET), due to the fact that the catalytic center of the enzyme, iron porphyrin ring, is deeply buried in the enzyme molecule and is not an electron transfer center itself, resulting in generally low enzyme electrocatalysis rate. In recent years, research on improving the enzyme electrocatalysis 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.), all of which aim to improve the microenvironment of the enzyme electrode. If we can start from the protein crystal structure and improve the enzyme electrocatalysis rate by rationally modifying key amino acid residues, it is expected to open up a new research direction for breaking through the bottleneck of P450 enzyme electrocatalysis efficiency. SUMMARY

[0004] The application provides a heme iron cytochrome enzyme mutant with improved enzyme electrocatalysis rate and application. The heme iron cytochrome enzyme with the amino acid sequence shown in SEQ ID NO: 1 as the parent is subjected to site-directed mutation, the leucine at the 82nd position of SEQ ID NO: 1 is mutated to glutamine, the phenylalanine at the 167th position is mutated to methionine, or the valine at the 280th position is mutated to threonine, to obtain a mutant with improved enzyme electrocatalysis rate, thereby solving the technical problem of low enzyme electrocatalysis rate of the heme iron cytochrome enzyme in the prior art.

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

[0006] According to another aspect of the present application, there is provided a nucleotide encoding the heme iron cytochrome P450 enzyme mutant.

[0007] According to another aspect of the present application, there is provided a recombinant plasmid comprising the nucleotide.

[0008] According to another aspect of the present application, there is provided a host cell comprising the recombinant plasmid.

[0009] According to another aspect of the present application, there is provided a lignin-degrading enzyme electrode comprising a metal electrode and the heme iron cytochrome P450 enzyme mutant coated on the surface of the metal electrode.

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

[0011] According to another aspect of the present application, there is provided a method for demethoxylation, wherein the lignin-degrading enzyme electrode or the enzyme electrocatalytic reactor is contacted with a reaction solution comprising a substrate, catalase and a buffer, and an electric current is generated, the substrate being 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.

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

[0013] Overall, the above technical solutions conceived by the present application have the following technical advantages compared with the prior art: (1) The mutant in the present application significantly improves the electron transfer rate. The present application optimizes the electron transfer path from the active center to the surface of the electrode by site-directed mutagenesis of key amino acid sites on the substrate channel, reduces the interface electron transfer resistance, and enables the enzyme electrode to obtain a higher electron transfer rate under the same conditions.

[0014] (2) The mutant in the application maintains the catalytic characteristics and stability of the enzyme. The amino acid mutation in the application is directed to the substrate channel rather than the active center, which optimizes the electron transfer without significantly changing the substrate recognition ability and overall stability of the enzyme, thereby prolonging the service life of the enzyme electrode.

[0015] (3) The catalytic efficiency of the P450 mutant provided by the application is maintained at 80-90% of the wild type, and the enzyme electrocatalytic efficiency is increased by 2-30 times. In the enzyme electrocatalytic reaction, the rate is mainly determined by the electron transfer rate and the enzyme catalytic ability, so the catalytic efficiency of the mutant obtained by the application is basically maintained, and the main reason for the increase of the enzyme electrocatalytic rate is the increase of the electron transfer rate. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The figure is the conversion rate result graph of the P450 substrate channel related amino acid residues.

[0017] Figure 2 The figure is the conversion rate result graph of the key amino acid residues of the P450 substrate channel.

[0018] Figure 3 The figure is the current efficiency result graph of the key amino acid residues of the P450 substrate channel. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical scheme and advantages of the application clearer, the application will be further described in detail below in combination with the drawings and examples. It should be understood that the specific examples described herein are only used to explain the application and not to limit the application. In addition, the technical features involved in each embodiment of the application described below can be combined with each other as long as they do not conflict with each other.

[0020] The application provides a P450 mutant derived from P450 of CYP102A1, which has improved enzyme electrocatalytic rate and stable enzyme catalytic activity. Rhodococcus jostii RHA1

[0021] The application discloses a heme iron cytochrome enzyme P450 mutant with improved enzyme electrocatalytic rate, which takes the amino acid sequence as shown in SEQ ID NO. 1 as a parent sequence, and specifically, leucine at the 82nd position of the parent sequence is mutated to glutamine Q (SEQ ID NO. 2), phenylalanine at the 167th position is mutated to methionine M (SEQ ID NO. 3), and valine at the 280th position is mutated to threonine T (SEQ ID NO. 4).

[0022] The application further discloses a recombinant plasmid containing the gene.

[0023] ​In an embodiment, the expression vector of the recombinant plasmid includes but is not limited to pET series, pACYC series or pGEX series, etc. In an embodiment, pET-28a is used as the vector.

[0024] The application also discloses the mutant or the host cell of the recombinant plasmid.

[0025] In an embodiment, the P450 mutant is expressed in Escherichia coli (E. coli) as the host. Escherichia coli, E. coli ) as the host.

[0026] The application also discloses the P450 mutant with improved enzyme electrocatalytic rate, the gene or the recombinant plasmid or the application of the P450 mutant in enzyme electrocatalysis.

[0027] The target mutant is obtained by plasmid amplification using a site-directed mutation primer.

[0028] Specifically, the host cell containing the recombinant plasmid is inoculated into 5 mL liquid LB medium at a single clone, and a seed liquid is obtained by culturing at 37°C overnight; The seed liquid is inoculated into LB medium at an inoculation amount of 1%, and the biomass OD 600 is cultured at 37°C to reach 0.6-0.8, then an inducer is added, and the culture is continued at 16°C for 12-16 h, and the bacterial cells are collected by centrifugation; The centrifugally collected cells are subjected to high-pressure homogenization crushing, and the crushing liquid is centrifuged at 8000 rpm at 4°C for 25 min to obtain a crude enzyme liquid; The crude enzyme liquid is subjected to nickel column affinity chromatography to obtain a P450 mutant pure enzyme.

[0029] The application finally obtains the P450 mutant with improved enzyme electrocatalytic rate by performing site-directed saturation mutation on the P450 enzyme from Rhodococcus.

[0030] The specific experiment and verification process are as follows.

[0031] Example 1 The key amino acid residues of the substrate channel of the P450 enzyme are predicted by using the Caver website (https: / / caver.cz / ), and the key amino acid residues L81, L82, F167, L230, L231, D236, T237, V280 and F283 are subjected to site-directed saturation mutation. The required cells and reagents are as follows: expression plasmid pET-28a is obtained from the laboratory, and E. coli Top 10, BL21(DE3) are self-made, and can also be purchased; nickel affinity chromatography filler is purchased from Sigma Company, and is columned according to the needs during protein purification; 5-amino levulinic acid (ALA) and β-nicotinamide adenine dinucleotide coenzyme reduced form (NADH) are purchased from Aladdin Company, and the rest of the reagents are domestic or imported analytical pure.

[0032] The target mutant is obtained by PCR amplification using a site-directed mutation primer and the mutant plasmid pET-28a-P450 containing the previous work of the laboratory as a template. The PCR amplification procedure is as follows: pre-denaturation 98℃ 2min; denaturation 98℃ 10s, annealing 56℃ 30s, extension 72℃ 90s, 25 cycles; extension 72℃ 5min. The above product is digested with DpnI at 37℃ for 1h, purified using a DNA reagent purification box, and then transformed into E. coli competent Top 10 to obtain positive clones, and after picking a single colony and sequencing correctly, different mutants are obtained, and the recombinant plasmids of 19 mutants are extracted for standby. The specific primer sequence is as shown in Table 1:

[0033] Example 2: Expression and screening of P450 mutants The P450 mutant plasmid obtained in Example 1 is transformed into E. coli BL21(DE3) for P450 mutant expression. The specific operation steps are as follows: (1) Preparation of E. coli strain for expressing P450 mutants. The positive strains screened are streaked on LB solid medium (100 μg / mL kanamycin) and cultured to grow single colonies, and a single colony is inoculated in liquid LB medium and cultured in a shaker at 37℃, 200 rpm for 12-16 h to obtain a seed liquid.

[0034] (2) Mutant enzyme induction expression and screening. The induction expression conditions of 57 mutant enzymes and the induction expression conditions of wild type P450 are consistent. The seed liquid of overnight culture is inoculated in LB medium according to 1% inoculation amount, and cultured at 37°C until the biomass OD600 reaches 0.6-0.8, then the inducer isopropyl-β-D-thiogalactoside (IPTG) is added with a final concentration of 0.2 mM, and induced at 16°C for 12-16 h, and the bacterial cells are collected by centrifugation at 4°C. The collected bacterial cells are added to the crushing liquid (50 mM PBS, pH 7.0) for high pressure homogenization crushing (pressure 1000 bar) for 1 min, then centrifuged at 8000 rpm at 4°C for 25 min to collect the supernatant. The supernatant is directly introduced into the nickel affinity chromatography column, and the impurity protein is eluted with the equilibrium buffer (50 mM PBS, pH 7.0 containing 20 mM imidazole), and then the target protein is eluted with the elution buffer (50 mM PBS, pH 7.0 containing 200 mM imidazole). The target protein is concentrated by centrifugation and the imidazole concentration is diluted to below 0.1 mM.

[0035] Example 3: Performance characterization of substrate conversion rate of P450 mutants The mutants in Example 2 were reacted with chaperone proteins (putidaredoxin reductase PdR and putidaredoxin Pdx), and the reaction conditions were as follows: 30 µM P450, 30 µM Pdx, 30 µM Pdr, 1000 U / l catalase, 1 mM 3,4-dimethoxybenzoic acid, 3 mM NADPH, 20 mM MOPS buffer solution. After 1 hour of reaction, the reaction was terminated with 10% acetonitrile, and 100 ul of the reaction solution was added to 100 ul of acetylacetone color developing liquid for color development at 100°C, and the absorbance value was read at 412 nm. One molecule of 3,4-dimethoxybenzoic acid is converted into one molecule of vanillic acid and one molecule of formaldehyde, and the substrate conversion rate is calculated by the yield of formaldehyde. Figure 1 and Figure 2 It can be known from Figure 1 that when the predicted substrate channel key amino acids are subjected to alanine scanning, the substrate conversion rates of L82, F167, and V280 are the lowest, indicating that the three sites may have important influence on enzyme catalytic activity, and then the three sites are subjected to site-directed saturation mutation, as shown in Figure 2 , the catalytic activity varies between 0-200% of the wild type.

[0036] Example 4: Electrochemical performance characterization of P450 mutants The glassy carbon electrode (GCE) was selected as a basic electrode, the glassy carbon electrode was polished with Al2O3, and then was cleaned with ultrapure water and ethanol for 5 min each time, and was dried by nitrogen blowing. 10 ul of 1 mg / mL graphene oxide (GO) solution was absorbed and was dropped on the surface of the GCE, and was marked as GO / GCE. After natural drying, 10 ul of 2 g / L of the above-mentioned active mutant was absorbed and was dropped on the surface of the GO / GCE electrode, and was naturally dried, and the unbound mutant was cleaned with ultrapure water, and was dried by N2 blowing, and was marked as P450 / GO / GCE. First, cyclic voltammetry scanning was performed on all mutant electrodes, the scanning range was-0.8 V to 0.6 V, the scanning rate was 1 mV / s, the electrolyte was 50 mM Tris-HCl (pH 7.0), 5 mM 3,4-dimethoxybenzoic acid was added, and the last circle was taken as the final analysis basis after stabilization. The reduction current of different mutant enzyme electrodes on 3,4-dimethoxybenzoic acid was compared, and the mutant electrode was screened according to the size of the reduction current Figure 3 ). It can be known from Figure 3 that the electrocatalytic rate of V280T is the highest, which is about 30 times of the wild type, and the catalytic activity is 80% of the wild type.

[0037] Those skilled in the art can easily understand that the above only describes the preferred embodiments of the present application and is not intended to limit the present application, and any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A mutant of heme iron cytochrome P450 enzyme with enhanced electrocatalytic rate, characterized in that, The heme iron cytochrome P450 enzyme mutant uses the amino acid sequence shown in SEQ ID NO:1 as the parent and performs site-directed mutations at the following positions: 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.

2. A nucleotide, characterized in that, The nucleotide encodes the heme iron cytochrome P450 enzyme mutant of claim 1.

3. A recombinant plasmid, characterized in that, The recombinant plasmid comprises the nucleotides described in claim 2.

4. A host cell, characterized in that, The host cell includes the recombinant plasmid as described in claim 3.

5. An enzyme electrode for degrading lignin, characterized in that, The invention includes a metal electrode and a heme iron cytochrome P450 enzyme mutant as described in claim 1, which covers the surface of the metal electrode.

6. An enzyme electrocatalytic reactor, characterized in that, The enzyme electrocatalytic reactor includes the enzyme electrode for degrading lignin as described in claim 5.

7. A method for demethoxylation, characterized in that, The enzyme electrode for lignin degradation according to claim 5 or the enzyme electrocatalytic reactor according to claim 6 is brought into contact with a reaction solution to generate an electric current for reaction. The reaction solution includes a substrate, catalase, and a buffer solution. The substrate is 3,4-dimethoxybenzoic acid. The 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.

8. The application of the heme iron cytochrome P450 enzyme mutant as described in claim 1 in the degradation of lignin.

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